Quantification of ligand density and stoichiometry on the surface of liposomes using single-molecule fluorescence imaging.

PubMed

Belfiore, Lisa; Spenkelink, Lisanne M; Ranson, Marie; van Oijen, Antoine M; Vine, Kara L

2018-05-28

Despite the longstanding existence of liposome technology in drug delivery applications, there have been no ligand-directed liposome formulations approved for clinical use to date. This lack of translation is due to several factors, one of which is the absence of molecular tools for the robust quantification of ligand density on the surface of liposomes. We report here for the first time the quantification of proteins attached to the surface of small unilamellar liposomes using single-molecule fluorescence imaging. Liposomes were surface-functionalized with fluorescently labeled human proteins previously validated to target the cancer cell surface biomarkers plasminogen activator inhibitor-2 (PAI-2) and trastuzumab (TZ, Herceptin®). These protein-conjugated liposomes were visualized using a custom-built wide-field fluorescence microscope with single-molecule sensitivity. By counting the photobleaching steps of the fluorescently labeled proteins, we calculated the number of attached proteins per liposome, which was 11 ± 4 proteins for single-ligand liposomes. Imaging of dual-ligand liposomes revealed stoichiometries of the two attached proteins in accordance with the molar ratios of protein added during preparation. Preparation of PAI-2/TZ dual-ligand liposomes via two different methods revealed that the post-insertion method generated liposomes with a more equal representation of the two differently sized proteins, demonstrating the ability of this preparation method to enable better control of liposome protein densities. We conclude that the single-molecule imaging method presented here is an accurate and reliable quantification tool for determining ligand density and stoichiometry on the surface of liposomes. This method has the potential to allow for comprehensive characterization of novel ligand-directed liposomes that should facilitate the translation of these nanotherapies through to the clinic. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

PubMed

Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

2015-01-01

Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

Protein quantification using a cleavable reporter peptide.

PubMed

Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

2015-02-06

Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described.

Quantification of Flavin-containing Monooxygenases 1, 3, and 5 in Human Liver Microsomes by UPLC-MRM-Based Targeted Quantitative Proteomics and Its Application to the Study of Ontogeny.

PubMed

Chen, Yao; Zane, Nicole R; Thakker, Dhiren R; Wang, Michael Zhuo

2016-07-01

Flavin-containing monooxygenases (FMOs) have a significant role in the metabolism of small molecule pharmaceuticals. Among the five human FMOs, FMO1, FMO3, and FMO5 are the most relevant to hepatic drug metabolism. Although age-dependent hepatic protein expression, based on immunoquantification, has been reported previously for FMO1 and FMO3, there is very little information on hepatic FMO5 protein expression. To overcome the limitations of immunoquantification, an ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-based targeted quantitative proteomic method was developed and optimized for the quantification of FMO1, FMO3, and FMO5 in human liver microsomes (HLM). A post-in silico product ion screening process was incorporated to verify LC-MRM detection of potential signature peptides before their synthesis. The developed method was validated by correlating marker substrate activity and protein expression in a panel of adult individual donor HLM (age 39-67 years). The mean (range) protein expression of FMO3 and FMO5 was 46 (26-65) pmol/mg HLM protein and 27 (11.5-49) pmol/mg HLM protein, respectively. To demonstrate quantification of FMO1, a panel of fetal individual donor HLM (gestational age 14-20 weeks) was analyzed. The mean (range) FMO1 protein expression was 7.0 (4.9-9.7) pmol/mg HLM protein. Furthermore, the ontogenetic protein expression of FMO5 was evaluated in fetal, pediatric, and adult HLM. The quantification of FMO proteins also was compared using two different calibration standards, recombinant proteins versus synthetic signature peptides, to assess the ratio between holoprotein versus total protein. In conclusion, a UPLC-MRM-based targeted quantitative proteomic method has been developed for the quantification of FMO enzymes in HLM. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Quantification of Flavin-containing Monooxygenases 1, 3, and 5 in Human Liver Microsomes by UPLC-MRM-Based Targeted Quantitative Proteomics and Its Application to the Study of Ontogeny

PubMed Central

Chen, Yao; Zane, Nicole R.; Thakker, Dhiren R.

2016-01-01

Flavin-containing monooxygenases (FMOs) have a significant role in the metabolism of small molecule pharmaceuticals. Among the five human FMOs, FMO1, FMO3, and FMO5 are the most relevant to hepatic drug metabolism. Although age-dependent hepatic protein expression, based on immunoquantification, has been reported previously for FMO1 and FMO3, there is very little information on hepatic FMO5 protein expression. To overcome the limitations of immunoquantification, an ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-based targeted quantitative proteomic method was developed and optimized for the quantification of FMO1, FMO3, and FMO5 in human liver microsomes (HLM). A post-in silico product ion screening process was incorporated to verify LC-MRM detection of potential signature peptides before their synthesis. The developed method was validated by correlating marker substrate activity and protein expression in a panel of adult individual donor HLM (age 39–67 years). The mean (range) protein expression of FMO3 and FMO5 was 46 (26–65) pmol/mg HLM protein and 27 (11.5–49) pmol/mg HLM protein, respectively. To demonstrate quantification of FMO1, a panel of fetal individual donor HLM (gestational age 14–20 weeks) was analyzed. The mean (range) FMO1 protein expression was 7.0 (4.9–9.7) pmol/mg HLM protein. Furthermore, the ontogenetic protein expression of FMO5 was evaluated in fetal, pediatric, and adult HLM. The quantification of FMO proteins also was compared using two different calibration standards, recombinant proteins versus synthetic signature peptides, to assess the ratio between holoprotein versus total protein. In conclusion, a UPLC-MRM-based targeted quantitative proteomic method has been developed for the quantification of FMO enzymes in HLM. PMID:26839369

Development and validation of a high-performance liquid chromatography method for the quantification of talazoparib in rat plasma: Application to plasma protein binding studies.

PubMed

Hidau, Mahendra Kumar; Kolluru, Srikanth; Palakurthi, Srinath

2018-02-01

A sensitive and selective RP-HPLC method has been developed and validated for the quantification of a highly potent poly ADP ribose polymerase inhibitor talazoparib (TZP) in rat plasma. Chromatographic separation was performed with isocratic elution method. Absorbance for TZP was measured with a UV detector (SPD-20A UV-vis) at a λ max of 227 nm. Protein precipitation was used to extract the drug from plasma samples using methanol-acetonitrile (65:35) as the precipitating solvent. The method proved to be sensitive and reproducible over a 100-2000 ng/mL linearity range with a lower limit of quantification (LLQC) of 100 ng/mL. TZP recovery was found to be >85%. Following analytical method development and validation, it was successfully employed to determine the plasma protein binding of TZP. TZP has a high level of protein binding in rat plasma (95.76 ± 0.38%) as determined by dialysis method. Copyright © 2017 John Wiley & Sons, Ltd.

Neutron-Encoded Protein Quantification by Peptide Carbamylation

NASA Astrophysics Data System (ADS)

Ulbrich, Arne; Merrill, Anna E.; Hebert, Alexander S.; Westphall, Michael S.; Keller, Mark P.; Attie, Alan D.; Coon, Joshua J.

2014-01-01

We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet.

New approach for the quantification of processed animal proteins in feed using light microscopy.

PubMed

Veys, P; Baeten, V

2010-07-01

A revision of European Union's total feed ban on animal proteins in feed will need robust quantification methods, especially for control analyses, if tolerance levels are to be introduced, as for fishmeal in ruminant feed. In 2006, a study conducted by the Community Reference Laboratory for Animal Proteins in feedstuffs (CRL-AP) demonstrated the deficiency of the official quantification method based on light microscopy. The study concluded that the method had to be revised. This paper puts forward an improved quantification method based on three elements: (1) the preparation of permanent slides with an optical adhesive preserving all morphological markers of bones necessary for accurate identification and precision counting; (2) the use of a counting grid eyepiece reticle; and (3) new definitions for correction factors for the estimated portions of animal particles in the sediment. This revised quantification method was tested on feeds adulterated at different levels with bovine meat and bone meal (MBM) and fishmeal, and it proved to be effortless to apply. The results obtained were very close to the expected values of contamination levels for both types of adulteration (MBM or fishmeal). Calculated values were not only replicable, but also reproducible. The advantages of the new approach, including the benefits of the optical adhesive used for permanent slide mounting and the experimental conditions that need to be met to implement the new method correctly, are discussed.

Quantitative interaction proteomics using mass spectrometry.

PubMed

Wepf, Alexander; Glatter, Timo; Schmidt, Alexander; Aebersold, Ruedi; Gstaiger, Matthias

2009-03-01

We present a mass spectrometry-based strategy for the absolute quantification of protein complex components isolated through affinity purification. We quantified bait proteins via isotope-labeled reference peptides corresponding to an affinity tag sequence and prey proteins by label-free correlational quantification using the precursor ion signal intensities of proteotypic peptides generated in reciprocal purifications. We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network.

Protein Quantification by Elemental Mass Spectrometry: An Experiment for Graduate Students

ERIC Educational Resources Information Center

Schwarz, Gunnar; Ickert, Stefanie; Wegner, Nina; Nehring, Andreas; Beck, Sebastian; Tiemann, Ruediger; Linscheid, Michael W.

2014-01-01

A multiday laboratory experiment was designed to integrate inductively coupled plasma-mass spectrometry (ICP-MS) in the context of protein quantification into an advanced practical course in analytical and environmental chemistry. Graduate students were familiar with the analytical methods employed, whereas the combination of bioanalytical assays…

A new dimethyl labeling-based SID-MRM-MS method and its application to three proteases involved in insulin maturation.

PubMed

Cheng, Dongwan; Zheng, Li; Hou, Junjie; Wang, Jifeng; Xue, Peng; Yang, Fuquan; Xu, Tao

2015-01-01

The absolute quantification of target proteins in proteomics involves stable isotope dilution coupled with multiple reactions monitoring mass spectrometry (SID-MRM-MS). The successful preparation of stable isotope-labeled internal standard peptides is an important prerequisite for the SID-MRM absolute quantification methods. Dimethyl labeling has been widely used in relative quantitative proteomics and it is fast, simple, reliable, cost-effective, and applicable to any protein sample, making it an ideal candidate method for the preparation of stable isotope-labeled internal standards. MRM mass spectrometry is of high sensitivity, specificity, and throughput characteristics and can quantify multiple proteins simultaneously, including low-abundance proteins in precious samples such as pancreatic islets. In this study, a new method for the absolute quantification of three proteases involved in insulin maturation, namely PC1/3, PC2 and CPE, was developed by coupling a stable isotope dimethyl labeling strategy for internal standard peptide preparation with SID-MRM-MS quantitative technology. This method offers a new and effective approach for deep understanding of the functional status of pancreatic β cells and pathogenesis in diabetes.

A new background subtraction method for Western blot densitometry band quantification through image analysis software.

PubMed

Gallo-Oller, Gabriel; Ordoñez, Raquel; Dotor, Javier

2018-06-01

Since its first description, Western blot has been widely used in molecular labs. It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex protein mixtures. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. Due to the technical knowledge required for densitometry analysis together with the resources availability, standard office scanners are often used for the imaging acquisition of developed Western blot films. Furthermore, the use of semi-quantitative software as ImageJ (Java-based image-processing and analysis software) is clearly increasing in different scientific fields. In this work, we describe the use of office scanner coupled with the ImageJ software together with a new image background subtraction method for accurate Western blot quantification. The proposed method represents an affordable, accurate and reproducible approximation that could be used in the presence of limited resources availability. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid capillary electrophoresis approach for the quantification of ewe milk adulteration with cow milk.

PubMed

Trimboli, Francesca; Morittu, Valeria Maria; Cicino, Caterina; Palmieri, Camillo; Britti, Domenico

2017-10-13

The substitution of ewe milk with more economic cow milk is a common fraud. Here we present a capillary electrophoresis method for the quantification of ewe milk in ovine/bovine milk mixtures, which allows for the rapid and inexpensive recognition of ewe milk adulteration with cow milk. We utilized a routine CE method for human blood and urine proteins analysis, which fulfilled the separation of skimmed milk proteins in alkaline buffer. Under this condition, ovine and bovine milk exhibited a recognizable and distinct CE protein profiles, with a specific ewe peak showing a reproducible migration zone in ovine/bovine mixtures. Based on ewe specific CE peak, we developed a method for ewe milk quantification in ovine/bovine skimmed milk mixtures, which showed good linearity, precision and accuracy, and a minimum amount of detectable fraudulent cow milk equal to 5%. Copyright © 2017 Elsevier B.V. All rights reserved.

Digital Quantification of Proteins and mRNA in Single Mammalian Cells.

PubMed

Albayrak, Cem; Jordi, Christian A; Zechner, Christoph; Lin, Jing; Bichsel, Colette A; Khammash, Mustafa; Tay, Savaş

2016-03-17

Absolute quantification of macromolecules in single cells is critical for understanding and modeling biological systems that feature cellular heterogeneity. Here we show extremely sensitive and absolute quantification of both proteins and mRNA in single mammalian cells by a very practical workflow that combines proximity ligation assay (PLA) and digital PCR. This digital PLA method has femtomolar sensitivity, which enables the quantification of very small protein concentration changes over its entire 3-log dynamic range, a quality necessary for accounting for single-cell heterogeneity. We counted both endogenous (CD147) and exogenously expressed (GFP-p65) proteins from hundreds of single cells and determined the correlation between CD147 mRNA and the protein it encodes. Using our data, a stochastic two-state model of the central dogma was constructed and verified using joint mRNA/protein distributions, allowing us to estimate transcription burst sizes and extrinsic noise strength and calculate the transcription and translation rate constants in single mammalian cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Ratiometric Raman Spectroscopy for Quantification of Protein Oxidative Damage

PubMed Central

Jiang, Dongping; Yanney, Michael; Zou, Sige; Sygula, Andrzej

2009-01-01

A novel ratiometric Raman spectroscopic (RMRS) method has been developed for quantitative determination of protein carbonyl levels. Oxidized bovine serum albumin (BSA) and oxidized lysozyme were used as model proteins to demonstrate this method. The technique involves conjugation of protein carbonyls with dinitrophenyl hydrazine (DNPH), followed by drop coating deposition Raman spectral acquisition (DCDR). The RMRS method is easy to implement as it requires only one conjugation reaction, a single spectral acquisition, and does not require sample calibration. Characteristic peaks from both protein and DNPH moieties are obtained in a single spectral acquisition, allowing the protein carbonyl level to be calculated from the peak intensity ratio. Detection sensitivity for the RMRS method is ~0.33 pmol carbonyl/measurement. Fluorescence and/or immunoassay based techniques only detect a signal from the labeling molecule and thus yield no structural or quantitative information for the modified protein while the RMRS technique provides for protein identification and protein carbonyl quantification in a single experiment. PMID:19457432

QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification.

PubMed

Simpson, Deborah M; Beynon, Robert J

2012-09-01

Systems biology requires knowledge of the absolute amounts of proteins in order to model biological processes and simulate the effects of changes in specific model parameters. Quantification concatamers (QconCATs) are established as a method to provide multiplexed absolute peptide standards for a set of target proteins in isotope dilution standard experiments. Two or more quantotypic peptides representing each of the target proteins are concatenated into a designer gene that is metabolically labelled with stable isotopes in Escherichia coli or other cellular or cell-free systems. Co-digestion of a known amount of QconCAT with the target proteins generates a set of labelled reference peptide standards for the unlabelled analyte counterparts, and by using an appropriate mass spectrometry platform, comparison of the intensities of the peptide ratios delivers absolute quantification of the encoded peptides and in turn the target proteins for which they are surrogates. In this review, we discuss the criteria and difficulties associated with surrogate peptide selection and provide examples in the design of QconCATs for quantification of the proteins of the nuclear factor κB pathway.

Detecting transitions in protein dynamics using a recurrence quantification analysis based bootstrap method.

PubMed

Karain, Wael I

2017-11-28

Proteins undergo conformational transitions over different time scales. These transitions are closely intertwined with the protein's function. Numerous standard techniques such as principal component analysis are used to detect these transitions in molecular dynamics simulations. In this work, we add a new method that has the ability to detect transitions in dynamics based on the recurrences in the dynamical system. It combines bootstrapping and recurrence quantification analysis. We start from the assumption that a protein has a "baseline" recurrence structure over a given period of time. Any statistically significant deviation from this recurrence structure, as inferred from complexity measures provided by recurrence quantification analysis, is considered a transition in the dynamics of the protein. We apply this technique to a 132 ns long molecular dynamics simulation of the β-Lactamase Inhibitory Protein BLIP. We are able to detect conformational transitions in the nanosecond range in the recurrence dynamics of the BLIP protein during the simulation. The results compare favorably to those extracted using the principal component analysis technique. The recurrence quantification analysis based bootstrap technique is able to detect transitions between different dynamics states for a protein over different time scales. It is not limited to linear dynamics regimes, and can be generalized to any time scale. It also has the potential to be used to cluster frames in molecular dynamics trajectories according to the nature of their recurrence dynamics. One shortcoming for this method is the need to have large enough time windows to insure good statistical quality for the recurrence complexity measures needed to detect the transitions.

Large scale systematic proteomic quantification from non-metastatic to metastatic colorectal cancer

NASA Astrophysics Data System (ADS)

Yin, Xuefei; Zhang, Yang; Guo, Shaowen; Jin, Hong; Wang, Wenhai; Yang, Pengyuan

2015-07-01

A systematic proteomic quantification of formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues from stage I to stage IIIC was performed in large scale. 1017 proteins were identified with 338 proteins in quantitative changes by label free method, while 341 proteins were quantified with significant expression changes among 6294 proteins by iTRAQ method. We found that proteins related to migration expression increased and those for binding and adherent decreased during the colorectal cancer development according to the gene ontology (GO) annotation and ingenuity pathway analysis (IPA). The integrin alpha 5 (ITA5) in integrin family was focused, which was consistent with the metastasis related pathway. The expression level of ITA5 decreased in metastasis tissues and the result has been further verified by Western blotting. Another two cell migration related proteins vitronectin (VTN) and actin-related protein (ARP3) were also proved to be up-regulated by both mass spectrometry (MS) based quantification results and Western blotting. Up to now, our result shows one of the largest dataset in colorectal cancer proteomics research. Our strategy reveals a disease driven omics-pattern for the metastasis colorectal cancer.

Overcoming biofluid protein complexity during targeted mass spectrometry detection and quantification of protein biomarkers by MRM cubed (MRM3).

PubMed

Jeudy, Jeremy; Salvador, Arnaud; Simon, Romain; Jaffuel, Aurore; Fonbonne, Catherine; Léonard, Jean-François; Gautier, Jean-Charles; Pasquier, Olivier; Lemoine, Jerome

2014-02-01

Targeted mass spectrometry in the so-called multiple reaction monitoring mode (MRM) is certainly a promising way for the precise, accurate, and multiplexed measurement of proteins and their genetic or posttranslationally modified isoforms. MRM carried out on a low-resolution triple quadrupole instrument faces a lack of specificity when addressing the quantification of weakly concentrated proteins. In this case, extensive sample fractionation or immunoenrichment alleviates signal contamination by interferences, but in turn decreases assay performance and throughput. Recently, MRM(3) was introduced as an alternative to MRM to improve the limit of quantification of weakly concentrated protein biomarkers. In the present work, we compare MRM and MRM(3) modes for the detection of biomarkers in plasma and urine. Calibration curves drawn with MRM and MRM(3) showed a similar range of linearity (R(2) > 0.99 for both methods) with protein concentrations above 1 μg/mL in plasma and a few nanogram per milliliter in urine. In contrast, optimized MRM(3) methods improve the limits of quantification by a factor of 2 to 4 depending on the targeted peptide. This gain arises from the additional MS(3) fragmentation step, which significantly removes or decreases interfering signals within the targeted transition channels.

Novel isotopic N, N-Dimethyl Leucine (iDiLeu) Reagents Enable Absolute Quantification of Peptides and Proteins Using a Standard Curve Approach

NASA Astrophysics Data System (ADS)

Greer, Tyler; Lietz, Christopher B.; Xiang, Feng; Li, Lingjun

2015-01-01

Absolute quantification of protein targets using liquid chromatography-mass spectrometry (LC-MS) is a key component of candidate biomarker validation. One popular method combines multiple reaction monitoring (MRM) using a triple quadrupole instrument with stable isotope-labeled standards (SIS) for absolute quantification (AQUA). LC-MRM AQUA assays are sensitive and specific, but they are also expensive because of the cost of synthesizing stable isotope peptide standards. While the chemical modification approach using mass differential tags for relative and absolute quantification (mTRAQ) represents a more economical approach when quantifying large numbers of peptides, these reagents are costly and still suffer from lower throughput because only two concentration values per peptide can be obtained in a single LC-MS run. Here, we have developed and applied a set of five novel mass difference reagents, isotopic N, N-dimethyl leucine (iDiLeu). These labels contain an amine reactive group, triazine ester, are cost effective because of their synthetic simplicity, and have increased throughput compared with previous LC-MS quantification methods by allowing construction of a four-point standard curve in one run. iDiLeu-labeled peptides show remarkably similar retention time shifts, slightly lower energy thresholds for higher-energy collisional dissociation (HCD) fragmentation, and high quantification accuracy for trypsin-digested protein samples (median errors <15%). By spiking in an iDiLeu-labeled neuropeptide, allatostatin, into mouse urine matrix, two quantification methods are validated. The first uses one labeled peptide as an internal standard to normalize labeled peptide peak areas across runs (<19% error), whereas the second enables standard curve creation and analyte quantification in one run (<8% error).

Simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by LC-MSE.

PubMed

Creskey, Marybeth C; Li, Changgui; Wang, Junzhi; Girard, Michel; Lorbetskie, Barry; Gravel, Caroline; Farnsworth, Aaron; Li, Xuguang; Smith, Daryl G S; Cyr, Terry D

2012-07-06

Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neuraminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1 μg/dose. The SRID assays require the availability of strain-specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neuraminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS(E), a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current manufacturing and quality control testing procedures. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Establishing Ion Ratio Thresholds Based on Absolute Peak Area for Absolute Protein Quantification using Protein Cleavage Isotope Dilution Mass Spectrometry

PubMed Central

Loziuk, Philip L.; Sederoff, Ronald R.; Chiang, Vincent L.; Muddiman, David C.

2014-01-01

Quantitative mass spectrometry has become central to the field of proteomics and metabolomics. Selected reaction monitoring is a widely used method for the absolute quantification of proteins and metabolites. This method renders high specificity using several product ions measured simultaneously. With growing interest in quantification of molecular species in complex biological samples, confident identification and quantitation has been of particular concern. A method to confirm purity or contamination of product ion spectra has become necessary for achieving accurate and precise quantification. Ion abundance ratio assessments were introduced to alleviate some of these issues. Ion abundance ratios are based on the consistent relative abundance (RA) of specific product ions with respect to the total abundance of all product ions. To date, no standardized method of implementing ion abundance ratios has been established. Thresholds by which product ion contamination is confirmed vary widely and are often arbitrary. This study sought to establish criteria by which the relative abundance of product ions can be evaluated in an absolute quantification experiment. These findings suggest that evaluation of the absolute ion abundance for any given transition is necessary in order to effectively implement RA thresholds. Overall, the variation of the RA value was observed to be relatively constant beyond an absolute threshold ion abundance. Finally, these RA values were observed to fluctuate significantly over a 3 year period, suggesting that these values should be assessed as close as possible to the time at which data is collected for quantification. PMID:25154770

Normalized Quantitative Western Blotting Based on Standardized Fluorescent Labeling.

PubMed

Faden, Frederik; Eschen-Lippold, Lennart; Dissmeyer, Nico

2016-01-01

Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein-epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable. To date, it is a standard procedure to normalize individual bands of immunodetected proteins of interest from a WB lane to other individual bands of so-called housekeeping proteins of the same sample lane. These are usually detected by an independent antibody or colorimetric detection and do not reflect the real total protein of a sample. Housekeeping proteins-assumed to be constitutively expressed mostly independent of developmental and environmental states-can greatly differ in their expression under these various conditions. Therefore, they actually do not represent a reliable reference to normalize the target protein's abundance to the total amount of protein contained in each lane of a blot.Here, we demonstrate the Smart Protein Layers (SPL) technology, a combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer via WB. SPL allows a rapid and highly sensitive protein visualization and quantification with a sensitivity comparable to conventional silver staining with a 1000-fold higher dynamic range. For normalization, standardization and quantification of protein gels and WBs, a sample-dependent bi-fluorescent standard reagent is applied and, for accurate quantification of data derived from different experiments, a second calibration standard is used. Together, the precise quantification of protein expression by lane-to-lane, gel-to-gel, and blot-to-blot comparisons is facilitated especially with respect to experiments in the area of proteostasis dealing with highly variable protein levels and involving protein degradation mutants and treatments modulating protein abundance.

Multiplex quantification of protein toxins in human biofluids and food matrices using immunoextraction and high-resolution targeted mass spectrometry.

PubMed

Dupré, Mathieu; Gilquin, Benoit; Fenaille, François; Feraudet-Tarisse, Cécile; Dano, Julie; Ferro, Myriam; Simon, Stéphanie; Junot, Christophe; Brun, Virginie; Becher, François

2015-08-18

The development of rapid methods for unambiguous identification and precise quantification of protein toxins in various matrices is essential for public health surveillance. Nowadays, analytical strategies classically rely on sensitive immunological assays, but mass spectrometry constitutes an attractive complementary approach thanks to direct measurement and protein characterization ability. We developed here an innovative multiplex immuno-LC-MS/MS method for the simultaneous and specific quantification of the three potential biological warfare agents, ricin, staphylococcal enterotoxin B, and epsilon toxin, in complex human biofluids and food matrices. At least 7 peptides were targeted for each toxin (43 peptides in total) with a quadrupole-Orbitrap high-resolution instrument for exquisite detection specificity. Quantification was performed using stable isotope-labeled toxin standards spiked early in the sample. Lower limits of quantification were determined at or close to 1 ng·mL(-1). The whole process was successfully applied to the quantitative analysis of toxins in complex samples such as milk, human urine, and plasma. Finally, we report new data on toxin stability with no evidence of toxin degradation in milk in a 48 h time frame, allowing relevant quantitative toxin analysis for samples collected in this time range.

Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins.

PubMed

Mesquita, Cristina S; Oliveira, Raquel; Bento, Fátima; Geraldo, Dulce; Rodrigues, João V; Marcos, João C

2014-08-01

This work proposes a modification of the 2,4-dinitrophenylhydrazine (DNPH) spectrophotometric assay commonly used to evaluate the concentration of carbonyl groups in oxidized proteins. In this approach NaOH is added to the protein solution after the addition of DNPH, shifting the maximum absorbance wavelength of the derivatized protein from 370 to 450nm. This reduces the interference of DNPH and allows the direct quantification in the sample solution without the need for the precipitation, washing, and resuspension steps that are carried out in the traditional DNPH method. The two methods were compared under various conditions and are statistically equivalent. Copyright © 2014 Elsevier Inc. All rights reserved.

A multi-center study benchmarks software tools for label-free proteome quantification

PubMed Central

Gillet, Ludovic C; Bernhardt, Oliver M.; MacLean, Brendan; Röst, Hannes L.; Tate, Stephen A.; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I.; Aebersold, Ruedi; Tenzer, Stefan

2016-01-01

The consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra), a method that uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test datasets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation windows setups. For consistent evaluation we developed LFQbench, an R-package to calculate metrics of precision and accuracy in label-free quantitative MS, and report the identification performance, robustness and specificity of each software tool. Our reference datasets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics. PMID:27701404

Establishing a reliable multiple reaction monitoring-based method for the quantification of obesity-associated comorbidities in serum and adipose tissue requires intensive clinical validation.

PubMed

Oberbach, Andreas; Schlichting, Nadine; Neuhaus, Jochen; Kullnick, Yvonne; Lehmann, Stefanie; Heinrich, Marco; Dietrich, Arne; Mohr, Friedrich Wilhelm; von Bergen, Martin; Baumann, Sven

2014-12-05

Multiple reaction monitoring (MRM)-based mass spectrometric quantification of peptides and their corresponding proteins has been successfully applied for biomarker validation in serum. The option of multiplexing offers the chance to analyze various proteins in parallel, which is especially important in obesity research. Here, biomarkers that reflect multiple comorbidities and allow monitoring of therapy outcomes are required. Besides the suitability of established MRM assays for serum protein quantification, it is also feasible for analysis of tissues secreting the markers of interest. Surprisingly, studies comparing MRM data sets with established methods are rare, and therefore the biological and clinical value of most analytes remains questionable. A MRM method using nano-UPLC-MS/MS for the quantification of obesity related surrogate markers for several comorbidities in serum, plasma, visceral and subcutaneous adipose tissue was established. Proteotypic peptides for complement C3, adiponectin, angiotensinogen, and plasma retinol binding protein (RBP4) were quantified using isotopic dilution analysis and compared to the standard ELISA method. MRM method variabilities were mainly below 10%. The comparison with other MS-based approaches showed a good correlation. However, large differences in absolute quantification for complement C3 and adiponectin were obtained compared to ELISA, while less marked differences were observed for angiotensinogen and RBP4. The verification of MRM in obesity was performed to discriminate first lean and obese phenotype and second to monitor excessive weight loss after gastric bypass surgery in a seven-month follow-up. The presented MRM assay was able to discriminate obese phenotype from lean and monitor weight loss related changes of surrogate markers. However, inclusion of additional biomarkers was necessary to interpret the MRM data on obesity phenotype properly. In summary, the development of disease-related MRMs should include a step of matching the MRM data with clinically approved standard methods and defining reference values in well-sized representative age, gender, and disease-matched cohorts.

Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies.

PubMed

Espinosa-de la Garza, Carlos E; Perdomo-Abúndez, Francisco C; Campos-García, Víctor R; Pérez, Néstor O; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

2013-09-01

In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Collagen Quantification in Tissue Specimens.

PubMed

Coentro, João Quintas; Capella-Monsonís, Héctor; Graceffa, Valeria; Wu, Zhuning; Mullen, Anne Maria; Raghunath, Michael; Zeugolis, Dimitrios I

2017-01-01

Collagen is the major extracellular protein in mammals. Accurate quantification of collagen is essential in the biomaterials (e.g., reproducible collagen scaffold fabrication), drug discovery (e.g., assessment of collagen in pathophysiologies, such as fibrosis), and tissue engineering (e.g., quantification of cell-synthesized collagen) fields. Although measuring hydroxyproline content is the most widely used method to quantify collagen in biological specimens, the process is very laborious. To this end, the Sircol™ Collagen Assay is widely used due to its inherent simplicity and convenience. However, this method leads to overestimation of collagen content due to the interaction of Sirius red with basic amino acids of non-collagenous proteins. Herein, we describe the addition of an ultrafiltration purification step in the process to accurately determine collagen content in tissues.

Towards absolute quantification of allergenic proteins in food--lysozyme in wine as a model system for metrologically traceable mass spectrometric methods and certified reference materials.

PubMed

Cryar, Adam; Pritchard, Caroline; Burkitt, William; Walker, Michael; O'Connor, Gavin; Burns, Duncan Thorburn; Quaglia, Milena

2013-01-01

Current routine food allergen quantification methods, which are based on immunochemistry, offer high sensitivity but can suffer from issues of specificity and significant variability of results. MS approaches have been developed, but currently lack metrological traceability. A feasibility study on the application of metrologically traceable MS-based reference procedures was undertaken. A proof of concept involving proteolytic digestion and isotope dilution MS for quantification of protein allergens in a food matrix was undertaken using lysozyme in wine as a model system. A concentration of lysozyme in wine of 0.95 +/- 0.03 microg/g was calculated based on the concentrations of two peptides, confirming that this type of analysis is viable at allergenically meaningful concentrations. The challenges associated with this promising method were explored; these included peptide stability, chemical modification, enzymatic digestion, and sample cleanup. The method is suitable for the production of allergen in food certified reference materials, which together with the achieved understanding of the effects of sample preparation and of the matrix on the final results, will assist in addressing the bias of the techniques routinely used and improve measurement confidence. Confirmation of the feasibility of MS methods for absolute quantification of an allergenic protein in a food matrix with results traceable to the International System of Units is a step towards meaningful comparison of results for allergen proteins among laboratories. This approach will also underpin risk assessment and risk management of allergens in the food industry, and regulatory compliance of the use of thresholds or action levels when adopted.

Absolute Quantification of Middle- to High-Abundant Plasma Proteins via Targeted Proteomics.

PubMed

Dittrich, Julia; Ceglarek, Uta

2017-01-01

The increasing number of peptide and protein biomarker candidates requires expeditious and reliable quantification strategies. The utilization of liquid chromatography coupled to quadrupole tandem mass spectrometry (LC-MS/MS) for the absolute quantitation of plasma proteins and peptides facilitates the multiplexed verification of tens to hundreds of biomarkers from smallest sample quantities. Targeted proteomics assays derived from bottom-up proteomics principles rely on the identification and analysis of proteotypic peptides formed in an enzymatic digestion of the target protein. This protocol proposes a procedure for the establishment of a targeted absolute quantitation method for middle- to high-abundant plasma proteins waiving depletion or enrichment steps. Essential topics as proteotypic peptide identification and LC-MS/MS method development as well as sample preparation and calibration strategies are described in detail.

[Building Mass Spectrometry Spectral Libraries of Human Cancer Cell Lines].

PubMed

Faktor, J; Bouchal, P

Cancer research often focuses on protein quantification in model cancer cell lines and cancer tissues. SWATH (sequential windowed acquisition of all theoretical fragment ion spectra), the state of the art method, enables the quantification of all proteins included in spectral library. Spectral library contains fragmentation patterns of each detectable protein in a sample. Thorough spectral library preparation will improve quantitation of low abundant proteins which usually play an important role in cancer. Our research is focused on the optimization of spectral library preparation aimed at maximizing the number of identified proteins in MCF-7 breast cancer cell line. First, we optimized the sample preparation prior entering the mass spectrometer. We examined the effects of lysis buffer composition, peptide dissolution protocol and the material of sample vial on the number of proteins identified in spectral library. Next, we optimized mass spectrometry (MS) method for spectral library data acquisition. Our thorough optimized protocol for spectral library building enabled the identification of 1,653 proteins (FDR < 1%) in 1 µg of MCF-7 lysate. This work contributed to the enhancement of protein coverage in SWATH digital biobanks which enable quantification of arbitrary protein from physically unavailable samples. In future, high quality spectral libraries could play a key role in preparing of patient proteome digital fingerprints.Key words: biomarker - mass spectrometry - proteomics - digital biobanking - SWATH - protein quantificationThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 7. 5. 2016Accepted: 9. 6. 2016.

Quantitative proteome analysis using isobaric peptide termini labeling (IPTL).

PubMed

Arntzen, Magnus O; Koehler, Christian J; Treumann, Achim; Thiede, Bernd

2011-01-01

The quantitative comparison of proteome level changes across biological samples has become an essential feature in proteomics that remains challenging. We have recently introduced isobaric peptide termini labeling (IPTL), a novel strategy for isobaric quantification based on the derivatization of peptide termini with complementary isotopically labeled reagents. Unlike non-isobaric quantification methods, sample complexity at the MS level is not increased, providing improved sensitivity and protein coverage. The distinguishing feature of IPTL when comparing it to more established isobaric labeling methods (iTRAQ and TMT) is the presence of quantification signatures in all sequence-determining ions in MS/MS spectra, not only in the low mass reporter ion region. This makes IPTL a quantification method that is accessible to mass spectrometers with limited capabilities in the low mass range. Also, the presence of several quantification points in each MS/MS spectrum increases the robustness of the quantification procedure.

Simultaneous quantification of α-lactalbumin and β-casein in human milk using ultra-performance liquid chromatography with tandem mass spectrometry based on their signature peptides and winged isotope internal standards.

PubMed

Chen, Qi; Zhang, Jingshun; Ke, Xing; Lai, Shiyun; Li, Duo; Yang, Jinchuan; Mo, Weimin; Ren, Yiping

2016-09-01

In recent years, there is an increasing need to measure the concentration of individual proteins in human milk, instead of total human milk proteins. Due to lack of human milk protein standards, there are only few quantification methods established. The objective of the present work was to develop a simple and rapid quantification method for simultaneous determination of α-lactalbumin and β-casein in human milk using signature peptides according to a modified quantitative proteomics strategy. The internal standards containing the signature peptide sequences were synthesized with isotope-labeled amino acids. The purity of synthesized peptides as standards was determined by amino acid analysis method and area normalization method. The contents of α-lactalbumin and β-casein in human milk were measured according to the equimolar relationship between the two proteins and their corresponding signature peptides. The method validation results showed a satisfied linearity (R(2)>0.99) and recoveries (97.2-102.5% for α-lactalbumin and 99.5-100.3% for β-casein). The limit of quantification for α-lactalbumin and β-casein was 8.0mg/100g and 1.2mg/100g, respectively. CVs for α-lactalbumin and β-casein in human milk were 5.2% and 3.0%. The contents of α-lactalbumin and β-casein in 147 human milk samples were successfully determined by the established method and their contents were 205.5-578.2mg/100g and 116.4-467.4mg/100g at different lactation stages. The developed method allows simultaneously determination of α-lactalbumin and β-casein in human milk. The quantitative strategy based on signature peptide should be applicable to other endogenous proteins in breast milk and other body fluids. Copyright © 2016 Elsevier B.V. All rights reserved.

Microvolume protein concentration determination using the NanoDrop 2000c spectrophotometer.

PubMed

Desjardins, Philippe; Hansen, Joel B; Allen, Michael

2009-11-04

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 microL. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

A novel nano-immunoassay method for quantification of proteins from CD138-purified myeloma cells: biological and clinical utility

PubMed Central

Misiewicz-Krzeminska, Irena; Corchete, Luis Antonio; Rojas, Elizabeta A.; Martínez-López, Joaquín; García-Sanz, Ramón; Oriol, Albert; Bladé, Joan; Lahuerta, Juan-José; Miguel, Jesús San; Mateos, María-Victoria; Gutiérrez, Norma C.

2018-01-01

Protein analysis in bone marrow samples from patients with multiple myeloma has been limited by the low concentration of proteins obtained after CD138+ cell selection. A novel approach based on capillary nano-immunoassay could make it possible to quantify dozens of proteins from each myeloma sample in an automated manner. Here we present a method for the accurate and robust quantification of the expression of multiple proteins extracted from CD138-purified multiple myeloma samples frozen in RLT Plus buffer, which is commonly used for nucleic acid preservation and isolation. Additionally, the biological and clinical value of this analysis for a panel of 12 proteins essential to the pathogenesis of multiple myeloma was evaluated in 63 patients with newly diagnosed multiple myeloma. The analysis of the prognostic impact of CRBN/Cereblon and IKZF1/Ikaros mRNA/protein showed that only the protein levels were able to predict progression-free survival of patients; mRNA levels were not associated with prognosis. Interestingly, high levels of Cereblon and Ikaros proteins were associated with longer progression-free survival only in patients who received immunomodulatory drugs and not in those treated with other drugs. In conclusion, the capillary nano-immunoassay platform provides a novel opportunity for automated quantification of the expression of more than 20 proteins in CD138+ primary multiple myeloma samples. PMID:29545347

A novel nano-immunoassay method for quantification of proteins from CD138-purified myeloma cells: biological and clinical utility.

PubMed

Misiewicz-Krzeminska, Irena; Corchete, Luis Antonio; Rojas, Elizabeta A; Martínez-López, Joaquín; García-Sanz, Ramón; Oriol, Albert; Bladé, Joan; Lahuerta, Juan-José; Miguel, Jesús San; Mateos, María-Victoria; Gutiérrez, Norma C

2018-05-01

Protein analysis in bone marrow samples from patients with multiple myeloma has been limited by the low concentration of proteins obtained after CD138 + cell selection. A novel approach based on capillary nano-immunoassay could make it possible to quantify dozens of proteins from each myeloma sample in an automated manner. Here we present a method for the accurate and robust quantification of the expression of multiple proteins extracted from CD138-purified multiple myeloma samples frozen in RLT Plus buffer, which is commonly used for nucleic acid preservation and isolation. Additionally, the biological and clinical value of this analysis for a panel of 12 proteins essential to the pathogenesis of multiple myeloma was evaluated in 63 patients with newly diagnosed multiple myeloma. The analysis of the prognostic impact of CRBN /Cereblon and IKZF1 /Ikaros mRNA/protein showed that only the protein levels were able to predict progression-free survival of patients; mRNA levels were not associated with prognosis. Interestingly, high levels of Cereblon and Ikaros proteins were associated with longer progression-free survival only in patients who received immunomodulatory drugs and not in those treated with other drugs. In conclusion, the capillary nano-immunoassay platform provides a novel opportunity for automated quantification of the expression of more than 20 proteins in CD138 + primary multiple myeloma samples. Copyright © 2018 Ferrata Storti Foundation.

A tool for selective inline quantification of co-eluting proteins in chromatography using spectral analysis and partial least squares regression.

PubMed

Brestrich, Nina; Briskot, Till; Osberghaus, Anna; Hubbuch, Jürgen

2014-07-01

Selective quantification of co-eluting proteins in chromatography is usually performed by offline analytics. This is time-consuming and can lead to late detection of irregularities in chromatography processes. To overcome this analytical bottleneck, a methodology for selective protein quantification in multicomponent mixtures by means of spectral data and partial least squares regression was presented in two previous studies. In this paper, a powerful integration of software and chromatography hardware will be introduced that enables the applicability of this methodology for a selective inline quantification of co-eluting proteins in chromatography. A specific setup consisting of a conventional liquid chromatography system, a diode array detector, and a software interface to Matlab® was developed. The established tool for selective inline quantification was successfully applied for a peak deconvolution of a co-eluting ternary protein mixture consisting of lysozyme, ribonuclease A, and cytochrome c on SP Sepharose FF. Compared to common offline analytics based on collected fractions, no loss of information regarding the retention volumes and peak flanks was observed. A comparison between the mass balances of both analytical methods showed, that the inline quantification tool can be applied for a rapid determination of pool yields. Finally, the achieved inline peak deconvolution was successfully applied to make product purity-based real-time pooling decisions. This makes the established tool for selective inline quantification a valuable approach for inline monitoring and control of chromatographic purification steps and just in time reaction on process irregularities. © 2014 Wiley Periodicals, Inc.

Quantification of protein concentration using UV absorbance and Coomassie dyes.

PubMed

Noble, James E

2014-01-01

The measurement of a solubilized protein concentration in solution is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantification assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Where multiple samples need measurement, and/or the sample volume and concentration is limited, preparations of the Coomassie dye commonly known as the Bradford assay can be used. © 2014 Elsevier Inc. All rights reserved.

Optimized approaches for quantification of drug transporters in tissues and cells by MRM proteomics.

PubMed

Prasad, Bhagwat; Unadkat, Jashvant D

2014-07-01

Drug transporter expression in tissues (in vivo) usually differs from that in cell lines used to measure transporter activity (in vitro). Therefore, quantification of transporter expression in tissues and cell lines is important to develop scaling factor for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated drug disposition. Since traditional immunoquantification methods are semiquantitative, targeted proteomics is now emerging as a superior method to quantify proteins, including membrane transporters. This superiority is derived from the selectivity, precision, accuracy, and speed of analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, LC-MS/MS proteomics has broader applicability because it does not require selective antibodies for individual proteins. There are a number of recent research and review papers that discuss the use of LC-MS/MS for transporter quantification. Here, we have compiled from the literature various elements of MRM proteomics to provide a comprehensive systematic strategy to quantify drug transporters. This review emphasizes practical aspects and challenges in surrogate peptide selection, peptide qualification, peptide synthesis and characterization, membrane protein isolation, protein digestion, sample preparation, LC-MS/MS parameter optimization, method validation, and sample analysis. In particular, bioinformatic tools used in method development and sample analysis are discussed in detail. Various pre-analytical and analytical sources of variability that should be considered during transporter quantification are highlighted. All these steps are illustrated using P-glycoprotein (P-gp) as a case example. Greater use of quantitative transporter proteomics will lead to a better understanding of the role of drug transporters in drug disposition.

Translational value of liquid chromatography coupled with tandem mass spectrometry-based quantitative proteomics for in vitro-in vivo extrapolation of drug metabolism and transport and considerations in selecting appropriate techniques.

PubMed

Al Feteisi, Hajar; Achour, Brahim; Rostami-Hodjegan, Amin; Barber, Jill

2015-01-01

Drug-metabolizing enzymes and transporters play an important role in drug absorption, distribution, metabolism and excretion and, consequently, they influence drug efficacy and toxicity. Quantification of drug-metabolizing enzymes and transporters in various tissues is therefore essential for comprehensive elucidation of drug absorption, distribution, metabolism and excretion. Recent advances in liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) have improved the quantification of pharmacologically relevant proteins. This report presents an overview of mass spectrometry-based methods currently used for the quantification of drug-metabolizing enzymes and drug transporters, mainly focusing on applications and cost associated with various quantitative strategies based on stable isotope-labeled standards (absolute quantification peptide standards, quantification concatemers, protein standards for absolute quantification) and label-free analysis. In mass spectrometry, there is no simple relationship between signal intensity and analyte concentration. Proteomic strategies are therefore complex and several factors need to be considered when selecting the most appropriate method for an intended application, including the number of proteins and samples. Quantitative strategies require appropriate mass spectrometry platforms, yet choice is often limited by the availability of appropriate instrumentation. Quantitative proteomics research requires specialist practical skills and there is a pressing need to dedicate more effort and investment to training personnel in this area. Large-scale multicenter collaborations are also needed to standardize quantitative strategies in order to improve physiologically based pharmacokinetic models.

Quantification of Major Royal Jelly Protein 1 in Fresh Royal Jelly by Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Lin, Na; Chen, Si; Zhang, Hong; Li, Junmin; Fu, Linglin

2018-02-07

Major royal jelly protein 1 (MRJP1) is the most abundant protein in royal jelly (RJ), and the level of MRJP1 has been suggested as a promising parameter for standardization and evaluation of RJ authenticity in quality. Here, a quantitative method was developed for the quantification of MRJP1 in RJ based on a signature peptide and a stable isotope-labeled internal standard peptide FFDYDFGSDER*(R*, 13 C 6 , 15 N 4 ) by ultraperformance liquid chromatography-tandem mass spectrometry. Recoveries of the established method ranged from 85.33 to 95.80%, and both the intra- and interday precision were RSD < 4.97%. Quantification results showed that content of MRJP1 in fresh RJ was 41.96-55.01 mg/g. Abnormal levels of MRJP1 were found in three commercial RJs and implied that these samples were of low quality and might be adulterated. Results of the present work suggested that the developed method could be successfully applied to quantify MRJP1 in RJ and also could evaluate the quality of RJ.

A multicenter study benchmarks software tools for label-free proteome quantification.

PubMed

Navarro, Pedro; Kuharev, Jörg; Gillet, Ludovic C; Bernhardt, Oliver M; MacLean, Brendan; Röst, Hannes L; Tate, Stephen A; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I; Aebersold, Ruedi; Tenzer, Stefan

2016-11-01

Consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH 2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from sequential window acquisition of all theoretical fragment-ion spectra (SWATH)-MS, which uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test data sets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation-window setups. For consistent evaluation, we developed LFQbench, an R package, to calculate metrics of precision and accuracy in label-free quantitative MS and report the identification performance, robustness and specificity of each software tool. Our reference data sets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics.

The impact of carbon-13 and deuterium on relative quantification of proteins using stable isotope diethyl labeling.

PubMed

Koehler, Christian J; Arntzen, Magnus Ø; Thiede, Bernd

2015-05-15

Stable isotopic labeling techniques are useful for quantitative proteomics. A cost-effective and convenient method for diethylation by reductive amination was established. The impact using either carbon-13 or deuterium on quantification accuracy and precision was investigated using diethylation. We established an effective approach for stable isotope labeling by diethylation of amino groups of peptides. The approach was validated using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nanospray liquid chromatography/electrospray ionization (nanoLC/ESI)-ion trap/orbitrap for mass spectrometric analysis as well as MaxQuant for quantitative data analysis. Reaction conditions with low reagent costs, high yields and minor side reactions were established for diethylation. Furthermore, we showed that diethylation can be applied to up to sixplex labeling. For duplex experiments, we compared diethylation in the analysis of the proteome of HeLa cells using acetaldehyde-(13) C(2)/(12) C(2) and acetaldehyde-(2) H(4)/(1) H(4). Equal numbers of proteins could be identified and quantified; however, (13) C(4)/(12) C(4) -diethylation revealed a lower variance of quantitative peptide ratios within proteins resulting in a higher precision of quantified proteins and less falsely regulated proteins. The results were compared with dimethylation showing minor effects because of the lower number of deuteriums. The described approach for diethylation of primary amines is a cost-effective and accurate method for up to sixplex relative quantification of proteomes. (13) C(4)/(12) C(4) -diethylation enables duplex quantification based on chemical labeling without using deuterium which reduces identification of false-negatives and increases the quality of the quantification results. Copyright © 2015 John Wiley & Sons, Ltd.

Stable isotope labelling methods in mass spectrometry-based quantitative proteomics.

PubMed

Chahrour, Osama; Cobice, Diego; Malone, John

2015-09-10

Mass-spectrometry based proteomics has evolved as a promising technology over the last decade and is undergoing a dramatic development in a number of different areas, such as; mass spectrometric instrumentation, peptide identification algorithms and bioinformatic computational data analysis. The improved methodology allows quantitative measurement of relative or absolute protein amounts, which is essential for gaining insights into their functions and dynamics in biological systems. Several different strategies involving stable isotopes label (ICAT, ICPL, IDBEST, iTRAQ, TMT, IPTL, SILAC), label-free statistical assessment approaches (MRM, SWATH) and absolute quantification methods (AQUA) are possible, each having specific strengths and weaknesses. Inductively coupled plasma mass spectrometry (ICP-MS), which is still widely recognised as elemental detector, has recently emerged as a complementary technique to the previous methods. The new application area for ICP-MS is targeting the fast growing field of proteomics related research, allowing absolute protein quantification using suitable elemental based tags. This document describes the different stable isotope labelling methods which incorporate metabolic labelling in live cells, ICP-MS based detection and post-harvest chemical label tagging for protein quantification, in addition to summarising their pros and cons. Copyright © 2015 Elsevier B.V. All rights reserved.

Update on Controls for Isolation and Quantification Methodology of Extracellular Vesicles Derived from Adipose Tissue Mesenchymal Stem Cells

PubMed Central

Franquesa, Marcella; Hoogduijn, Martin J.; Ripoll, Elia; Luk, Franka; Salih, Mahdi; Betjes, Michiel G. H.; Torras, Juan; Baan, Carla C.; Grinyó, Josep M.; Merino, Ana Maria

2014-01-01

The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV. The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 μm). As a control, non-conditioned culture medium was used (control medium). To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV. PMID:25374572

Advances in targeted proteomics and applications to biomedical research

PubMed Central

Shi, Tujin; Song, Ehwang; Nie, Song; Rodland, Karin D.; Liu, Tao; Qian, Wei-Jun; Smith, Richard D.

2016-01-01

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications in human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed. PMID:27302376

Advances in targeted proteomics and applications to biomedical research

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Song, Ehwang; Nie, Song

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity (Shi et al., Proteomics, 12, 1074–1092, 2012) herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications inmore » human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed.« less

Evaluation of Direct Infusion-Multiple Reaction Monitoring Mass Spectrometry for Quantification of Heat Shock Proteins

PubMed Central

Xiang, Yun; Koomen, John M.

2012-01-01

Protein quantification with liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has emerged as a powerful platform for assessing panels of biomarkers. In this study, direct infusion, using automated, chip-based nanoelectrospray ionization, coupled with MRM (DI-MRM) is used for protein quantification. Removal of the LC separation step increases the importance of evaluating the ratios between the transitions. Therefore, the effects of solvent composition, analyte concentration, spray voltage, and quadrupole resolution settings on fragmentation patterns have been studied using peptide and protein standards. After DI-MRM quantification was evaluated for standards, quantitative assays for the expression of heat shock proteins (HSPs) were translated from LC-MRM to DI-MRM for implementation in cell line models of multiple myeloma. Requirements for DI-MRM assay development are described. Then, the two methods are compared; criteria for effective DI-MRM analysis are reported based on the analysis of HSP expression in digests of whole cell lysates. The increased throughput of DI-MRM analysis is useful for rapid analysis of large batches of similar samples, such as time course measurements of cellular responses to therapy. PMID:22293045

Accurate Quantification of Cardiovascular Biomarkers in Serum Using Protein Standard Absolute Quantification (PSAQ™) and Selected Reaction Monitoring*

PubMed Central

Huillet, Céline; Adrait, Annie; Lebert, Dorothée; Picard, Guillaume; Trauchessec, Mathieu; Louwagie, Mathilde; Dupuis, Alain; Hittinger, Luc; Ghaleh, Bijan; Le Corvoisier, Philippe; Jaquinod, Michel; Garin, Jérôme; Bruley, Christophe; Brun, Virginie

2012-01-01

Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies. PMID:22080464

Fast quantification of bovine milk proteins employing external cavity-quantum cascade laser spectroscopy.

PubMed

Schwaighofer, Andreas; Kuligowski, Julia; Quintás, Guillermo; Mayer, Helmut K; Lendl, Bernhard

2018-06-30

Analysis of proteins in bovine milk is usually tackled by time-consuming analytical approaches involving wet-chemical, multi-step sample clean-up procedures. The use of external cavity-quantum cascade laser (EC-QCL) based IR spectroscopy was evaluated as an alternative screening tool for direct and simultaneous quantification of individual proteins (i.e. casein and β-lactoglobulin) and total protein content in commercial bovine milk samples. Mid-IR spectra of protein standard mixtures were used for building partial least squares (PLS) regression models. A sample set comprising different milk types (pasteurized; differently processed extended shelf life, ESL; ultra-high temperature, UHT) was analysed and results were compared to reference methods. Concentration values of the QCL-IR spectroscopy approach obtained within several minutes are in good agreement with reference methods involving multiple sample preparation steps. The potential application as a fast screening method for estimating the heat load applied to liquid milk is demonstrated. Copyright © 2018 Elsevier Ltd. All rights reserved.

Classification of Dynamical Diffusion States in Single Molecule Tracking Microscopy

PubMed Central

Bosch, Peter J.; Kanger, Johannes S.; Subramaniam, Vinod

2014-01-01

Single molecule tracking of membrane proteins by fluorescence microscopy is a promising method to investigate dynamic processes in live cells. Translating the trajectories of proteins to biological implications, such as protein interactions, requires the classification of protein motion within the trajectories. Spatial information of protein motion may reveal where the protein interacts with cellular structures, because binding of proteins to such structures often alters their diffusion speed. For dynamic diffusion systems, we provide an analytical framework to determine in which diffusion state a molecule is residing during the course of its trajectory. We compare different methods for the quantification of motion to utilize this framework for the classification of two diffusion states (two populations with different diffusion speed). We found that a gyration quantification method and a Bayesian statistics-based method are the most accurate in diffusion-state classification for realistic experimentally obtained datasets, of which the gyration method is much less computationally demanding. After classification of the diffusion, the lifetime of the states can be determined, and images of the diffusion states can be reconstructed at high resolution. Simulations validate these applications. We apply the classification and its applications to experimental data to demonstrate the potential of this approach to obtain further insights into the dynamics of cell membrane proteins. PMID:25099798

Optimizing total reflection X-ray fluorescence for direct trace element quantification in proteins I: Influence of sample homogeneity and reflector type

NASA Astrophysics Data System (ADS)

Wellenreuther, G.; Fittschen, U. E. A.; Achard, M. E. S.; Faust, A.; Kreplin, X.; Meyer-Klaucke, W.

2008-12-01

Total reflection X-ray fluorescence (TXRF) is a very promising method for the direct, quick and reliable multi-elemental quantification of trace elements in protein samples. With the introduction of an internal standard consisting of two reference elements, scandium and gallium, a wide range of proteins can be analyzed, regardless of their salt content, buffer composition, additives and amino acid composition. This strategy also enables quantification of matrix effects. Two potential issues associated with drying have been considered in this study: (1) Formation of heterogeneous residues of varying thickness and/or density; and (2) separation of the internal standard and protein during drying (which has to be prevented to allow accurate quantification). These issues were investigated by microbeam X-ray fluorescence (μXRF) with special emphasis on (I) the influence of sample support and (II) the protein / buffer system used. In the first part, a model protein was studied on well established sample supports used in TXRF, PIXE and XRF (Mylar, siliconized quartz, Plexiglas and silicon). In the second part we imaged proteins of different molecular weight, oligomerization state, bound metals and solubility. A partial separation of protein and internal standard was only observed with untreated silicon, suggesting it may not be an adequate support material. Siliconized quartz proved to be the least prone to heterogeneous drying of the sample and yielded the most reliable results.

Prediction of protein structural classes by recurrence quantification analysis based on chaos game representation.

PubMed

Yang, Jian-Yi; Peng, Zhen-Ling; Yu, Zu-Guo; Zhang, Rui-Jie; Anh, Vo; Wang, Desheng

2009-04-21

In this paper, we intend to predict protein structural classes (alpha, beta, alpha+beta, or alpha/beta) for low-homology data sets. Two data sets were used widely, 1189 (containing 1092 proteins) and 25PDB (containing 1673 proteins) with sequence homology being 40% and 25%, respectively. We propose to decompose the chaos game representation of proteins into two kinds of time series. Then, a novel and powerful nonlinear analysis technique, recurrence quantification analysis (RQA), is applied to analyze these time series. For a given protein sequence, a total of 16 characteristic parameters can be calculated with RQA, which are treated as feature representation of protein sequences. Based on such feature representation, the structural class for each protein is predicted with Fisher's linear discriminant algorithm. The jackknife test is used to test and compare our method with other existing methods. The overall accuracies with step-by-step procedure are 65.8% and 64.2% for 1189 and 25PDB data sets, respectively. With one-against-others procedure used widely, we compare our method with five other existing methods. Especially, the overall accuracies of our method are 6.3% and 4.1% higher for the two data sets, respectively. Furthermore, only 16 parameters are used in our method, which is less than that used by other methods. This suggests that the current method may play a complementary role to the existing methods and is promising to perform the prediction of protein structural classes.

Assessment of Recovery of Milk Protein Allergens from Processed Food for Mass Spectrometry Quantification.

PubMed

Groves, Kate; Cryar, Adam; Walker, Michael; Quaglia, Milena

2018-01-01

Assessing the recovery of food allergens from solid processed matrixes is one of the most difficult steps that needs to be overcome to enable the accurate quantification of protein allergens by immunoassay and MS. A feasibility study is described herein applying International System of Units (SI)-traceably quantified milk protein solutions to assess recovery by an improved extraction method. Untargeted MS analysis suggests that this novel extraction method can be further developed to provide high recoveries for a broad range of food allergens. A solution of α-casein was traceably quantified to the SI for the content of α-S1 casein. Cookie dough was prepared by spiking a known amount of the SI-traceable quantified solution into a mixture of flour, sugar, and soya spread, followed by baking. A novel method for the extraction of protein food allergens from solid matrixes based on proteolytic digestion was developed, and its performance was compared with the performance of methods reported in the literature.

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

PubMed

Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

2012-01-01

Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein. Copyright © 2011. Published by Elsevier B.V.

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

PubMed Central

Desjardins, Philippe; Hansen, Joel B.; Allen, Michael

2009-01-01

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 μL. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay. PMID:19890248

Biochemical Fractionation and Stable Isotope Dilution Liquid Chromatography-mass Spectrometry for Targeted and Microdomain-specific Protein Quantification in Human Postmortem Brain Tissue*

PubMed Central

MacDonald, Matthew L.; Ciccimaro, Eugene; Prakash, Amol; Banerjee, Anamika; Seeholzer, Steven H.; Blair, Ian A.; Hahn, Chang-Gyu

2012-01-01

Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease. PMID:22942359

A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide-concatenated standards.

PubMed

Kito, Keiji; Okada, Mitsuhiro; Ishibashi, Yuko; Okada, Satoshi; Ito, Takashi

2016-05-01

The accurate and precise absolute abundance of proteins can be determined using mass spectrometry by spiking the sample with stable isotope-labeled standards. In this study, we developed a strategy of hierarchical use of peptide-concatenated standards (PCSs) to quantify more proteins over a wider dynamic range. Multiple primary PCSs were used for quantification of many target proteins. Unique "ID-tag peptides" were introduced into individual primary PCSs, allowing us to monitor the exact amounts of individual PCSs using a "secondary PCS" in which all "ID-tag peptides" were concatenated. Furthermore, we varied the copy number of the "ID-tag peptide" in each PCS according to a range of expression levels of target proteins. This strategy accomplished absolute quantification over a wider range than that of the measured ratios. The quantified abundance of budding yeast proteins showed a high reproducibility for replicate analyses and similar copy numbers per cell for ribosomal proteins, demonstrating the accuracy and precision of this strategy. A comparison with the absolute abundance of transcripts clearly indicated different post-transcriptional regulation of expression for specific functional groups. Thus, the approach presented here is a faithful method for the absolute quantification of proteomes and provides insights into biological mechanisms, including the regulation of expressed protein abundance. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms.

PubMed

Randrianjatovo, I; Girbal-Neuhauser, E; Marcato-Romain, C-E

2015-06-01

Biofilms are ecosystems of closely associated bacteria encapsulated in an extracellular matrix mainly composed of polysaccharides and proteins. A novel approach was developed for in situ quantification of extracellular proteins (ePNs) in various bacterial biofilms using epicocconone, a natural, fluorescent compound that binds amine residues of proteins. Six commercial proteins were tested for their reaction with epicocconone, and bovine serum albumin (BSA) was selected for assay optimization. The optimized protocol, performed as a microassay, allowed protein amounts as low as 0.7 μg to as high as 50 μg per well to be detected. Addition of monosaccharides or polysaccharides (glucose, dextran or alginate) to the standard BSA solutions (0 to 250 μg ml(-1)) showed little or no sugar interference up to 2000 μg ml(-1), thus providing an assessment of the specificity of epicocconone for proteins. The optimized protocol was then applied to three different biofilms, and in situ quantification of ePN showed contrasted protein amounts of 22.1 ± 3.1, 38.3 ± 7.1 and 0.3 ± 0.1 μg equivalent BSA of proteins for 48-h biofilms of Pseudomonas aeruginosa, Bacillus licheniformis and Weissella confusa, respectively. Possible interference due to global matrix compounds on the in situ quantification of proteins was also investigated by applying the standard addition method (SAM). Low error percentages were obtained, indicating a correct quantification of both the ePN and the added proteins. For the first time, a specific and sensitive assay has been developed for in situ determination of ePN produced by bacterial cells. This advance should lead to an accurate, rapid tool for further protein labelling and microscopic observation of the extracellular matrix of biofilms.

Protein 3-Nitrotyrosine in Complex Biological Samples: Quantification by High-Pressure Liquid Chromatography/Electrochemical Detection and Emergence of Proteomic Approaches for Unbiased Identification of Modification Sites

PubMed Central

Nuriel, Tal; Deeb, Ruba S.; Hajjar, David P.; Gross, Steven S.

2008-01-01

Nitration of tyrosine residues by nitric oxide (NO)-derived species results in the accumulation of 3-nitrotyrosine in proteins, a hallmark of nitrosative stress in cells and tissues. Tyrosine nitration is recognized as one of the multiple signaling modalities used by NO-derived species for the regulation of protein structure and function in health and disease. Various methods have been described for the quantification of protein 3-nitrotyrosine residues, and several strategies have been presented toward the goal of proteome-wide identification of protein tyrosine modification sites. This chapter details a useful protocol for the quantification of 3-nitrotyrosine in cells and tissues using high-pressure liquid chromatography with electrochemical detection. Additionally, this chapter describes a novel biotin-tagging strategy for specific enrichment of 3-nitrotyrosine-containing peptides. Application of this strategy, in conjunction with high-throughput MS/MS-based peptide sequencing, is anticipated to fuel efforts in developing comprehensive inventories of nitrosative stress-induced protein-tyrosine modification sites in cells and tissues. PMID:18554526

Accurate proteome-wide protein quantification from high-resolution 15N mass spectra

PubMed Central

2011-01-01

In quantitative mass spectrometry-based proteomics, the metabolic incorporation of a single source of 15N-labeled nitrogen has many advantages over using stable isotope-labeled amino acids. However, the lack of a robust computational framework for analyzing the resulting spectra has impeded wide use of this approach. We have addressed this challenge by introducing a new computational methodology for analyzing 15N spectra in which quantification is integrated with identification. Application of this method to an Escherichia coli growth transition reveals significant improvement in quantification accuracy over previous methods. PMID:22182234

Dielectrophoretic immobilization of proteins: Quantification by atomic force microscopy.

PubMed

Laux, Eva-Maria; Knigge, Xenia; Bier, Frank F; Wenger, Christian; Hölzel, Ralph

2015-09-01

The combination of alternating electric fields with nanometer-sized electrodes allows the permanent immobilization of proteins by dielectrophoretic force. Here, atomic force microscopy is introduced as a quantification method, and results are compared with fluorescence microscopy. Experimental parameters, for example the applied voltage and duration of field application, are varied systematically, and the influence on the amount of immobilized proteins is investigated. A linear correlation to the duration of field application was found by atomic force microscopy, and both microscopical methods yield a square dependence of the amount of immobilized proteins on the applied voltage. While fluorescence microscopy allows real-time imaging, atomic force microscopy reveals immobilized proteins obscured in fluorescence images due to low S/N. Furthermore, the higher spatial resolution of the atomic force microscope enables the visualization of the protein distribution on single nanoelectrodes. The electric field distribution is calculated and compared to experimental results with very good agreement to atomic force microscopy measurements. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly multiplexed simultaneous detection of RNAs and proteins in single cells.

PubMed

Frei, Andreas P; Bava, Felice-Alessio; Zunder, Eli R; Hsieh, Elena W Y; Chen, Shih-Yu; Nolan, Garry P; Gherardini, Pier Federico

2016-03-01

To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.

Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry.

PubMed

Seibert, Cathrin; Davidson, Brian R; Fuller, Barry J; Patterson, Laurence H; Griffiths, William J; Wang, Yuqin

2009-04-01

Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel protein separation and 2D-LC peptide separation gave comparable results. In total, 18 CYP isoforms were unambiguously identified based on unique peptide matches. Further, we have determined the absolute quantity of two CYP enzymes (2E1 and 1A2) in human liver microsomes using stable-isotope dilution mass spectrometry, where microsomal proteins were separated by 1D-gel electrophoresis, digested with trypsin in the presence of either a CYP2E1- or 1A2-specific stable-isotope labeled tryptic peptide and analyzed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labeled tryptic peptides and their natural unlabeled analogues quantification could be performed over the range of 0.1-1.5 pmol on column. Liver microsomes from four individuals were analyzed for CYP2E1 giving values of 88-200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 to 263 pmol/mg microsomal protein. Although, in this proof-of-concept study for CYP quantification, the two CYP isoforms were quantified from different samples, there are no practical reasons to prevent multiplexing the method to allow the quantification of multiple CYP isoforms in a single sample.

Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry

PubMed Central

Seibert, Cathrin; Davidson, Brian R.; Fuller, Barry J.; Patterson, Laurence H.; Griffiths, William J.; Wang, Yuqin

2009-01-01

Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel protein separation and 2D-LC peptide separation gave comparable results. In total 18 CYP isoforms were unambiguously identified based on unique peptide matches. Further, we have determined the absolute quantity of two CYP enzymes (2E1 and 1A2) in human liver microsomes using stable-isotope dilution mass spectrometry, where microsomal proteins were separated by 1D-gel electrophoresis, digested with trypsin in the presence of either a CYP2E1- or 1A2-specific stable-isotope labelled tryptic peptide and analysed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labelled tryptic peptides and their natural unlabelled analogues quantification could be performed over the range of 0.1 – 1.5 pmol on column. Liver microsomes from four individuals were analysed for CYP2E1 giving values of 88 - 200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 – 263 pmol/mg microsomal protein. Although, in this proof-of-concept study for CYP quantification, the two CYP-isoforms were quantified from different samples, there are no practical reasons to prevent multiplexing the method to allow the quantification of multiple CYP-isoforms in a single sample. PMID:19714871

An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

PubMed

Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

2015-12-07

As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

A proteomic insight into vitellogenesis during tick ovary maturation.

PubMed

Xavier, Marina Amaral; Tirloni, Lucas; Pinto, Antônio F M; Diedrich, Jolene K; Yates, John R; Mulenga, Albert; Logullo, Carlos; da Silva Vaz, Itabajara; Seixas, Adriana; Termignoni, Carlos

2018-03-16

Ticks are arthropod ectoparasites of importance for public and veterinary health. The understanding of tick oogenesis and embryogenesis could contribute to the development of novel control methods. However, to date, studies on the temporal dynamics of proteins during ovary development were not reported. In the present study we followed protein profile during ovary maturation. Proteomic analysis of ovary extracts was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using shotgun strategy, in addition to dimethyl labelling-based protein quantification. A total of 3,756 proteins were identified, which were functionally annotated into 30 categories. Circa 80% of the annotated proteins belong to categories related to basal metabolism, such as protein synthesis and modification machineries, nuclear regulation, cytoskeleton, proteasome machinery, transcriptional machinery, energetic metabolism, extracellular matrix/cell adhesion, immunity, oxidation/detoxification metabolism, signal transduction, and storage. The abundance of selected proteins involved in yolk uptake and degradation, as well as vitellin accumulation during ovary maturation, was assessed using dimethyl-labelling quantification. In conclusion, proteins identified in this study provide a framework for future studies to elucidate tick development and validate candidate targets for novel control methods.

A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells

PubMed Central

Forment, Josep V.; Jackson, Stephen P.

2016-01-01

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, make it easier to quantify and allow a stream-lined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry1. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell-cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy. PMID:26226461

Serum protein measurement using a tapered fluorescent fibre-optic evanescent wave-based biosensor

NASA Astrophysics Data System (ADS)

Preejith, P. V.; Lim, C. S.; Chia, T. F.

2006-12-01

A novel method to measure the total serum protein concentration is described in this paper. The method is based on the principles of fibre-optic evanescent wave spectroscopy. The biosensor applies a fluorescent dye-immobilized porous glass coating on a multi-mode optical fibre. The evanescent wave's intensity at the fibre-optic core-cladding interface is used to monitor the protein-induced changes in the sensor element. The sensor offers a rapid, single-step method for quantifying protein concentrations without destroying the sample. This unique sensing method presents a sensitive and accurate platform for the quantification of protein.

Integrated analyses of proteins and their glycans in a magnetic bead-based multiplex assay format.

PubMed

Li, Danni; Chiu, Hanching; Chen, Jing; Zhang, Hui; Chan, Daniel W

2013-01-01

Well-annotated clinical samples are valuable resources for biomarker discovery and validation. Multiplex and integrated methods that simultaneously measure multiple analytes and generate integrated information about these analytes from a single measurement are desirable because these methods help conserve precious samples. We developed a magnetic bead-based system for multiplex and integrated glycoprotein quantification by immunoassays and glycan detection by lectin immunosorbent assays (LISAs). Magnetic beads coupled with antibodies were used for capturing proteins of interest. Biotinylated antibodies in combination with streptavidin-labeled phycoerythrin were used for protein quantification. In the LISAs, biotinylated detection antibodies were replaced by biotinylated lectins for glycan detection. Using tissue inhibitor of metallopeptidase 1 (TIMP-1), tissue plasminogen activator, membrane metallo-endopeptidase, and dipeptidyl peptidase-IV (DPP-4) as models, we found that the multiplex integrated system was comparable to single immunoassays in protein quantification and LISAs in glycan detection. The merits of this system were demonstrated when applied to well-annotated prostate cancer tissues for validation of biomarkers in aggressive prostate cancer. Because of the system's multiplex ability, we used only 300 ng of tissue protein for the integrated detection of glycans in these proteins. Fucosylated TIMP-1 and DPP-4 offered improved performance over the proteins in distinguishing aggressive and nonaggressive prostate cancer. The multiplex and integrated system conserves samples and is a useful tool for validation of glycoproteins and their glycoforms as biomarkers. © 2012 American Association for Clinical Chemistry

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers*

PubMed Central

Chen, Hsiao-Wei; Wu, Chun-Feng; Chu, Lichieh Julie; Chiang, Wei-Fang; Wu, Chih-Ching; Yu, Jau-Song; Tsai, Cheng-Han; Liang, Kung-Hao; Chang, Yu-Sun; Wu, Maureen; Ou Yang, Wei-Ting

2017-01-01

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research. PMID:28235782

Development of a novel method for quantification of autophagic protein degradation by AHA labeling.

PubMed

Zhang, Jianbin; Wang, Jigang; Ng, Shukie; Lin, Qingsong; Shen, Han-Ming

2014-05-01

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.

Development of a novel method for quantification of autophagic protein degradation by AHA labeling

PubMed Central

Zhang, Jianbin; Wang, Jigang; Ng, Shukie; Lin, Qingsong; Shen, Han-Ming

2014-01-01

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems. PMID:24675368

Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

PubMed Central

Russell, Jason D.; Scalf, Mark; Book, Adam J.; Ladror, Daniel T.; Vierstra, Richard D.; Smith, Lloyd M.; Coon, Joshua J.

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1. PMID:23536786

Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.

PubMed

Russell, Jason D; Scalf, Mark; Book, Adam J; Ladror, Daniel T; Vierstra, Richard D; Smith, Lloyd M; Coon, Joshua J

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.

Characterization and elimination of undesirable protein residues in plant cell walls for enhancing lignin analysis by solution-state 2D gel-NMR methods

USDA-ARS?s Scientific Manuscript database

Proteins exist in every plant cell wall. Certain protein residues interfere with lignin characterization and quantification. The current solution-state 2D-NMR technique (gel-NMR) for whole plant cell wall structural profiling provides detailed information regarding cell walls and proteins. However, ...

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

PubMed Central

Xenopoulos, Alex; Fadgen, Keith; Murphy, Jim; Skilton, St. John; Prentice, Holly; Stapels, Martha

2012-01-01

Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a “discovery” assay, the latter as a “monitoring” assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique (“Hi3” method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry. PMID:22327428

Recommendations for standardized reporting of protein electrophoresis in Australia and New Zealand.

PubMed

Tate, Jillian; Caldwell, Grahame; Daly, James; Gillis, David; Jenkins, Margaret; Jovanovich, Sue; Martin, Helen; Steele, Richard; Wienholt, Louise; Mollee, Peter

2012-05-01

Although protein electrophoresis of serum (SPEP) and urine (UPEP) specimens is a well-established laboratory technique, the reporting of results using this important method varies considerably between laboratories. The Australasian Association of Clinical Biochemists recognized a need to adopt a standardized approach to reporting SPEP and UPEP by clinical laboratories. A Working Party considered available data including published literature and clinical studies, together with expert opinion in order to establish optimal reporting practices. A position paper was produced, which was subsequently revised through a consensus process involving scientists and pathologists with expertise in the field throughout Australia and New Zealand. Recommendations for standardized reporting of protein electrophoresis have been produced. These cover analytical requirements: detection systems; serum protein and albumin quantification; fractionation into alpha-1, alpha-2, beta and gamma fractions; paraprotein quantification; urine Bence Jones protein quantification; paraprotein characterization; and laboratory performance, expertise and staffing. The recommendations also include general interpretive commenting and commenting for specimens with paraproteins and small bands together with illustrative examples of reports. Recommendations are provided for standardized reporting of protein electrophoresis in Australia and New Zealand. It is expected that such standardized reporting formats will reduce both variation between laboratories and the risk of misinterpretation of results.

High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.

PubMed

Razavi, Morteza; Leigh Anderson, N; Pope, Matthew E; Yip, Richard; Pearson, Terry W

2016-09-25

Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Uncertainty Quantification in Alchemical Free Energy Methods.

PubMed

Bhati, Agastya P; Wan, Shunzhou; Hu, Yuan; Sherborne, Brad; Coveney, Peter V

2018-06-12

Alchemical free energy methods have gained much importance recently from several reports of improved ligand-protein binding affinity predictions based on their implementation using molecular dynamics simulations. A large number of variants of such methods implementing different accelerated sampling techniques and free energy estimators are available, each claimed to be better than the others in its own way. However, the key features of reproducibility and quantification of associated uncertainties in such methods have barely been discussed. Here, we apply a systematic protocol for uncertainty quantification to a number of popular alchemical free energy methods, covering both absolute and relative free energy predictions. We show that a reliable measure of error estimation is provided by ensemble simulation-an ensemble of independent MD simulations-which applies irrespective of the free energy method. The need to use ensemble methods is fundamental and holds regardless of the duration of time of the molecular dynamics simulations performed.

Hyperplex-MRM: a hybrid multiple reaction monitoring method using mTRAQ/iTRAQ labeling for multiplex absolute quantification of human colorectal cancer biomarker.

PubMed

Yin, Hong-Rui; Zhang, Lei; Xie, Li-Qi; Huang, Li-Yong; Xu, Ye; Cai, San-Jun; Yang, Peng-Yuan; Lu, Hao-Jie

2013-09-06

Novel biomarker verification assays are urgently required to improve the efficiency of biomarker development. Benefitting from lower development costs, multiple reaction monitoring (MRM) has been used for biomarker verification as an alternative to immunoassay. However, in general MRM analysis, only one sample can be quantified in a single experiment, which restricts its application. Here, a Hyperplex-MRM quantification approach, which combined mTRAQ for absolute quantification and iTRAQ for relative quantification, was developed to increase the throughput of biomarker verification. In this strategy, equal amounts of internal standard peptides were labeled with mTRAQ reagents Δ0 and Δ8, respectively, as double references, while 4-plex iTRAQ reagents were used to label four different samples as an alternative to mTRAQ Δ4. From the MRM trace and MS/MS spectrum, total amounts and relative ratios of target proteins/peptides of four samples could be acquired simultaneously. Accordingly, absolute amounts of target proteins/peptides in four different samples could be achieved in a single run. In addition, double references were used to increase the reliability of the quantification results. Using this approach, three biomarker candidates, ademosylhomocysteinase (AHCY), cathepsin D (CTSD), and lysozyme C (LYZ), were successfully quantified in colorectal cancer (CRC) tissue specimens of different stages with high accuracy, sensitivity, and reproducibility. To summarize, we demonstrated a promising quantification method for high-throughput verification of biomarker candidates.

Nondestructive Analysis of Tumor-Associated Membrane Protein Integrating Imaging and Amplified Detection in situ Based on Dual-Labeled DNAzyme.

PubMed

Chen, Xiaoxia; Zhao, Jing; Chen, Tianshu; Gao, Tao; Zhu, Xiaoli; Li, Genxi

2018-01-01

Comprehensive analysis of the expression level and location of tumor-associated membrane proteins (TMPs) is of vital importance for the profiling of tumor cells. Currently, two kinds of independent techniques, i.e. ex situ detection and in situ imaging, are usually required for the quantification and localization of TMPs respectively, resulting in some inevitable problems. Methods: Herein, based on a well-designed and fluorophore-labeled DNAzyme, we develop an integrated and facile method, in which imaging and quantification of TMPs in situ are achieved simultaneously in a single system. The labeled DNAzyme not only produces localized fluorescence for the visualization of TMPs but also catalyzes the cleavage of a substrate to produce quantitative fluorescent signals that can be collected from solution for the sensitive detection of TMPs. Results: Results from the DNAzyme-based in situ imaging and quantification of TMPs match well with traditional immunofluorescence and western blotting. In addition to the advantage of two-in-one, the DNAzyme-based method is highly sensitivity, allowing the detection of TMPs in only 100 cells. Moreover, the method is nondestructive. Cells after analysis could retain their physiological activity and could be cultured for other applications. Conclusion: The integrated system provides solid results for both imaging and quantification of TMPs, making it a competitive method over some traditional techniques for the analysis of TMPs, which offers potential application as a toolbox in the future.

Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)

NASA Astrophysics Data System (ADS)

Meyer, Jesse G.; D'Souza, Alexandria K.; Sorensen, Dylan J.; Rardin, Matthew J.; Wolfe, Alan J.; Gibson, Bradford W.; Schilling, Birgit

2016-11-01

Post-translational modification of lysine residues by NƐ-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy.

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers.

PubMed

Chen, Yi-Ting; Chen, Hsiao-Wei; Wu, Chun-Feng; Chu, Lichieh Julie; Chiang, Wei-Fang; Wu, Chih-Ching; Yu, Jau-Song; Tsai, Cheng-Han; Liang, Kung-Hao; Chang, Yu-Sun; Wu, Maureen; Ou Yang, Wei-Ting

2017-05-01

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Assessment of heat treatment of dairy products by MALDI-TOF-MS.

PubMed

Meltretter, Jasmin; Birlouez-Aragon, Inès; Becker, Cord-Michael; Pischetsrieder, Monika

2009-12-01

The formation of the Amadori product from lactose (protein lactosylation) is a major parameter to evaluate the quality of processed milk. Here, MALDI-TOF-MS was used for the relative quantification of lactose-adducts in heated milk. Milk was heated at a temperature of 70, 80, and 100 degrees C between 0 and 300 min, diluted, and subjected directly to MALDI-TOF-MS. The lactosylation rate of alpha-lactalbumin increased with increasing reaction temperature and time. The results correlated well with established markers for heat treatment of milk (concentration of total soluble protein, soluble alpha-lactalbumin and beta-lactoglobulin at pH 4.6, and fluorescence of advanced Maillard products and soluble tryptophan index; r=0.969-0.997). The method was also applied to examine commercially available dairy products. In severely heated products, protein pre-purification by immobilized metal affinity chromatography improved spectra quality. Relative quantification of protein lactosylation by MALDI-TOF-MS proved to be a very fast and reliable method to monitor early Maillard reaction during milk processing.

Spot quantification in two dimensional gel electrophoresis image analysis: comparison of different approaches and presentation of a novel compound fitting algorithm

PubMed Central

2014-01-01

Background Various computer-based methods exist for the detection and quantification of protein spots in two dimensional gel electrophoresis images. Area-based methods are commonly used for spot quantification: an area is assigned to each spot and the sum of the pixel intensities in that area, the so-called volume, is used a measure for spot signal. Other methods use the optical density, i.e. the intensity of the most intense pixel of a spot, or calculate the volume from the parameters of a fitted function. Results In this study we compare the performance of different spot quantification methods using synthetic and real data. We propose a ready-to-use algorithm for spot detection and quantification that uses fitting of two dimensional Gaussian function curves for the extraction of data from two dimensional gel electrophoresis (2-DE) images. The algorithm implements fitting using logical compounds and is computationally efficient. The applicability of the compound fitting algorithm was evaluated for various simulated data and compared with other quantification approaches. We provide evidence that even if an incorrect bell-shaped function is used, the fitting method is superior to other approaches, especially when spots overlap. Finally, we validated the method with experimental data of urea-based 2-DE of Aβ peptides andre-analyzed published data sets. Our methods showed higher precision and accuracy than other approaches when applied to exposure time series and standard gels. Conclusion Compound fitting as a quantification method for 2-DE spots shows several advantages over other approaches and could be combined with various spot detection methods. The algorithm was scripted in MATLAB (Mathworks) and is available as a supplemental file. PMID:24915860

Antibody-free PRISM-SRM for multiplexed protein quantification: Is this the new competition for immunoassays in bioanalysis?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Qian, Weijun

2013-02-01

Highly sensitive technologies for multiplexed quantification of a large number of candidate proteins will play an increasingly important role in clinical biomarker discovery, systems biology, and general biomedical research. Herein we introduce the new PRISM-SRM technology, which represents a highly sensitive multiplexed quantification technology capable of simultaneous quantification of many low-abundance proteins without the need of affinity reagents. The versatility of antibody-free PRISM-SRM for quantifying various types of targets including protein isoforms, protein modifications, metabolites, and others, thus offering new competition with immunoassays.

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

PubMed

Hoofnagle, Andrew N; Whiteaker, Jeffrey R; Carr, Steven A; Kuhn, Eric; Liu, Tao; Massoni, Sam A; Thomas, Stefani N; Townsend, R Reid; Zimmerman, Lisa J; Boja, Emily; Chen, Jing; Crimmins, Daniel L; Davies, Sherri R; Gao, Yuqian; Hiltke, Tara R; Ketchum, Karen A; Kinsinger, Christopher R; Mesri, Mehdi; Meyer, Matthew R; Qian, Wei-Jun; Schoenherr, Regine M; Scott, Mitchell G; Shi, Tujin; Whiteley, Gordon R; Wrobel, John A; Wu, Chaochao; Ackermann, Brad L; Aebersold, Ruedi; Barnidge, David R; Bunk, David M; Clarke, Nigel; Fishman, Jordan B; Grant, Russ P; Kusebauch, Ulrike; Kushnir, Mark M; Lowenthal, Mark S; Moritz, Robert L; Neubert, Hendrik; Patterson, Scott D; Rockwood, Alan L; Rogers, John; Singh, Ravinder J; Van Eyk, Jennifer E; Wong, Steven H; Zhang, Shucha; Chan, Daniel W; Chen, Xian; Ellis, Matthew J; Liebler, Daniel C; Rodland, Karin D; Rodriguez, Henry; Smith, Richard D; Zhang, Zhen; Zhang, Hui; Paulovich, Amanda G

2016-01-01

For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care. © 2015 American Association for Clinical Chemistry.

Determination of free polysaccharide in Vi glycoconjugate vaccine against typhoid fever.

PubMed

Giannelli, C; Cappelletti, E; Di Benedetto, R; Pippi, F; Arcuri, M; Di Cioccio, V; Martin, L B; Saul, A; Micoli, F

2017-05-30

Glycoconjugate vaccines based on the Vi capsular polysaccharide directed against Salmonella enterica serovar Typhi are licensed or in development against typhoid fever, an important cause of morbidity and mortality in developing countries. Quantification of free polysaccharide in conjugate vaccines is an important quality control for release, to monitor vaccine stability and to ensure appropriate immune response. However, we found that existing separation methods based on size are not appropriate as free Vi non-specifically binds to unconjugated and conjugated protein. We developed a method based on free Vi separation by Capto Adhere resin and quantification by HPAEC-PAD. The method has been tested for conjugates of Vi derived from Citrobacter freundii with different carrier proteins such as CRM 197 , Tetanus Toxoid and Diphtheria Toxoid. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

An MRM-based workflow for quantifying cardiac mitochondrial protein phosphorylation in murine and human tissue.

PubMed

Lam, Maggie P Y; Scruggs, Sarah B; Kim, Tae-Young; Zong, Chenggong; Lau, Edward; Wang, Ding; Ryan, Christopher M; Faull, Kym F; Ping, Peipei

2012-08-03

The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology. This article is part of a Special Issue entitled: Translational Proteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

A direct, ratiometric, and quantitative MALDI–MS assay for protein methyltransferases and acetyltransferases

PubMed Central

Richardson, Stacie L.; Hanjra, Pahul; Zhang, Gang; Mackie, Brianna D.; Peterson, Darrell L.; Huang, Rong

2016-01-01

Protein methylation and acetylation play important roles in biological processes, and misregulation of these modifications is involved in various diseases. Therefore, it is critical to understand the activities of the enzymes responsible for these modifications. Herein we describe a sensitive method for ratiometric quantification of methylated and acetylated peptides via MALDI-MS by direct spotting of enzymatic methylation and acetylation reaction mixtures without tedious purification procedures. The quantifiable detection limit for peptides with our method is approximately 10 fmol. This is achieved by increasing the signal-to-noise ratio through the addition of NH4H2PO4 to the matrix solution and reduction of the matrix α-cyanohydroxycinnamic acid concentration to 2 mg/ml. We have demonstrated the application of this method in enzyme kinetic analysis and inhibition studies. The unique feature of this method is the simultaneous quantification of multiple peptide species for investigation of processivity mechanisms. Its wide buffer compatibility makes it possible to be adapted to investigate the activity of any protein methyltransferase or acetyltransferase. PMID:25778392

LC-MS/MS strategies for therapeutic antibodies and investigation into the quantitative impact of antidrug-antibodies.

PubMed

Ewles, Matthew; Mannu, Ranbir; Fox, Chris; Stanta, Johannes; Evans, Graeme; Goodwin, Lee; Duffy, James; Bell, Len; Estdale, Sian; Firth, David

2016-12-01

We aimed to establish novel, high-throughput LC-MS/MS strategies for quantification of monoclonal antibodies in human serum and examine the potential impact of antidrug antibodies. We present two strategies using a thermally stable immobilized trypsin. The first strategy uses whole serum digestion and the second introduces Protein G enrichment to improve the selectivity. The impact of anti-trastuzumab antibodies on the methods was tested. Whole serum digestion has been validated for trastuzumab (LLOQ 0.25 µg/ml). Protein G enrichment has been validated for trastuzumab (LLOQ 0.1 µg/ml), bevacizumab (LLOQ 0.1 µg/ml) and adalimumab (LLOQ 0.25 µg/ml). We have shown the potential for anti-drug antibodies to impact on the quantification and we have subsequently established a strategy to overcome this impact where total quantification is desired.

Development of a Protein Standard Absolute Quantification (PSAQ™) assay for the quantification of Staphylococcus aureus enterotoxin A in serum.

PubMed

Adrait, Annie; Lebert, Dorothée; Trauchessec, Mathieu; Dupuis, Alain; Louwagie, Mathilde; Masselon, Christophe; Jaquinod, Michel; Chevalier, Benoît; Vandenesch, François; Garin, Jérôme; Bruley, Christophe; Brun, Virginie

2012-06-06

Enterotoxin A (SEA) is a staphylococcal virulence factor which is suspected to worsen septic shock prognosis. However, the presence of SEA in the blood of sepsis patients has never been demonstrated. We have developed a mass spectrometry-based assay for the targeted and absolute quantification of SEA in serum. To enhance sensitivity and specificity, we combined an immunoaffinity-based sample preparation with mass spectrometry analysis in the selected reaction monitoring (SRM) mode. Absolute quantification of SEA was performed using the PSAQ™ method (Protein Standard Absolute Quantification), which uses a full-length isotope-labeled SEA as internal standard. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were estimated at 352pg/mL and 1057pg/mL, respectively. SEA recovery after immunocapture was determined to be 7.8±1.4%. Therefore, we assumed that less than 1femtomole of each SEA proteotypic peptide was injected on the liquid chromatography column before SRM analysis. From a 6-point titration experiment, quantification accuracy was determined to be 77% and precision at LLOQ was lower than 5%. With this sensitive PSAQ-SRM assay, we expect to contribute to decipher the pathophysiological role of SEA in severe sepsis. This article is part of a Special Issue entitled: Proteomics: The clinical link. Copyright © 2011 Elsevier B.V. All rights reserved.

Linearization of the bradford protein assay.

PubMed

Ernst, Orna; Zor, Tsaffrir

2010-04-12

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

Epsilon-Q: An Automated Analyzer Interface for Mass Spectral Library Search and Label-Free Protein Quantification.

PubMed

Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Paik, Young-Ki

2017-12-01

Mass spectrometry (MS) is a widely used proteome analysis tool for biomedical science. In an MS-based bottom-up proteomic approach to protein identification, sequence database (DB) searching has been routinely used because of its simplicity and convenience. However, searching a sequence DB with multiple variable modification options can increase processing time, false-positive errors in large and complicated MS data sets. Spectral library searching is an alternative solution, avoiding the limitations of sequence DB searching and allowing the detection of more peptides with high sensitivity. Unfortunately, this technique has less proteome coverage, resulting in limitations in the detection of novel and whole peptide sequences in biological samples. To solve these problems, we previously developed the "Combo-Spec Search" method, which uses manually multiple references and simulated spectral library searching to analyze whole proteomes in a biological sample. In this study, we have developed a new analytical interface tool called "Epsilon-Q" to enhance the functions of both the Combo-Spec Search method and label-free protein quantification. Epsilon-Q performs automatically multiple spectral library searching, class-specific false-discovery rate control, and result integration. It has a user-friendly graphical interface and demonstrates good performance in identifying and quantifying proteins by supporting standard MS data formats and spectrum-to-spectrum matching powered by SpectraST. Furthermore, when the Epsilon-Q interface is combined with the Combo-Spec search method, called the Epsilon-Q system, it shows a synergistic function by outperforming other sequence DB search engines for identifying and quantifying low-abundance proteins in biological samples. The Epsilon-Q system can be a versatile tool for comparative proteome analysis based on multiple spectral libraries and label-free quantification.

Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)

DOE PAGES

Meyer, Jesse G.; D’Souza, Alexandria K.; Sorensen, Dylan J.; ...

2016-09-02

Post-translational modification of lysine residues by N ε-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods canmore » overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy.« less

Rapid flow cytometric measurement of protein inclusions and nuclear trafficking

PubMed Central

Whiten, D. R.; San Gil, R.; McAlary, L.; Yerbury, J. J.; Ecroyd, H.; Wilson, M. R.

2016-01-01

Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules. PMID:27516358

Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

PubMed Central

Hesse, Almut

2016-01-01

Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

Highly abundant defense proteins in human sweat as revealed by targeted proteomics and label-free quantification mass spectrometry.

PubMed

Csősz, É; Emri, G; Kalló, G; Tsaprailis, G; Tőzsér, J

2015-10-01

The healthy human skin with its effective antimicrobial defense system forms an efficient barrier against invading pathogens. There is evidence suggesting that the composition of this chemical barrier varies between diseases, making the easily collected sweat an ideal candidate for biomarker discoveries. Our aim was to provide information about the normal composition of the sweat, and to study the chemical barrier found at the surface of skin. Sweat samples from healthy individuals were collected during sauna bathing, and the global protein panel was analysed by label-free mass spectrometry. SRM-based targeted proteomic methods were designed and stable isotope labelled reference peptides were used for method validation. Ninety-five sweat proteins were identified, 20 of them were novel proteins. It was shown that dermcidin is the most abundant sweat protein, and along with apolipoprotein D, clusterin, prolactin-inducible protein and serum albumin, they make up 91% of secreted sweat proteins. The roles of these highly abundant proteins were reviewed; all of which have protective functions, highlighting the importance of sweat glands in composing the first line of innate immune defense system, and maintaining the epidermal barrier integrity. Our findings with regard to the proteins forming the chemical barrier of the skin as determined by label-free quantification and targeted proteomics methods are in accordance with previous studies, and can be further used as a starting point for non-invasive sweat biomarker research. © 2015 European Academy of Dermatology and Venereology.

MSblender: A probabilistic approach for integrating peptide identifications from multiple database search engines.

PubMed

Kwon, Taejoon; Choi, Hyungwon; Vogel, Christine; Nesvizhskii, Alexey I; Marcotte, Edward M

2011-07-01

Shotgun proteomics using mass spectrometry is a powerful method for protein identification but suffers limited sensitivity in complex samples. Integrating peptide identifications from multiple database search engines is a promising strategy to increase the number of peptide identifications and reduce the volume of unassigned tandem mass spectra. Existing methods pool statistical significance scores such as p-values or posterior probabilities of peptide-spectrum matches (PSMs) from multiple search engines after high scoring peptides have been assigned to spectra, but these methods lack reliable control of identification error rates as data are integrated from different search engines. We developed a statistically coherent method for integrative analysis, termed MSblender. MSblender converts raw search scores from search engines into a probability score for every possible PSM and properly accounts for the correlation between search scores. The method reliably estimates false discovery rates and identifies more PSMs than any single search engine at the same false discovery rate. Increased identifications increment spectral counts for most proteins and allow quantification of proteins that would not have been quantified by individual search engines. We also demonstrate that enhanced quantification contributes to improve sensitivity in differential expression analyses.

MSblender: a probabilistic approach for integrating peptide identifications from multiple database search engines

PubMed Central

Kwon, Taejoon; Choi, Hyungwon; Vogel, Christine; Nesvizhskii, Alexey I.; Marcotte, Edward M.

2011-01-01

Shotgun proteomics using mass spectrometry is a powerful method for protein identification but suffers limited sensitivity in complex samples. Integrating peptide identifications from multiple database search engines is a promising strategy to increase the number of peptide identifications and reduce the volume of unassigned tandem mass spectra. Existing methods pool statistical significance scores such as p-values or posterior probabilities of peptide-spectrum matches (PSMs) from multiple search engines after high scoring peptides have been assigned to spectra, but these methods lack reliable control of identification error rates as data are integrated from different search engines. We developed a statistically coherent method for integrative analysis, termed MSblender. MSblender converts raw search scores from search engines into a probability score for all possible PSMs and properly accounts for the correlation between search scores. The method reliably estimates false discovery rates and identifies more PSMs than any single search engine at the same false discovery rate. Increased identifications increment spectral counts for all detected proteins and allow quantification of proteins that would not have been quantified by individual search engines. We also demonstrate that enhanced quantification contributes to improve sensitivity in differential expression analyses. PMID:21488652

Molecular Approaches for High Throughput Detection and Quantification of Genetically Modified Crops: A Review

PubMed Central

Salisu, Ibrahim B.; Shahid, Ahmad A.; Yaqoob, Amina; Ali, Qurban; Bajwa, Kamran S.; Rao, Abdul Q.; Husnain, Tayyab

2017-01-01

As long as the genetically modified crops are gaining attention globally, their proper approval and commercialization need accurate and reliable diagnostic methods for the transgenic content. These diagnostic techniques are mainly divided into two major groups, i.e., identification of transgenic (1) DNA and (2) proteins from GMOs and their products. Conventional methods such as PCR (polymerase chain reaction) and enzyme-linked immunosorbent assay (ELISA) were routinely employed for DNA and protein based quantification respectively. Although, these Techniques (PCR and ELISA) are considered as significantly convenient and productive, but there is need for more advance technologies that allow for high throughput detection and the quantification of GM event as the production of more complex GMO is increasing day by day. Therefore, recent approaches like microarray, capillary gel electrophoresis, digital PCR and next generation sequencing are more promising due to their accuracy and precise detection of transgenic contents. The present article is a brief comparative study of all such detection techniques on the basis of their advent, feasibility, accuracy, and cost effectiveness. However, these emerging technologies have a lot to do with detection of a specific event, contamination of different events and determination of fusion as well as stacked gene protein are the critical issues to be addressed in future. PMID:29085378

Fast quantification of endogenous carbohydrates in plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.

PubMed

Zhu, Bangjie; Liu, Feng; Li, Xituo; Wang, Yan; Gu, Xue; Dai, Jieyu; Wang, Guiming; Cheng, Yu; Yan, Chao

2015-01-01

Endogenous carbohydrates in biosamples are frequently highlighted as the most differential metabolites in many metabolomics studies. A simple, fast, simultaneous quantitative method for 16 endogenous carbohydrates in plasma has been developed using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. In order to quantify 16 endogenous carbohydrates in plasma, various conditions, including columns, chromatographic conditions, mass spectrometry conditions, and plasma preparation methods, were investigated. Different conditions in this quantified analysis were performed and optimized. The reproducibility, precision, recovery, matrix effect, and stability of the method were verified. The results indicated that a methanol/acetonitrile (50:50, v/v) mixture could effectively and reproducibly precipitate rat plasma proteins. Cold organic solvents coupled with vortex for 1 min and incubated at -20°C for 20 min were the most optimal conditions for protein precipitation and extraction. The results, according to the linearity, recovery, precision, matrix effect, and stability, showed that the method was satisfactory in the quantification of endogenous carbohydrates in rat plasma. The quantified analysis of endogenous carbohydrates in rat plasma performed excellently in terms of sensitivity, high throughput, and simple sample preparation, which met the requirement of quantification in specific expanded metabolomic studies after the global metabolic profiling research. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Data-Independent MS/MS Quantification of Neuropeptides for Determination of Putative Feeding-Related Neurohormones in Microdialysate

PubMed Central

2015-01-01

Food consumption is an important behavior that is regulated by an intricate array of neuropeptides (NPs). Although many feeding-related NPs have been identified in mammals, precise mechanisms are unclear and difficult to study in mammals, as current methods are not highly multiplexed and require extensive a priori knowledge about analytes. New advances in data-independent acquisition (DIA) MS/MS and the open-source quantification software Skyline have opened up the possibility to identify hundreds of compounds and quantify them from a single DIA MS/MS run. An untargeted DIA MSE quantification method using Skyline software for multiplexed, discovery-driven quantification was developed and found to produce linear calibration curves for peptides at physiologically relevant concentrations using a protein digest as internal standard. By using this method, preliminary relative quantification of the crab Cancer borealis neuropeptidome (<2 kDa, 137 peptides from 18 families) was possible in microdialysates from 8 replicate feeding experiments. Of these NPs, 55 were detected with an average mass error below 10 ppm. The time-resolved profiles of relative concentration changes for 6 are shown, and there is great potential for the use of this method in future experiments to aid in correlation of NP changes with behavior. This work presents an unbiased approach to winnowing candidate NPs related to a behavior of interest in a functionally relevant manner, and demonstrates the success of such a UPLC-MSE quantification method using the open source software Skyline. PMID:25552291

Data-independent MS/MS quantification of neuropeptides for determination of putative feeding-related neurohormones in microdialysate.

PubMed

Schmerberg, Claire M; Liang, Zhidan; Li, Lingjun

2015-01-21

Food consumption is an important behavior that is regulated by an intricate array of neuropeptides (NPs). Although many feeding-related NPs have been identified in mammals, precise mechanisms are unclear and difficult to study in mammals, as current methods are not highly multiplexed and require extensive a priori knowledge about analytes. New advances in data-independent acquisition (DIA) MS/MS and the open-source quantification software Skyline have opened up the possibility to identify hundreds of compounds and quantify them from a single DIA MS/MS run. An untargeted DIA MS(E) quantification method using Skyline software for multiplexed, discovery-driven quantification was developed and found to produce linear calibration curves for peptides at physiologically relevant concentrations using a protein digest as internal standard. By using this method, preliminary relative quantification of the crab Cancer borealis neuropeptidome (<2 kDa, 137 peptides from 18 families) was possible in microdialysates from 8 replicate feeding experiments. Of these NPs, 55 were detected with an average mass error below 10 ppm. The time-resolved profiles of relative concentration changes for 6 are shown, and there is great potential for the use of this method in future experiments to aid in correlation of NP changes with behavior. This work presents an unbiased approach to winnowing candidate NPs related to a behavior of interest in a functionally relevant manner, and demonstrates the success of such a UPLC-MS(E) quantification method using the open source software Skyline.

Quantitation of Human Cytochrome P450 2D6 Protein with Immunoblot and Mass Spectrometry Analysis

PubMed Central

Yu, Ai-Ming; Qu, Jun; Felmlee, Melanie A.; Cao, Jin; Jiang, Xi-Ling

2009-01-01

Accurate quantification of cytochrome P450 (P450) protein contents is essential for reliable assessment of drug safety, including the prediction of in vivo clearance from in vitro metabolism data, which may be hampered by the use of uncharacterized standards and existence of unknown allelic isozymes. Therefore, this study aimed to delineate the variability in absolute quantification of polymorphic CYP2D6 drug-metabolizing enzyme and compare immunoblot and nano liquid chromatography coupled to mass spectrometry (nano-LC/MS) methods in identification and relative quantification of CYP2D6.1 and CYP2D6.2 allelic isozymes. Holoprotein content of in-house purified CYP2D6 isozymes was determined according to carbon monoxide difference spectrum, and total protein was quantified with bicinchoninic acid protein assay. Holoprotein/total CYP2D6 protein ratio was markedly higher for purified CYP2D6.1 (71.0%) than that calculated for CYP2D6.1 Supersomes (35.5%), resulting in distinct linear calibration range (0.05–0.50 versus 0.025–0.25 pmol) that was determined by densitometric analysis of immunoblot bands. Likewise, purified CYP2D6.2 and CYP2D6.10 and the CYP2D6.10 Supersomes all showed different holoprotein/total CYP2D6 protein ratios and distinct immunoblot linear calibration ranges. In contrast to immunoblot, nano-LC/MS readily distinguished CYP2D6.2 (R296C and S486T) from CYP2D6.1 by isoform-specific proteolytic peptides that contain the altered amino acid residues. In addition, relative quantitation of the two allelic isozymes was successfully achieved with label-free protein quantification, consistent with the nominated ratio. Because immunoblot and nano-LC/MS analyses measure total P450 protein (holoprotein and apoprotein) in a sample, complete understanding of holoprotein and apoprotein contents in P450 standards is desired toward reliable quantification. Our data also suggest that nano-LC/MS not only facilitates P450 quantitation but also provides genotypic information. PMID:18832475

A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.

PubMed

Redmile-Gordon, M A; Armenise, E; White, R P; Hirsch, P R; Goulding, K W T

2013-12-01

Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.

Find Pairs: The Module for Protein Quantification of the PeakQuant Software Suite

PubMed Central

Eisenacher, Martin; Kohl, Michael; Wiese, Sebastian; Hebeler, Romano; Meyer, Helmut E.

2012-01-01

Abstract Accurate quantification of proteins is one of the major tasks in current proteomics research. To address this issue, a wide range of stable isotope labeling techniques have been developed, allowing one to quantitatively study thousands of proteins by means of mass spectrometry. In this article, the FindPairs module of the PeakQuant software suite is detailed. It facilitates the automatic determination of protein abundance ratios based on the automated analysis of stable isotope-coded mass spectrometric data. Furthermore, it implements statistical methods to determine outliers due to biological as well as technical variance of proteome data obtained in replicate experiments. This provides an important means to evaluate the significance in obtained protein expression data. For demonstrating the high applicability of FindPairs, we focused on the quantitative analysis of proteome data acquired in 14N/15N labeling experiments. We further provide a comprehensive overview of the features of the FindPairs software, and compare these with existing quantification packages. The software presented here supports a wide range of proteomics applications, allowing one to quantitatively assess data derived from different stable isotope labeling approaches, such as 14N/15N labeling, SILAC, and iTRAQ. The software is publicly available at http://www.medizinisches-proteom-center.de/software and free for academic use. PMID:22909347

Accuracy of a method based on atomic absorption spectrometry to determine inorganic arsenic in food: Outcome of the collaborative trial IMEP-41.

PubMed

Fiamegkos, I; Cordeiro, F; Robouch, P; Vélez, D; Devesa, V; Raber, G; Sloth, J J; Rasmussen, R R; Llorente-Mirandes, T; Lopez-Sanchez, J F; Rubio, R; Cubadda, F; D'Amato, M; Feldmann, J; Raab, A; Emteborg, H; de la Calle, M B

2016-12-15

A collaborative trial was conducted to determine the performance characteristics of an analytical method for the quantification of inorganic arsenic (iAs) in food. The method is based on (i) solubilisation of the protein matrix with concentrated hydrochloric acid to denature proteins and allow the release of all arsenic species into solution, and (ii) subsequent extraction of the inorganic arsenic present in the acid medium using chloroform followed by back-extraction to acidic medium. The final detection and quantification is done by flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS). The seven test items used in this exercise were reference materials covering a broad range of matrices: mussels, cabbage, seaweed (hijiki), fish protein, rice, wheat, mushrooms, with concentrations ranging from 0.074 to 7.55mgkg(-1). The relative standard deviation for repeatability (RSDr) ranged from 4.1 to 10.3%, while the relative standard deviation for reproducibility (RSDR) ranged from 6.1 to 22.8%. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Identification of MAPK Substrates Using Quantitative Phosphoproteomics.

PubMed

Zhang, Tong; Schneider, Jacqueline D; Zhu, Ning; Chen, Sixue

2017-01-01

Activation of mitogen-activated protein kinases (MAPKs) under diverse biotic and abiotic factors and identification of an array of downstream MAPK target proteins are hot topics in plant signal transduction. Through interactions with a plethora of substrate proteins, MAPK cascades regulate many physiological processes in the course of plant growth, development, and response to environmental factors. Identification and quantification of potential MAPK substrates are essential, but have been technically challenging. With the recent advancement in phosphoproteomics, here we describe a method that couples metal dioxide for phosphopeptide enrichment with tandem mass tags (TMT) mass spectrometry (MS) for large-scale MAPK substrate identification and quantification. We have applied this method to a transient expression system carrying a wild type (WT) and a constitutive active (CA) version of a MAPK. This combination of genetically engineered MAPKs and phosphoproteomics provides a high-throughput, unbiased analysis of MAPK-triggered phosphorylation changes on the proteome scale. Therefore, it is a robust method for identifying potential MAPK substrates and should be applicable in the study of other kinase cascades in plants as well as in other organisms.

Recent advances in stable isotope labeling based techniques for proteome relative quantification.

PubMed

Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

2014-10-24

The large scale relative quantification of all proteins expressed in biological samples under different states is of great importance for discovering proteins with important biological functions, as well as screening disease related biomarkers and drug targets. Therefore, the accurate quantification of proteins at proteome level has become one of the key issues in protein science. Herein, the recent advances in stable isotope labeling based techniques for proteome relative quantification were reviewed, from the aspects of metabolic labeling, chemical labeling and enzyme-catalyzed labeling. Furthermore, the future research direction in this field was prospected. Copyright © 2014 Elsevier B.V. All rights reserved.

Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer*

PubMed Central

Gallien, Sebastien; Duriez, Elodie; Crone, Catharina; Kellmann, Markus; Moehring, Thomas; Domon, Bruno

2012-01-01

There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies. PMID:22962056

Proteomic Identification and Quantification of S-glutathionylation in Mouse Macrophages Using Resin-Assisted Enrichment and Isobaric Labeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Dian; Gaffrey, Matthew J.; Guo, Jia

2014-02-11

Protein S-glutathionylation (SSG) is an important regulatory posttranslational modification of protein cysteine (Cys) thiol redox switches, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols and enrichment using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was validatedmore » by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys-sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG-modification compared to controls.. This approach was extended to identify potential SSG modified Cys-sites in response to H2O2, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys-sites from 265 proteins that were sensitive to S-glutathionylation in response to H2O2 treatment. These proteins covered a range of molecular types and molecular functions with free radical scavenging, and cell death and survival included as the most significantly enriched functional categories. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of S-glutathionylated proteins. The analytical strategy also provides a unique approach to determining the major pathways and cell processes most susceptible to glutathionylation at a proteome-wide scale.« less

Simultaneous quantification of protein phosphorylation sites using liquid chromatography-tandem mass spectrometry-based targeted proteomics: a linear algebra approach for isobaric phosphopeptides.

PubMed

Xu, Feifei; Yang, Ting; Sheng, Yuan; Zhong, Ting; Yang, Mi; Chen, Yun

2014-12-05

As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples.

TAPAS: tools to assist the targeted protein quantification of human alternative splice variants.

PubMed

Yang, Jae-Seong; Sabidó, Eduard; Serrano, Luis; Kiel, Christina

2014-10-15

In proteomes of higher eukaryotes, many alternative splice variants can only be detected by their shared peptides. This makes it highly challenging to use peptide-centric mass spectrometry to distinguish and to quantify protein isoforms resulting from alternative splicing events. We have developed two complementary algorithms based on linear mathematical models to efficiently compute a minimal set of shared and unique peptides needed to quantify a set of isoforms and splice variants. Further, we developed a statistical method to estimate the splice variant abundances based on stable isotope labeled peptide quantities. The algorithms and databases are integrated in a web-based tool, and we have experimentally tested the limits of our quantification method using spiked proteins and cell extracts. The TAPAS server is available at URL http://davinci.crg.es/tapas/. luis.serrano@crg.eu or christina.kiel@crg.eu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Non-perturbative Quantification of Ionic Charge Transfer through Nm-Scale Protein Pores Using Graphene Microelectrodes

NASA Astrophysics Data System (ADS)

Ping, Jinglei; Johnson, A. T. Charlie; A. T. Charlie Johnson Team

Conventional electrical methods for detecting charge transfer through protein pores perturb the electrostatic condition of the solution and chemical reactivity of the pore, and are not suitable to be used for complex biofluids. We developed a non-perturbative methodology ( fW input power) for quantifying trans-pore electrical current and detecting the pore status (i.e., open vs. closes) via graphene microelectrodes. Ferritin was used as a model protein featuring a large interior compartment, well-separated from the exterior solution with discrete pores as charge commuting channels. The charge flowing through the ferritin pores transfers into the graphene microelectrode and is recorded by an electrometer. In this example, our methodology enables the quantification of an inorganic nanoparticle-protein nanopore interaction in complex biofluids. The authors acknowledge the support from the Defense Advanced Research Projects Agency (DARPA) and the U.S. Army Research Office under Grant Number W911NF1010093.

Multiplexed Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Cancer Signaling Proteins

PubMed Central

Chen, Yi; Fisher, Kate J.; Lloyd, Mark; Wood, Elizabeth R.; Coppola, Domenico; Siegel, Erin; Shibata, David; Chen, Yian A.; Koomen, John M.

2017-01-01

Quantitative evaluation of protein expression across multiple cancer-related signaling pathways (e.g. Wnt/β-catenin, TGF-β, receptor tyrosine kinases (RTK), MAP kinases, NF-κB, and apoptosis) in tumor tissues may enable the development of a molecular profile for each individual tumor that can aid in the selection of appropriate targeted cancer therapies. Here, we describe the development of a broadly applicable protocol to develop and implement quantitative mass spectrometry assays using cell line models and frozen tissue specimens from colon cancer patients. Cell lines are used to develop peptide-based assays for protein quantification, which are incorporated into a method based on SDS-PAGE protein fractionation, in-gel digestion, and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). This analytical platform is then applied to frozen tumor tissues. This protocol can be broadly applied to the study of human disease using multiplexed LC-MRM assays. PMID:28808993

Comparative and Quantitative Global Proteomics Approaches: An Overview

PubMed Central

Deracinois, Barbara; Flahaut, Christophe; Duban-Deweer, Sophie; Karamanos, Yannis

2013-01-01

Proteomics became a key tool for the study of biological systems. The comparison between two different physiological states allows unravelling the cellular and molecular mechanisms involved in a biological process. Proteomics can confirm the presence of proteins suggested by their mRNA content and provides a direct measure of the quantity present in a cell. Global and targeted proteomics strategies can be applied. Targeted proteomics strategies limit the number of features that will be monitored and then optimise the methods to obtain the highest sensitivity and throughput for a huge amount of samples. The advantage of global proteomics strategies is that no hypothesis is required, other than a measurable difference in one or more protein species between the samples. Global proteomics methods attempt to separate quantify and identify all the proteins from a given sample. This review highlights only the different techniques of separation and quantification of proteins and peptides, in view of a comparative and quantitative global proteomics analysis. The in-gel and off-gel quantification of proteins will be discussed as well as the corresponding mass spectrometry technology. The overview is focused on the widespread techniques while keeping in mind that each approach is modular and often recovers the other. PMID:28250403

Nitric Oxide Analyzer Quantification of Plant S-Nitrosothiols.

PubMed

Hussain, Adil; Yun, Byung-Wook; Loake, Gary J

2018-01-01

Nitric oxide (NO) is a small diatomic molecule that regulates multiple physiological processes in animals, plants, and microorganisms. In animals, it is involved in vasodilation and neurotransmission and is present in exhaled breath. In plants, it regulates both plant immune function and numerous developmental programs. The high reactivity and short half-life of NO and cross-reactivity of its various derivatives make its quantification difficult. Different methods based on calorimetric, fluorometric, and chemiluminescent detection of NO and its derivatives are available, but all of them have significant limitations. Here we describe a method for the chemiluminescence-based quantification of NO using ozone-chemiluminescence technology in plants. This approach provides a sensitive, robust, and flexible approach for determining the levels of NO and its signaling products, protein S-nitrosothiols.

18O-labeled proteome reference as global internal standards for targeted quantification by selected reaction monitoring-mass spectrometry.

PubMed

Kim, Jong-Seo; Fillmore, Thomas L; Liu, Tao; Robinson, Errol; Hossain, Mahmud; Champion, Boyd L; Moore, Ronald J; Camp, David G; Smith, Richard D; Qian, Wei-Jun

2011-12-01

Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.

Multiple products monitoring as a robust approach for peptide quantification.

PubMed

Baek, Je-Hyun; Kim, Hokeun; Shin, Byunghee; Yu, Myeong-Hee

2009-07-01

Quantification of target peptides and proteins is crucial for biomarker discovery. Approaches such as selected reaction monitoring (SRM) and multiple reaction monitoring (MRM) rely on liquid chromatography and mass spectrometric analysis of defined peptide product ions. These methods are not very widespread because the determination of quantifiable product ion using either SRM or MRM is a very time-consuming process. We developed a novel approach for quantifying target peptides without such an arduous process of ion selection. This method is based on monitoring multiple product ions (multiple products monitoring: MpM) from full-range MS2 spectra of a target precursor. The MpM method uses a scoring system that considers both the absolute intensities of product ions and the similarities between the query MS2 spectrum and the reference MS2 spectrum of the target peptide. Compared with conventional approaches, MpM greatly improves sensitivity and selectivity of peptide quantification using an ion-trap mass spectrometer.

Quantification of Inflammasome Adaptor Protein ASC in Biological Samples by Multiple-Reaction Monitoring Mass Spectrometry.

PubMed

Ulke-Lemée, Annegret; Lau, Arthur; Nelson, Michelle C; James, Matthew T; Muruve, Daniel A; MacDonald, Justin A

2018-06-09

Inflammation is an integral component of many diseases, including chronic kidney disease (CKD). ASC (apoptosis-associated speck-like protein containing CARD, also PYCARD) is the key inflammasome adaptor protein in the innate immune response. Since ASC specks, a macromolecular condensate of ASC protein, can be released by inflammasome-activated cells into the extracellular space to amplify inflammatory responses, the ASC protein could be an important biomarker in diagnostic applications. Herein, we describe the development and validation of a multiple reaction monitoring mass spectrometry (MRM-MS) assay for the accurate quantification of ASC in human biospecimens. Limits of detection and quantification for the signature DLLLQALR peptide (used as surrogate for the target ASC protein) were determined by the method of standard addition using synthetic isotope-labeled internal standard (SIS) peptide and urine matrix from a healthy donor (LOQ was 8.25 pM, with a ~ 1000-fold linear range). We further quantified ASC in the urine of CKD patients (8.4 ± 1.3 ng ASC/ml urine, n = 13). ASC was positively correlated with proteinuria and urinary IL-18 in CKD samples but not with urinary creatinine. Unfortunately, the ASC protein is susceptible to degradation, and patient urine that was thawed and refrozen lost 85% of the ASC signal. In summary, the MRM-MS assay provides a robust means to quantify ASC in biological samples, including clinical biospecimens; however, sample collection and storage conditions will have a critical impact on assay reliability.

Mapping proteins in the presence of paralogs using units of coevolution

PubMed Central

2013-01-01

Background We study the problem of mapping proteins between two protein families in the presence of paralogs. This problem occurs as a difficult subproblem in coevolution-based computational approaches for protein-protein interaction prediction. Results Similar to prior approaches, our method is based on the idea that coevolution implies equal rates of sequence evolution among the interacting proteins, and we provide a first attempt to quantify this notion in a formal statistical manner. We call the units that are central to this quantification scheme the units of coevolution. A unit consists of two mapped protein pairs and its score quantifies the coevolution of the pairs. This quantification allows us to provide a maximum likelihood formulation of the paralog mapping problem and to cast it into a binary quadratic programming formulation. Conclusion CUPID, our software tool based on a Lagrangian relaxation of this formulation, makes it, for the first time, possible to compute state-of-the-art quality pairings in a few minutes of runtime. In summary, we suggest a novel alternative to the earlier available approaches, which is statistically sound and computationally feasible. PMID:24564758

PEPA test: fast and powerful differential analysis from relative quantitative proteomics data using shared peptides.

PubMed

Jacob, Laurent; Combes, Florence; Burger, Thomas

2018-06-18

We propose a new hypothesis test for the differential abundance of proteins in mass-spectrometry based relative quantification. An important feature of this type of high-throughput analyses is that it involves an enzymatic digestion of the sample proteins into peptides prior to identification and quantification. Due to numerous homology sequences, different proteins can lead to peptides with identical amino acid chains, so that their parent protein is ambiguous. These so-called shared peptides make the protein-level statistical analysis a challenge and are often not accounted for. In this article, we use a linear model describing peptide-protein relationships to build a likelihood ratio test of differential abundance for proteins. We show that the likelihood ratio statistic can be computed in linear time with the number of peptides. We also provide the asymptotic null distribution of a regularized version of our statistic. Experiments on both real and simulated datasets show that our procedures outperforms state-of-the-art methods. The procedures are available via the pepa.test function of the DAPAR Bioconductor R package.

Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

PubMed

Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

2014-01-01

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry

PubMed Central

Gil, Geun-Cheol; Iliff, Bryce; Cerny, Ron; Velander, William H.; Van Cott, Kevin E.

2010-01-01

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method. PMID:20586471

Qualitative and quantitative comparison of brand name and generic protein pharmaceuticals using isotope tags for relative and absolute quantification and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.

PubMed

Ye, Hongping; Hill, John; Kauffman, John; Han, Xianlin

2010-05-01

The capability of iTRAQ (isotope tags for relative and absolute quantification) reagents coupled with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) as a qualitative and quantitative technique for the analysis of complicated protein pharmaceutical mixtures was evaluated. Mixtures of Somavert and Miacalcin with a small amount of bovine serum albumin (BSA) as an impurity were analyzed. Both Somavert and Miacalcin were qualitatively identified, and BSA was detected at levels as low as 0.8mol%. Genotropin and Somavert were compared in a single experiment, and all of the distinct amino acid residues from the two proteins were readily identified. Four somatropin drug products (Genotropin, Norditropin, Jintropin, and Omnitrope) were compared using the iTRAQ/MALDI-MS method to determine the similarity between their primary structures and quantify the amount of protein in each product. All four product samples were well labeled and successfully compared when a filtration cleanup step preceded iTRAQ labeling. The quantitative accuracy of the iTRAQ method was evaluated. In all cases, the accuracy of experimentally determined protein ratios was higher than 90%, and the relative standard deviation (RSD) was less than 10%. The iTRAQ and global internal standard technology (GIST) methods were compared, and the iTRAQ method provided both higher sequence coverage and enhanced signal intensity. Published by Elsevier Inc.

The parallel reaction monitoring method contributes to a highly sensitive polyubiquitin chain quantification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsuchiya, Hikaru; Tanaka, Keiji, E-mail: tanaka-kj@igakuken.or.jp; Saeki, Yasushi, E-mail: saeki-ys@igakuken.or.jp

2013-06-28

Highlights: •The parallel reaction monitoring method was applied to ubiquitin quantification. •The ubiquitin PRM method is highly sensitive even in biological samples. •Using the method, we revealed that Ufd4 assembles the K29-linked ubiquitin chain. -- Abstract: Ubiquitylation is an essential posttranslational protein modification that is implicated in a diverse array of cellular functions. Although cells contain eight structurally distinct types of polyubiquitin chains, detailed function of several chain types including K29-linked chains has remained largely unclear. Current mass spectrometry (MS)-based quantification methods are highly inefficient for low abundant atypical chains, such as K29- and M1-linked chains, in complex mixtures thatmore » typically contain highly abundant proteins. In this study, we applied parallel reaction monitoring (PRM), a quantitative, high-resolution MS method, to quantify ubiquitin chains. The ubiquitin PRM method allows us to quantify 100 attomole amounts of all possible ubiquitin chains in cell extracts. Furthermore, we quantified ubiquitylation levels of ubiquitin-proline-β-galactosidase (Ub-P-βgal), a historically known model substrate of the ubiquitin fusion degradation (UFD) pathway. In wild-type cells, Ub-P-βgal is modified with ubiquitin chains consisting of 21% K29- and 78% K48-linked chains. In contrast, K29-linked chains are not detected in UFD4 knockout cells, suggesting that Ufd4 assembles the K29-linked ubiquitin chain(s) on Ub-P-βgal in vivo. Thus, the ubiquitin PRM is a novel, useful, quantitative method for analyzing the highly complicated ubiquitin system.« less

Protein precipitation of diluted samples in SDS-containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach.

PubMed

Santa, Cátia; Anjo, Sandra I; Manadas, Bruno

2016-07-01

Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest, especially because of the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work, two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in very diluted samples solubilized in a strong buffer (containing SDS). The amount of protein recovered after acetone and TCA/acetone precipitation was assessed, as well as the protein identification and relative quantification by SWATH-MS yields were compared with the results from the same sample without precipitation. From this study, it was possible to conclude that in the case of diluted samples in denaturing buffers, the use of cold acetone as precipitation protocol is more favourable than the use of TCA/acetone in terms of reproducibility in protein recovery and number of identified and quantified proteins. Furthermore, the reproducibility in relative quantification of the proteins is even higher in samples precipitated with acetone compared with the original sample. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A quasi-static continuum model describing interactions between plasmons and non-absorbing biomolecules

NASA Astrophysics Data System (ADS)

Salary, Mohammad Mahdi; Mosallaei, Hossein

2015-06-01

Interactions between the plasmons of noble metal nanoparticles and non-absorbing biomolecules forms the basis of the plasmonic sensors, which have received much attention. Studying these interactions can help to exploit the full potentials of plasmonic sensors in quantification and analysis of biomolecules. Here, a quasi-static continuum model is adopted for this purpose. We present a boundary-element method for computing the optical response of plasmonic particles to the molecular binding events by solving the Poisson equation. The model represents biomolecules with their molecular surfaces, thus accurately accounting for the influence of exact binding conformations as well as structural differences between different proteins on the response of plasmonic nanoparticles. The linear systems arising in the method are solved iteratively with Krylov generalized minimum residual algorithm, and the acceleration is achieved by applying precorrected-Fast Fourier Transformation technique. We apply the developed method to investigate interactions of biotinylated gold nanoparticles (nanosphere and nanorod) with four different types of biotin-binding proteins. The interactions are studied at both ensemble and single-molecule level. Computational results demonstrate the ability of presented model for analyzing realistic nanoparticle-biomolecule configurations. The method can provide comprehensive study for wide variety of applications, including protein structures, monitoring structural and conformational transitions, and quantification of protein concentrations. In addition, it is suitable for design and optimization of the nano-plasmonic sensors.

Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway.

PubMed

Yi, Lian; Shi, Tujin; Gritsenko, Marina A; X'avia Chan, Chi-Yuet; Fillmore, Thomas L; Hess, Becky M; Swensen, Adam C; Liu, Tao; Smith, Richard D; Wiley, H Steven; Qian, Wei-Jun

2018-04-17

Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway as our model. A total of 43 phosphopeptides from the EGFR-MAPK pathway were selected for the study. The recovery and sensitivity of two commonly used enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO 2 ), combined with selected reaction monitoring (SRM)-MS were evaluated. The recovery of phosphopeptides by IMAC and TiO 2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1 to 100 μg starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 μg peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25 μg range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 10 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.

Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi, Lian; Shi, Tujin; Gritsenko, Marina A.

2018-03-27

Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway as our model. 43 phosphopeptides from the EGFR–MAPK pathway were selected for the study. The recovery and sensitivity of a workflow consisted of two commonly used enrichmentmore » methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2), combined with selected reaction monitoring (SRM)-MS, were evaluated. The recovery of phosphopeptides by IMAC and TiO2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1-100 g starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 g peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25g range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 3 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.« less

Quantification of RNA Content in Reconstituted Ebola Virus Nucleocapsids by Immunoprecipitation.

PubMed

Banadyga, Logan; Ebihara, Hideki

2017-01-01

Immunoprecipitations are commonly used to isolate proteins or protein complexes and assess protein-protein interactions; however, they can also be used to assess protein-RNA complexes. Here we describe an adapted RNA immunoprecipitation technique that permits the quantification of RNA content in Ebola virus nucleocapsids that have been reconstituted in vitro by transient transfection.

Metallothionein quantification in clams by reversed-phase high-performance liquid chromatography coupled to fluorescence detection after monobromobimane derivatization.

PubMed

Alhama, José; Romero-Ruiz, Antonio; López-Barea, Juan

2006-02-24

In this paper, we describe a highly specific, sensitive and reliable method for total metallothionein (MT) quantification by RP-HPLC coupled to fluorescence detection following reaction with monobromobimane of thiols from metal-depleted MT after heat-denaturation of extracts in the presence of sodium dodecyl sulphate (SDS). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the identity of the peak resolved (t(R)=16.44) with MT: a highly fluorescent protein of approximately 8.3 kDa, in agreement with the high thiol content and low MT size. Other heat-resistant and Cys-containing proteins of 35 kDa were efficiently separated. The new method was successfully used to quantify MT content in digestive gland of clams from southern Spanish coastal sites with different metal levels, and is proposed as a tool for using MTs as biomarker in monitoring programmes.

Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics

PubMed Central

2012-01-01

Multiple reaction monitoring mass spectrometry (MRM-MS) with stable isotope dilution (SID) is increasingly becoming a widely accepted assay for the quantification of proteins and peptides. These assays have shown great promise in relatively high throughput verification of candidate biomarkers. While the use of MRM-MS assays is well established in the small molecule realm, their introduction and use in proteomics is relatively recent. As such, statistical and computational methods for the analysis of MRM-MS data from proteins and peptides are still being developed. Based on our extensive experience with analyzing a wide range of SID-MRM-MS data, we set forth a methodology for analysis that encompasses significant aspects ranging from data quality assessment, assay characterization including calibration curves, limits of detection (LOD) and quantification (LOQ), and measurement of intra- and interlaboratory precision. We draw upon publicly available seminal datasets to illustrate our methods and algorithms. PMID:23176545

Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics.

PubMed

Mani, D R; Abbatiello, Susan E; Carr, Steven A

2012-01-01

Multiple reaction monitoring mass spectrometry (MRM-MS) with stable isotope dilution (SID) is increasingly becoming a widely accepted assay for the quantification of proteins and peptides. These assays have shown great promise in relatively high throughput verification of candidate biomarkers. While the use of MRM-MS assays is well established in the small molecule realm, their introduction and use in proteomics is relatively recent. As such, statistical and computational methods for the analysis of MRM-MS data from proteins and peptides are still being developed. Based on our extensive experience with analyzing a wide range of SID-MRM-MS data, we set forth a methodology for analysis that encompasses significant aspects ranging from data quality assessment, assay characterization including calibration curves, limits of detection (LOD) and quantification (LOQ), and measurement of intra- and interlaboratory precision. We draw upon publicly available seminal datasets to illustrate our methods and algorithms.

Tryptophan and kynurenine determination in human hair by liquid chromatography.

PubMed

Dario, Michelli F; Freire, Thamires Batello; Pinto, Claudinéia Aparecida Sales de Oliveira; Prado, María Segunda Aurora; Baby, André R; Velasco, Maria Valéria R

2017-10-15

Tryptophan, an amino acid found in hair proteinaceous structure is used as a marker of hair photodegradation. Also, protein loss caused by several chemical/physical treatments can be inferred by tryptophan quantification. Kynurenine is a photo-oxidation product of tryptophan, expected to be detected when hair is exposed mainly to UVB (290-320nm) radiation range. Tryptophan from hair is usually quantified directly as a solid or after alkaline hydrolysis, spectrofluorimetrically. However, these types of measure are not sufficiently specific and present several interfering substances. Thus, this work aimed to propose a quantification method for both tryptophan and kynurenine in hair samples, after alkali hydrolysis process, by using high-performance liquid chromatography (HPLC) with fluorimetric and UV detection. The tryptophan and kynurenine quantification method was developed and validated. Black, white, bleached and dyed (blond and auburn) hair tresses were used in this study. Tryptophan and kynurenine were separated within ∼9min by HPLC. Both black and white virgin hair samples presented similar concentrations of tryptophan, while bleaching caused a reduction in the tryptophan content as well as dyeing process. Unexpectedly, UV/vis radiation did not promote significantly the conversion of tryptophan into its photo-oxidation product and consequently, kynurenine was not detected. Thus, this works presented an acceptable method for quantification of tryptophan and its photooxidation metabolite kynurenine in hair samples. Also, the results indicated that bleaching and dyeing processes promoted protein/amino acids loss but tryptophan is not extensively degraded in human hair by solar radiation. Copyright © 2017 Elsevier B.V. All rights reserved.

Volumetric adsorptive microsampling-liquid chromatography tandem mass spectrometry assay for the simultaneous quantification of four antibiotics in human blood: Method development, validation and comparison with dried blood spot.

PubMed

Barco, Sebastiano; Castagnola, Elio; Moscatelli, Andrea; Rudge, James; Tripodi, Gino; Cangemi, Giuliana

2017-10-25

In this paper we show the development and validation of a volumetric absorptive microsampling (VAMS™)-LC-MS/MS method for the simultaneous quantification of four antibiotics: piperacillin-tazobactam, meropenem, linezolid and ceftazidime in 10μL human blood. The novel VAMS-LC-MS/MS method has been compared with a dried blood spot (DBS)-based method in terms of impact of hematocrit (HCT) on accuracy, reproducibility, recovery and matrix effect. Antibiotics were extracted from VAMS and DBS by protein precipitation with methanol after a re-hydration step at 37°C for 10min. LC-MS/MS was carried out on a Thermo Scientific™ TSQ Quantum™ Access MAX triple quadrupole coupled to an Accela ™UHPLC system. The VAMS-LC-MS/MS method is selective, precise and reproducible. In contrast to DBS, it allows an accurate quantification without any HCT influence. It has been applied to samples derived from pediatric patients under therapy. VAMS is a valid alternative sampling strategy for the quantification of antibiotics and is valuable in support of clinical PK/PD studies and consequently therapeutic drug monitoring (TDM) in pediatrics. Copyright © 2017 Elsevier B.V. All rights reserved.

In situ visualization of carbonylation and its co-localization with proteins, lipids, DNA and RNA in Caenorhabditis elegans.

PubMed

Kuzmic, Mira; Javot, Hélène; Bonzom, Jean-Marc; Lecomte-Pradines, Catherine; Radman, Miroslav; Garnier-Laplace, Jacqueline; Frelon, Sandrine

2016-12-01

All key biological macromolecules are susceptible to carbonylation - an irreparable oxidative damage with deleterious biological consequences. Carbonyls in proteins, lipids and DNA from cell extracts have been used as a biomarker of oxidative stress and aging, but formation of insoluble aggregates by carbonylated proteins precludes quantification. Since carbonylated proteins correlate with and become a suspected cause of morbidity and mortality in some organisms, there is a need for their accurate quantification and localization. Using appropriate fluorescent probes, we have developed an in situ detection of total proteins, DNA, RNA, lipids and carbonyl groups at the level of the whole organism. In C. elegans, we found that after UV irradiation carbonylation co-localizes mainly with proteins and, to a lesser degree, with DNA, RNA and lipids. The method efficiency was illustrated by carbonylation induction assessment over 5 different UV doses. The procedure enables the monitoring of carbonylation in the nematode C. elegans during stress, aging and disease along its life cycle including the egg stage. Copyright © 2016 Elsevier Inc. All rights reserved.

Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nie, Song; Shi, Tujin; Fillmore, Thomas L.

Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low ng/mL to sub-ng/mL level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundant but biologically important proteins (e.g., ≤100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging. To address this need, we have developed an antibody-independent Deep-Dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide enrichment combined withmore » precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ~5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue has been demonstrated to enable precise quantification of endogenous proteins at ~10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibody is not available.« less

Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

PubMed Central

Shi, Tujin; Sun, Xuefei; Gao, Yuqian; Fillmore, Thomas L.; Schepmoes, Athena A.; Zhao, Rui; He, Jintang; Moore, Ronald J.; Kagan, Jacob; Rodland, Karin D.; Liu, Tao; Liu, Alvin Y.; Smith, Richard D.; Tang, Keqi; Camp, David G.; Qian, Wei-Jun

2013-01-01

We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50–100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ~12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successful quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies. PMID:23763644

A novel TaqMan® assay for Nosema ceranae quantification in honey bee, based on the protein coding gene Hsp70.

PubMed

Cilia, Giovanni; Cabbri, Riccardo; Maiorana, Giacomo; Cardaio, Ilaria; Dall'Olio, Raffaele; Nanetti, Antonio

2018-04-01

Nosema ceranae is now a widespread honey bee pathogen with high incidence in apiculture. Rapid and reliable detection and quantification methods are a matter of concern for research community, nowadays mainly relying on the use of biomolecular techniques such as PCR, RT-PCR or HRMA. The aim of this technical paper is to provide a new qPCR assay, based on the highly-conserved protein coding gene Hsp70, to detect and quantify the microsporidian Nosema ceranae affecting the western honey bee Apis mellifera. The validation steps to assess efficiency, sensitivity, specificity and robustness of the assay are described also. Copyright © 2018 Elsevier GmbH. All rights reserved.

Separation and quantification of monoclonal-antibody aggregates by hollow-fiber-flow field-flow fractionation.

PubMed

Fukuda, Jun; Iwura, Takafumi; Yanagihara, Shigehiro; Kano, Kenji

2014-10-01

Hollow-fiber-flow field-flow fractionation (HF5) separates protein molecules on the basis of the difference in the diffusion coefficient, and can evaluate the aggregation ratio of proteins. However, HF5 is still a minor technique because information on the separation conditions is limited. We examined in detail the effect of different settings, including the main-flow rate, the cross-flow rate, the focus point, the injection amount, and the ionic strength of the mobile phase, on fractographic characteristics. On the basis of the results, we proposed optimized conditions of the HF5 method for quantification of monoclonal antibody in sample solutions. The HF5 method was qualified regarding the precision, accuracy, linearity of the main peak, and quantitation limit. In addition, the HF5 method was applied to non-heated Mab A and heat-induced-antibody-aggregate-containing samples to evaluate the aggregation ratio and the distribution extent. The separation performance was comparable with or better than that of conventional methods including analytical ultracentrifugation-sedimentation velocity and asymmetric-flow field-flow fractionation.

Selected reaction monitoring (SRM) mass spectrometry without isotope labeling can be used for rapid protein quantification

PubMed Central

Zhi, Wenbo; Wang, Meiyao

2014-01-01

The validation of putative biomarker candidates has become the major bottle-neck in protein biomarker development. Conventional immunoaffinity methods are limited by the availability of antibodies and kits. Here we demonstrated the feasibility of using the selected reaction monitoring (SRM) without isotope labeling to achieve fast and reproducible quantification of serum proteins. The SRM/MRM assays for three standard serum proteins, including ceruloplasmin (CP), serum aymloid A (SAA) and sex hormone binding globulin (SHBG) have good linear ranges, generally 103 – 104. There are almost perfect correlations between SRM intensities and the loaded peptide amounts (R2 is usually ~0.99). Our data suggest that SRM/MRM is able to quantify proteins at 0.2 – 2 fmol level, which are comparable to the commercial ELISA/LUMINEX kits for these proteins. Excellent correlations between SRM/MRM and ELISA/LUMINEX assays were observed for SAA and SHBG (R2 = 0.928 and 0.851 respectively). The correlation between SRM/MRM and ELISA for CP is less desirable (R2 = 0.565). The reproducibility for SRM/MRM assays is generally very good but may depend on the proteins/peptides (R2 = 0.931 and 0.882 for SAA and SHBG, and 0.723 for CP). SRM/MRM assay without isotope labeling is a rapid and useful method for protein biomarker validation in a modest number of samples and is especially useful when other assays such as ELISA or Luminex beads are not available. PMID:21594933

WGA-based lectin affinity gel electrophoresis: A novel method for the detection of O-GlcNAc-modified proteins.

PubMed

Kubota, Yuji; Fujioka, Ko; Takekawa, Mutsuhiro

2017-01-01

Post-translational modification with O-linked β-N-acetylglucosamine (O-GlcNAc) occurs selectively on serine and/or threonine residues of cytoplasmic and nuclear proteins, and dynamically regulates their molecular functions. Since conventional strategies to evaluate the O-GlcNAcylation level of a specific protein require time-consuming steps, the development of a rapid and easy method for the detection and quantification of an O-GlcNAcylated protein has been a challenging issue. Here, we describe a novel method in which O-GlcNAcylated and non-O-GlcNAcylated forms of proteins are separated by lectin affinity gel electrophoresis using wheat germ agglutinin (WGA), which primarily binds to N-acetylglucosamine residues. Electrophoresis of cell lysates through a gel containing copolymerized WGA selectively induced retardation of the mobility of O-GlcNAcylated proteins, thereby allowing the simultaneous visualization of both the O-GlcNAcylated and the unmodified forms of proteins. This method is therefore useful for the quantitative detection of O-GlcNAcylated proteins.

Covariation of Peptide Abundances Accurately Reflects Protein Concentration Differences*

PubMed Central

Pirmoradian, Mohammad

2017-01-01

Most implementations of mass spectrometry-based proteomics involve enzymatic digestion of proteins, expanding the analysis to multiple proteolytic peptides for each protein. Currently, there is no consensus of how to summarize peptides' abundances to protein concentrations, and such efforts are complicated by the fact that error control normally is applied to the identification process, and do not directly control errors linking peptide abundance measures to protein concentration. Peptides resulting from suboptimal digestion or being partially modified are not representative of the protein concentration. Without a mechanism to remove such unrepresentative peptides, their abundance adversely impacts the estimation of their protein's concentration. Here, we present a relative quantification approach, Diffacto, that applies factor analysis to extract the covariation of peptides' abundances. The method enables a weighted geometrical average summarization and automatic elimination of incoherent peptides. We demonstrate, based on a set of controlled label-free experiments using standard mixtures of proteins, that the covariation structure extracted by the factor analysis accurately reflects protein concentrations. In the 1% peptide-spectrum match-level FDR data set, as many as 11% of the peptides have abundance differences incoherent with the other peptides attributed to the same protein. If not controlled, such contradicting peptide abundance have a severe impact on protein quantifications. When adding the quantities of each protein's three most abundant peptides, we note as many as 14% of the proteins being estimated as having a negative correlation with their actual concentration differences between samples. Diffacto reduced the amount of such obviously incorrectly quantified proteins to 1.6%. Furthermore, by analyzing clinical data sets from two breast cancer studies, our method revealed the persistent proteomic signatures linked to three subtypes of breast cancer. We conclude that Diffacto can facilitate the interpretation and enhance the utility of most types of proteomics data. PMID:28302922

High sensitivity mass spectrometric quantification of serum growth hormone by amphiphilic peptide conjugation

NASA Astrophysics Data System (ADS)

Arsene, Cristian G.; Schulze, Dirk; Kratzsch, Jürgen; Henrion, André

2012-12-01

Amphiphilic peptide conjugation affords a significant increase in sensitivity with protein quantification by electrospray-ionization mass spectrometry. This has been demonstrated here for human growth hormone in serum using N-(3-iodopropyl)-N,N,N-dimethyloctylammonium iodide (IPDOA-iodide) as derivatizing reagent. The signal enhancement achieved in comparison to the method without derivatization enables extension of the applicable concentration range down to the very low concentrations as encountered with clinical glucose suppression tests for patients with acromegaly. The method has been validated using a set of serum samples spiked with known amounts of recombinant 22 kDa growth hormone in the range of 0.48 to 7.65 \\mug/L. The coefficient of variation (CV) calculated, based on the deviation of results from the expected concentrations, was 3.5% and the limit of quantification (LoQ) was determined as 0.4 \\mug/L. The potential of the method as a tool in clinical practice has been demonstrated with patient samples of about 1 \\mug/L.

Proteomic Profiling of De Novo Protein Synthesis in Starvation-Induced Autophagy Using Bioorthogonal Noncanonical Amino Acid Tagging.

PubMed

Zhang, J; Wang, J; Lee, Y-M; Lim, T-K; Lin, Q; Shen, H-M

2017-01-01

Autophagy is an intracellular degradation process activated by stress factors such as nutrient starvation to maintain cellular homeostasis. There is emerging evidence demonstrating that de novo protein synthesis is involved in the autophagic process. However, up-to-date characterizing of these de novo proteins is technically difficult. In this chapter, we describe a novel method to identify newly synthesized proteins during starvation-mediated autophagy by bioorthogonal noncanonical amino acid tagging (BONCAT), in conjunction with isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics. l-azidohomoalanine (AHA) is an analog of methionine, and it can be readily incorporated into the newly synthesized proteins. The AHA-containing proteins can be enriched with avidin beads after a "click" reaction between alkyne-bearing biotin and the azide moiety of AHA. The enriched proteins are then subjected to iTRAQ™ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By using this technique, we have successfully profiled more than 700 proteins that are synthesized during starvation-induced autophagy. We believe that this approach is effective in identification of newly synthesized proteins in the process of autophagy and provides useful insights to the molecular mechanisms and biological functions of autophagy. © 2017 Elsevier Inc. All rights reserved.

A Comparative Analysis of Computational Approaches to Relative Protein Quantification Using Peptide Peak Intensities in Label-free LC-MS Proteomics Experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matzke, Melissa M.; Brown, Joseph N.; Gritsenko, Marina A.

2013-02-01

Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used for discovery studies. Typically, the objective of these discovery studies is to identify proteins that are affected by some condition of interest (e.g. disease, exposure). However, for complex biological samples, label-free LC-MS proteomics experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred frommore » one or more measured peptides. In recent years, many computational approaches to relative protein quantification of label-free LC-MS data have been published. In this review, we examine the most commonly employed quantification approaches to relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.« less

Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification

PubMed Central

2010-01-01

Background Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level. Results A new antibody-mediated signal amplification (AMSA) strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins. Conclusions Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range. PMID:20569466

Decision peptide-driven: a free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry.

PubMed

Santos, Hugo M; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Nunes-Miranda, J D; Fdez-Riverola, Florentino; Carvallo, R; Capelo, J L

2010-09-15

The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase. Copyright (c) 2010 Elsevier B.V. All rights reserved.

ICan: An Optimized Ion-Current-Based Quantification Procedure with Enhanced Quantitative Accuracy and Sensitivity in Biomarker Discovery

PubMed Central

2015-01-01

The rapidly expanding availability of high-resolution mass spectrometry has substantially enhanced the ion-current-based relative quantification techniques. Despite the increasing interest in ion-current-based methods, quantitative sensitivity, accuracy, and false discovery rate remain the major concerns; consequently, comprehensive evaluation and development in these regards are urgently needed. Here we describe an integrated, new procedure for data normalization and protein ratio estimation, termed ICan, for improved ion-current-based analysis of data generated by high-resolution mass spectrometry (MS). ICan achieved significantly better accuracy and precision, and lower false-positive rate for discovering altered proteins, over current popular pipelines. A spiked-in experiment was used to evaluate the performance of ICan to detect small changes. In this study E. coli extracts were spiked with moderate-abundance proteins from human plasma (MAP, enriched by IgY14-SuperMix procedure) at two different levels to set a small change of 1.5-fold. Forty-five (92%, with an average ratio of 1.71 ± 0.13) of 49 identified MAP protein (i.e., the true positives) and none of the reference proteins (1.0-fold) were determined as significantly altered proteins, with cutoff thresholds of ≥1.3-fold change and p ≤ 0.05. This is the first study to evaluate and prove competitive performance of the ion-current-based approach for assigning significance to proteins with small changes. By comparison, other methods showed remarkably inferior performance. ICan can be broadly applicable to reliable and sensitive proteomic survey of multiple biological samples with the use of high-resolution MS. Moreover, many key features evaluated and optimized here such as normalization, protein ratio determination, and statistical analyses are also valuable for data analysis by isotope-labeling methods. PMID:25285707

Absolute quantification of Pru av 2 in sweet cherry fruit by liquid chromatography/tandem mass spectrometry with the use of a stable isotope-labelled peptide.

PubMed

Ippoushi, Katsunari; Sasanuma, Motoe; Oike, Hideaki; Kobori, Masuko; Maeda-Yamamoto, Mari

2016-08-01

Pru av 2, a pathogenesis-related (PR) protein present in the sweet cherry (Prunus avium L.) fruit, is the principal allergen of cherry and one of the chief causes of pollen food syndrome (oral allergy syndrome). In this study, a quantitative assay for this protein was developed with the use of the protein absolute quantification (AQUA) method, which consists of liquid chromatography/tandem mass spectrometry (LC/MS/MS) employing TGC[CAM]STDASGK[(13)C6,(15)N2], a stable isotope-labelled internal standard (SIIS) peptide. This assay gave a linear relationship (r(2)>0.99) in a concentration range (2.3-600fmol/μL), and the overall coefficient of variation (CV) for multiple tests was 14.6%. Thus, the contents of this allergenic protein in sweet cherry products could be determined using this assay. This assay should be valuable for allergological investigations of Pru av 2 in sweet cherry and detection of protein contamination in foods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ultrasensitive HIV-1 p24 Assay Detects Single Infected Cells and Differences in Reservoir Induction by Latency Reversal Agents.

PubMed

Passaes, Caroline Pereira Bittencourt; Bruel, Timothée; Decalf, Jérémie; David, Annie; Angin, Mathieu; Monceaux, Valerie; Muller-Trutwin, Michaela; Noel, Nicolas; Bourdic, Katia; Lambotte, Olivier; Albert, Matthew L; Duffy, Darragh; Schwartz, Olivier; Sáez-Cirión, Asier

2017-03-15

The existence of HIV reservoirs in infected individuals under combined antiretroviral therapy (cART) represents a major obstacle toward cure. Viral reservoirs are assessed by quantification of HIV nucleic acids, a method which does not discriminate between infectious and defective viruses, or by viral outgrowth assays, which require large numbers of cells and long-term cultures. Here, we used an ultrasensitive p24 digital assay, which we report to be 1,000-fold more sensitive than classical enzyme-linked immunosorbent assays (ELISAs) in the quantification of HIV-1 Gag p24 production in samples from HIV-infected individuals. Results from ultrasensitive p24 assays were compared to those from conventional viral RNA reverse transcription-quantitative PCR (RT-qPCR)-based assays and from outgrowth assay readout by flow cytometry. Using serial dilutions and flow-based single-cell sorting, we show that viral proteins produced by a single infected cell can be detected by the ultrasensitive p24 assay. This unique sensitivity allowed the early (as soon as day 1 in 43% of cases) and more efficient detection and quantification of p24 in phytohemagglutinin-L (PHA)-stimulated CD4 + T cells from individuals under effective cART. When seven different classes of latency reversal agents (LRA) in resting CD4 + T cells from HIV-infected individuals were tested, the ultrasensitive p24 assay revealed differences in the extent of HIV reactivation. Of note, HIV RNA production was infrequently accompanied by p24 protein production (19%). Among the drugs tested, prostratin showed a superior capacity in inducing viral protein production. In summary, the ultrasensitive p24 assay allows the detection and quantification of p24 produced by single infected CD4 + T cells and provides a unique tool to assess early reactivation of infectious virus from reservoirs in HIV-infected individuals. IMPORTANCE The persistence of HIV reservoirs in infected individuals under effective antiretroviral treatment represents a major obstacle toward cure. Different methods to estimate HIV reservoirs exist, but there is currently no optimal assay to measure HIV reservoirs in HIV eradication interventions. In the present study, we report an ultrasensitive digital ELISA platform for quantification of the HIV-1 protein p24. This method was employed to assess the early reactivation of infectious virus from reservoirs in HIV-1-infected individuals. We found that viral proteins produced by a single infected cell can be detected by an ultrasensitive p24 assay. This unprecedented resolution showed major advantages in comparison to other techniques currently used to assess viral replication in reactivation studies. In addition, such a highly sensitive assay allows discrimination of drug-induced reactivation of productive HIV based on protein expression. The present study heralds new opportunities to evaluate the HIV reservoir and the efficacy of drugs used to target it. Copyright © 2017 American Society for Microbiology.

Interferon-alpha 2b quantification in inclusion bodies using reversed phase-ultra performance liquid chromatography (RP-UPLC).

PubMed

Cueto-Rojas, H F; Pérez, N O; Pérez-Sánchez, G; Ocampo-Juárez, I; Medina-Rivero, E

2010-04-15

Interferon-alpha 2b (IFN-alpha 2b) is a recombinant therapeutic cytokine produced as inclusion bodies using a strain of Escherichia coli as expression system. After fermentation and recovery, it is necessary to know the amount of recombinant IFN-alpha 2b, in order to determine the yield and the load for solubilization, and chromatographic protein purification steps. The present work details the validation of a new short run-time and fast sample-preparation method to quantify IFN-alpha 2b in inclusion bodies using Reversed Phase-Ultra Performance Liquid Chromatography (RP-UPLC). The developed method demonstrated an accuracy of 100.28%; the relative standard deviations for method precision, repeatability and inter-day precision tests were found to be 0.57%, 1.54% and 1.83%, respectively. Linearity of the method was assessed in the range of concentrations from 0.05 mg/mL to 0.5 mg/mL, the curve obtained had a determination coefficient (r(2)) of 0.9989. Detection and quantification limits were found to be 0.008 mg/mL and 0.025 mg/mL, respectively. The method also demonstrated robustness for changes in column temperature, and specificity against host proteins and other recombinant protein expressed in the same E. coli strain. Copyright 2010 Elsevier B.V. All rights reserved.

LC-MS/MS quantification of next-generation biotherapeutics: a case study for an IgE binding Nanobody in cynomolgus monkey plasma.

PubMed

Sandra, Koen; Mortier, Kjell; Jorge, Lucie; Perez, Luis C; Sandra, Pat; Priem, Sofie; Poelmans, Sofie; Bouche, Marie-Paule

2014-05-01

Nanobodies(®) are therapeutic proteins derived from the smallest functional fragments of heavy chain-only antibodies. The development and validation of an LC-MS/MS-based method for the quantification of an IgE binding Nanobody in cynomolgus monkey plasma is presented. Nanobody quantification was performed making use of a proteotypic tryptic peptide chromatographically enriched prior to LC-MS/MS analysis. The validated LLOQ at 36 ng/ml was measured with an intra- and inter-assay precision and accuracy <20%. The required sensitivity could be obtained based on the selectivity of 2D LC combined with MS/MS. No analyte specific tools for affinity purification were used. Plasma samples originating from a PK/PD study were analyzed and compared with the results obtained with a traditional ligand-binding assay. Excellent correlations between the two techniques were obtained, and similar PK parameters were estimated. A 2D LC-MS/MS method was successfully developed and validated for the quantification of a next generation biotherapeutic.

A proteomics method using immunoaffinity fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) to identify a set of interacting proteins.

PubMed

Nakata, Katsunori; Saitoh, Ryoichi; Ishigai, Masaki; Imai, Kazuhiro

2018-02-01

Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein. Copyright © 2017 John Wiley & Sons, Ltd.

Quantification of chitinase and thaumatin-like proteins in grape juices and wines.

PubMed

Le Bourse, D; Conreux, A; Villaume, S; Lameiras, P; Nuzillard, J-M; Jeandet, P

2011-09-01

Chitinases and thaumatin-like proteins are important grape proteins as they have a great influence on wine quality. The quantification of these proteins in grape juices and wines, along with their purification, is therefore crucial to study their intrinsic characteristics and the exact role they play in wines. The main isoforms of these two proteins from Chardonnay grape juice were thus purified by liquid chromatography. Two fast protein liquid chromatography (FLPC) steps allowed the fractionation and purification of the juice proteins, using cation exchange and hydrophobic interaction media. A further high-performance liquid chromatography (HPLC) step was used to achieve higher purity levels. Fraction assessment was achieved by mass spectrometry. Fraction purity was determined by HPLC to detect the presence of protein contaminants, and by nuclear magnetic resonance (NMR) spectroscopy to detect the presence of organic contaminants. Once pure fractions of lyophilized chitinase and thaumatin-like protein were obtained, ultra-HPLC (UHPLC) and enzyme-linked immunosorbent assay (ELISA) calibration curves were constructed. The quantification of these proteins in different grape juice and wine samples was thus achieved for the first time with both techniques through comparison with the purified protein calibration curve. UHPLC and ELISA showed very consistent results (less than 16% deviation for both proteins) and either could be considered to provide an accurate and reliable quantification of proteins in the oenology field.

Technical advances in proteomics: new developments in data-independent acquisition.

PubMed

Hu, Alex; Noble, William S; Wolf-Yadlin, Alejandro

2016-01-01

The ultimate aim of proteomics is to fully identify and quantify the entire complement of proteins and post-translational modifications in biological samples of interest. For the last 15 years, liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-dependent acquisition (DDA) mode has been the standard for proteomics when sampling breadth and discovery were the main objectives; multiple reaction monitoring (MRM) LC-MS/MS has been the standard for targeted proteomics when precise quantification, reproducibility, and validation were the main objectives. Recently, improvements in mass spectrometer design and bioinformatics algorithms have resulted in the rediscovery and development of another sampling method: data-independent acquisition (DIA). DIA comprehensively and repeatedly samples every peptide in a protein digest, producing a complex set of mass spectra that is difficult to interpret without external spectral libraries. Currently, DIA approaches the identification breadth of DDA while achieving the reproducible quantification characteristic of MRM or its newest version, parallel reaction monitoring (PRM). In comparative de novo identification and quantification studies in human cell lysates, DIA identified up to 89% of the proteins detected in a comparable DDA experiment while providing reproducible quantification of over 85% of them. DIA analysis aided by spectral libraries derived from prior DIA experiments or auxiliary DDA data produces identification and quantification as reproducible and precise as that achieved by MRM/PRM, except on low‑abundance peptides that are obscured by stronger signals. DIA is still a work in progress toward the goal of sensitive, reproducible, and precise quantification without external spectral libraries. New software tools applied to DIA analysis have to deal with deconvolution of complex spectra as well as proper filtering of false positives and false negatives. However, the future outlook is positive, and various researchers are working on novel bioinformatics techniques to address these issues and increase the reproducibility, fidelity, and identification breadth of DIA.

Quantitative Targeted Absolute Proteomics (QTAP)-based Pharmacoproteomics: The Importance of International Collaboration.

PubMed

Terasaki, Tetsuya

2017-01-01

Proteins such as membrane transporters, enzymes, receptors and channels play key roles in drug absorption, distribution, metabolism, and elimination, and also influence efficacy and the likelihood of adverse reactions. Therefore, if we can quantify the activities of these molecules, it may be possible to predict the behavior of candidate drugs in humans in disease states; such methodology would be extremely helpful for efficient drug development. We have developed an in silico method to select appropriate peptides within amino acid sequences in order to quantify targeted proteins by LC-MS/MS in selected reaction monitoring (SRM) mode. We have applied this method for the quantification of functional proteins in order to validate various in vitro and in vivo models. We found fairly good correlation between protein amounts and the enzymatic activities of microsomal cytochrome P450 (CYP) isoforms and uridine 5'-diphospho-glucuronosyltransferase (UGT) in human liver, as well as between protein amounts and the transport activities of multiple transporters in human lung cells. These results suggest that protein quantification can be useful in predicting activity. We have applied this approach to evaluate the usefulness and limitations of an immortalized human brain capillary endothelial cell line (D3 cells) and a P-glycoprotein humanized (hMDR1) mouse model by comparing the amounts of functional proteins in the models with those in isolated capillaries from human brain. In order to obtain sufficient human tissue specimens for further studies leading to clinical applications, we believe that international collaboration will be crucial.

Identification and quantification of ricin in biomedical samples by magnetic immunocapture enrichment and liquid chromatography electrospray ionization tandem mass spectrometry.

PubMed

Ma, Xiaoxi; Tang, Jijun; Li, Chunzheng; Liu, Qin; Chen, Jia; Li, Hua; Guo, Lei; Xie, Jianwei

2014-08-01

Ricin is a toxic protein derived from castor beans and composed of a cytotoxic A chain and a galactose-binding B chain linked by a disulfide bond, which can inhibit protein synthesis and cause cell death. Owing to its high toxicity, ease of preparation, and lack of medical countermeasures, ricin has been listed as both chemical and biological warfare agents. For homeland security or public safety, the unambiguous, sensitive, and rapid methods for identification and quantification of ricin in complicated matrices are of urgent need. Mass spectrometric analysis, which provides specific and sensitive characterization of protein, can be applied to confirm and quantify ricin. Here, we report a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method in which ricin was extracted and enriched from serum by immunocapture using anti-ricin monoclonal antibody 3D74 linked to magnetic beads, then digested by trypsin, and analyzed by LC-ESI-MS/MS. Among 19 distinct peptides observed in LC-quadrupole/time of flight-MS (LC-QTOF-MS), two specific and sensitive peptides, T7A ((49)VGLPINQR(56)) and T14B ((188)DNCLTSDSNIR(198)), were chosen, and a highly sensitive determination of ricin was established in LC-triple quadrupole-MS (LC-QqQ-MS) operating in multiple reaction monitoring mode. These specific peptides can definitely distinguish ricin from the homologous protein Ricinus communis agglutinin (RCA120), even though the amino acid sequence homology of the A-chain of ricin and RCA120 is up to ca. 93% and that of B-chain is ca. 85%. Furthermore, peptide T7A was preferred in the quantification of ricin because its sensitivity was at least one order of magnitude higher than that of the peptide T14B. Combined with immunocapture enrichment, this method provided a limit of detection of ca. 2.5 ng/mL and the limit of quantification was ca. 5 ng/mL of ricin in serum, respectively. Both precision and accuracy of this method were determined and the RSD was less than 15%. This established method was then applied to measure ricin in serum samples collected from rats exposed to ricin at the dosage of 50 μg/kg in an intravenous injection manner. The results showed that ca. 10 ng/mL of the residual ricin in poisoned rats serum could be detected even at 12 h after exposure.

Development and validation of a rapid and simple LC-MS/MS method for quantification of vemurafenib in human plasma: application to a human pharmacokinetic study.

PubMed

Bihan, Kevin; Sauzay, Chloé; Goldwirt, Lauriane; Charbonnier-Beaupel, Fanny; Hulot, Jean-Sebastien; Funck-Brentano, Christian; Zahr, Noël

2015-02-01

Vemurafenib (Zelboraf) is a new tyrosine kinase inhibitor that selectively targets activated BRAF V600E gene and is indicated for the treatment of advanced BRAF mutation-positive melanoma. We developed a simple method for vemurafenib quantification using liquid chromatography-tandem mass spectrometry. A stability study of vemurafenib in human plasma was also performed. (13)C(6)-vemurafenib was used as the internal standard. A single-step protein precipitation was used for plasma sample preparation. Chromatography was performed on an Acquity UPLC system (Waters) with chromatographic separation by the use of an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7-mm particle size; Waters). Quantification was performed using the monitoring of multiple reactions of following transitions: m/z 488.2 → 381.0 for vemurafenib and m/z 494.2 → 387.0 for internal standard. This method was linear over the range from 1.0 to 100.0 mcg/mL. The lower limit of quantification was 0.1 mcg/mL for vemurafenib in plasma. Vemurafenib remained stable for 1 month at all levels tested, when stored indifferently at room temperature (20 °C), at +4 °C, or at -20 °C. This method was used successfully to perform a plasma pharmacokinetic study of vemurafenib in a patient after oral administration at a steady state. This liquid chromatography-tandem mass spectrometry method for vemurafenib quantification in human plasma is simple, rapid, specific, sensitive, accurate, precise, and reliable.

Development of monoclonal antibody-based sensitive ELISA for the determination of Cry1Ie protein in transgenic plant.

PubMed

Zhang, Yuwen; Zhang, Wei; Liu, Yan; Wang, Jianhua; Wang, Guoying; Liu, Yunjun

2016-11-01

Cry1Ie is a kind of Bacillus thuringiensis (Bt) toxin protein which has a different action model than the Cry1Ab and Cry1Ac protein. The transgenic maize expressing Cry1Ie might be commercially used in the near future and it is urgent to develop a method to detect Cry1Ie protein in transgenic plants and their products. To develop an ELISA method, Cry1Ie protein was expressed in Escherichia coli strain Transetta DE3, purified with the Ni-NTA spin columns, and then validated by sequencing. Bioassay results showed that the purified Cry1Ie protein was highly toxic to the Asian corn borer. The polyclonal antibody (pAb) and the specific monoclonal antibody (mAb) 1G 4 2D 6 were generated from rabbit and mice which were immunized with Cry1Ie protein, respectively. Western blotting of crude Cry1Ie protein extracts was established by employing mAb 1G 4 2D 6 , whereas the mAb 1G 4 2D 6 negligibly recognized other Bt proteins. Sandwich ELISA against Cry1Ie protein was established by coating with pAb and detecting with mAb 1G 4 2D 6 . The limit of detection (LOD), the limit of quantification (LOQ), and the quantification range of the assay in different matrices of maize plant were determined as 0.27-0.51, 0.29-0.78, and 0.45-15.71 ng/mL, respectively. Recoveries of Cry1Ie protein spiked in different maize tissues ranged from 75.1 to 99.5 %. The established sandwich ELISA was verified using transgenic maize overexpressing Cry1Ie. The results in this study suggested that the established ELISA method is effective for detecting Cry1Ie protein in transgenic plants.

Quantification of a biomarker of acetaminophen protein adducts in human serum by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry: clinical and animal model applications.

PubMed

Cook, Sarah F; King, Amber D; Chang, Yan; Murray, Gordon J; Norris, Hye-Ryun K; Dart, Richard C; Green, Jody L; Curry, Steven C; Rollins, Douglas E; Wilkins, Diana G

2015-03-15

The aims of this study were to develop, validate, and apply a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantification of protein-derived 3-(cystein-S-yl)-acetaminophen (APAP-Cys) in human serum. Formation of acetaminophen (APAP) protein adducts is thought to be a critical, early event in the development of APAP-induced hepatotoxicity, and quantification of these protein adducts in human serum represents a valuable tool for assessment of APAP exposure, metabolism, and toxicity. In the reported procedure, serum samples were first dialyzed or passed through gel filtration columns to remove APAP-Cys not covalently bound to proteins. Serum eluates were then subjected to enzymatic protease digestion to liberate protein-bound APAP-Cys. Norbuprenorphine-D3 was utilized as an internal standard (IS). APAP-Cys and IS were recovered from digested serum by protein precipitation with acetonitrile, and sample extracts were analyzed by HPLC-ESI-MS/MS. The method was validated by assessment of intra- and inter-assay accuracy and imprecision on two different analytical instrument platforms. APAP-Cys could be accurately quantified from 0.010 to 10μM, and intra- and inter-assay imprecision were <15% on both analytical instruments. APAP-Cys was stable in human serum for three freeze-thaw cycles and for 24h at ambient temperature. Extracted samples were stable when stored in refrigerated autosamplers for the typical duration of analysis or when stored at -20°C for six days. Results from process efficiency and matrix effect experiments indicated adequate recovery from human serum and insignificant ion suppression or enhancement. The utility and sensitivity of the reported procedure were illustrated by analysis of clinical samples collected from subjects taking chronic, therapeutic doses of APAP. Applicability to other biological matrices was also demonstrated by measurement of protein-derived APAP-Cys in plasma collected from APAP-treated mice, a common animal model of APAP-induced hepatotoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

Use of capillary Western immunoassay (Wes) for quantification of dystrophin levels in skeletal muscle of healthy controls and individuals with Becker and Duchenne muscular dystrophy.

PubMed

Beekman, Chantal; Janson, Anneke A; Baghat, Aabed; van Deutekom, Judith C; Datson, Nicole A

2018-01-01

Duchenne muscular dystrophy (DMD) is a neuromuscular disease characterized by progressive weakness of the skeletal and cardiac muscles. This X-linked disorder is caused by open reading frame disrupting mutations in the DMD gene, resulting in strong reduction or complete absence of dystrophin protein. In order to use dystrophin as a supportive or even surrogate biomarker in clinical studies on investigational drugs aiming at correcting the primary cause of the disease, the ability to reliably quantify dystrophin expression in muscle biopsies of DMD patients pre- and post-treatment is essential. Here we demonstrate the application of the ProteinSimple capillary immunoassay (Wes) method, a gel- and blot-free method requiring less sample, antibody and time to run than conventional Western blot assay. We optimized dystrophin quantification by Wes using 2 different antibodies and found it to be highly sensitive, reproducible and quantitative over a large dynamic range. Using a healthy control muscle sample as a reference and α-actinin as a protein loading/muscle content control, a panel of skeletal muscle samples consisting of 31 healthy controls, 25 Becker Muscle dystrophy (BMD) and 17 DMD samples was subjected to Wes analysis. In healthy controls dystrophin levels varied 3 to 5-fold between the highest and lowest muscle samples, with the reference sample representing the average of all 31 samples. In BMD muscle samples dystrophin levels ranged from 10% to 90%, with an average of 33% of the healthy muscle average, while for the DMD samples the average dystrophin level was 1.3%, ranging from 0.7% to 7% of the healthy muscle average. In conclusion, Wes is a suitable, efficient and reliable method for quantification of dystrophin expression as a biomarker in DMD clinical drug development.

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Applications in Quantitative Proteomics.

PubMed

Chahrour, Osama; Malone, John

2017-01-01

Recent advances in inductively coupled plasma mass spectrometry (ICP-MS) hyphenated to different separation techniques have promoted it as a valuable tool in protein/peptide quantification. These emerging ICP-MS applications allow absolute quantification by measuring specific elemental responses. One approach quantifies elements already present in the structure of the target peptide (e.g. phosphorus and sulphur) as natural tags. Quantification of these natural tags allows the elucidation of the degree of protein phosphorylation in addition to absolute protein quantification. A separate approach is based on utilising bi-functional labelling substances (those containing ICP-MS detectable elements), that form a covalent chemical bond with the protein thus creating analogs which are detectable by ICP-MS. Based on the previously established stoichiometries of the labelling reagents, quantification can be achieved. This technique is very useful for the design of precise multiplexed quantitation schemes to address the challenges of biomarker screening and discovery. This review discusses the capabilities and different strategies to implement ICP-MS in the field of quantitative proteomics. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Development of Glycoprotein Capture-Based Label-Free Method for the High-throughput Screening of Differential Glycoproteins in Hepatocellular Carcinoma*

PubMed Central

Chen, Rui; Tan, Yexiong; Wang, Min; Wang, Fangjun; Yao, Zhenzhen; Dong, Liwei; Ye, Mingliang; Wang, Hongyang; Zou, Hanfa

2011-01-01

A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including α-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers. PMID:21474793

Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Sun, Xuefei; Gao, Yuqian

2013-07-05

We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion and the multiplexing potential of this technique. Limits of quantification (LOQs) at low ng/mL levels with a medianmore » CV of ~12% were achieved for proteins spiked into human female serum using as little as 2 µL serum. PRISM-SRM provided up to ~1000-fold improvement in the LOQ when compared to conventional SRM measurements. Multiplexing capability of PRISM-SRM was also evaluated by two sets of serum samples with 6 and 21 target peptides spiked at the low attomole/µL levels. The results from SRM measurements for pooled or post-concatenated samples were comparable to those obtained from individual peptide fractions in terms of signal-to-noise ratios and SRM peak area ratios of light to heavy peptides. PRISM-SRM was applied to measure several ng/mL-level endogenous plasma proteins, including prostate-specific antigen, in clinical patient sera where correlation coefficients > 0.99 were observed between the results from PRISM-SRM and ELISA assays. Our results demonstrate that PRISM-SRM can be successfully used for quantification of low-abundance endogenous proteins in highly complex samples. Moderate throughput (50 samples/week) can be achieved by applying the post-concatenation or fraction multiplexing strategies. We anticipate broad applications for targeted PRISM-SRM quantification of low-abundance cellular proteins in systems biology studies as well as candidate biomarkers in biofluids.« less

Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

PubMed

Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

2016-08-01

Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Automated Online Solid-Phase Derivatization for Sensitive Quantification of Endogenous S-Nitrosoglutathione and Rapid Capture of Other Low-Molecular-Mass S-Nitrosothiols.

PubMed

Wang, Xin; Garcia, Carlos T; Gong, Guanyu; Wishnok, John S; Tannenbaum, Steven R

2018-02-06

S-Nitrosothiols (RSNOs) constitute a circulating endogenous reservoir of nitric oxide and have important biological activities. In this study, an online coupling of solid-phase derivatization (SPD) with liquid chromatography-mass spectrometry (LC-MS) was developed and applied in the analysis of low-molecular-mass RSNOs. A derivatizing-reagent-modified polymer monolithic column was prepared and adapted for online SPD-LC-MS. Analytes from the LC autosampler flowed through the monolithic column for derivatization and then directly into the LC-MS for analysis. This integration of the online derivatization, LC separation, and MS detection facilitated system automation, allowing rapid, laborsaving, and sensitive detection of RSNOs. S-Nitrosoglutathione (GSNO) was quantified using this automated online method with good linearity (R 2 = 0.9994); the limit of detection was 0.015 nM. The online SPD-LC-MS method has been used to determine GSNO levels in mouse samples, 138 ± 13.2 nM of endogenous GSNO was detected in mouse plasma. Besides, the GSNO concentrations in liver (64.8 ± 11.3 pmol/mg protein), kidney (47.2 ± 6.1 pmol/mg protein), heart (8.9 ± 1.8 pmol/mg protein), muscle (1.9 ± 0.3 pmol/mg protein), hippocampus (5.3 ± 0.9 pmol/mg protein), striatum (6.7 ± 0.6 pmol/mg protein), cerebellum (31.4 ± 6.5 pmol/mg protein), and cortex (47.9 ± 4.6 pmol/mg protein) were also successfully quantified. When the derivatization was performed within 8 min, followed by LC-MS detection, samples could be rapidly analyzed compared with the offline manual method. Other low-molecular-mass RSNOs, such as S-nitrosocysteine and S-nitrosocysteinylglycine, were captured by rapid precursor-ion scanning, showing that the proposed method is a potentially powerful tool for capture, identification, and quantification of RSNOs in biological samples.

Quantification of Histidine-Rich Protein 3 of Plasmodium falciparum.

PubMed

Palani, Balraj

2018-04-01

Malaria is a life-threatening infectious disease and continues to be a major public health crisis in many parts of the tropical world. Plasmodium falciparum is responsible for the majority of mortality and morbidity associated with malaria. During the intraerythrocytic cycle, P. falciparum releases three proteins with high histidine content as follows: histidine-rich protein 1 (HRP1), histidine-rich protein 2 (HRP2), and histidine-rich protein 3 (HRP3). Currently, most of the diagnostic tests of P. falciparum infection target HRP2, and a number of monoclonal antibodies (mAbs) against HRP2 have been developed for use in HRP2 detection and quantification. When parasites have HRP2 deletions, the detection of HRP3 could augment the sensitivity of the detection system. The combination of both HRP2 and HRP3 mAbs in the detection system will enhance the test sensitivity. In the HRP quantitative enzyme-linked immunosorbent assay (ELISA), both HRP2 and HRP3 contribute to the result, but the relative contribution of HRP2 and HRP3 was unable to investigate, because of the nonavailability of HRP3 specific antibody ELISA. Hence an ELISA test system based on HRP3 is also essential for detection and quantification. There is not much documented in the literature on HRP3 antigen and HRP3 specific mAbs and polyclonal antibodies (pAbs). In the present study, recombinant HRP3 was expressed in Escherichia coli and purified with Ni-NTA agarose column. The purified rHRP3 was used for the generation and characterization of monoclonal and pAbs. The purification of monoclonal and pAbs was done using a mixed-mode chromatography sorbent, phenylpropylamine HyperCel™. With the purified antibodies, a sandwich ELISA was developed. The sandwich ELISA method was explored to detect and quantify HRP3 of P. falciparum in the spent medium. The generated mAbs could be potentially used for the detection and quantification of P. falciparum HRP3.

Sensitive Analysis of Protein Adsorption to Colloidal Gold by Differential Centrifugal Sedimentation

PubMed Central

2017-01-01

It is demonstrated that the adsorption of bovine serum albumin (BSA) to aqueous gold colloids can be quantified with molecular resolution by differential centrifugal sedimentation (DCS). This method separates colloidal particles of comparable density by mass. When proteins adsorb to the nanoparticles, both their mass and their effective density change, which strongly affects the sedimentation time. A straightforward analysis allows quantification of the adsorbed layer. Most importantly, unlike many other methods, DCS can be used to detect chemisorbed proteins (“hard corona”) as well as physisorbed proteins (“soft corona”). The results for BSA on gold colloid nanoparticles can be modeled in terms of Langmuir-type adsorption isotherms (Hill model). The effects of surface modification with small thiol-PEG ligands on protein adsorption are also demonstrated. PMID:28513153

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.

PubMed

Kleifeld, Oded; Doucet, Alain; Prudova, Anna; auf dem Keller, Ulrich; Gioia, Magda; Kizhakkedathu, Jayachandran N; Overall, Christopher M

2011-09-22

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.

Determination and Quantification of Molecular Interactions in Protein Films: A Review.

PubMed

Hammann, Felicia; Schmid, Markus

2014-12-10

Protein based films are nowadays also prepared with the aim of replacing expensive, crude oil-based polymers as environmentally friendly and renewable alternatives. The protein structure determines the ability of protein chains to form intra- and intermolecular bonds, whereas the degree of cross-linking depends on the amino acid composition and molecular weight of the protein, besides the conditions used in film preparation and processing. The functionality varies significantly depending on the type of protein and affects the resulting film quality and properties. This paper reviews the methods used in examination of molecular interactions in protein films and discusses how these intermolecular interactions can be quantified. The qualitative determination methods can be distinguished by structural analysis of solutions (electrophoretic analysis, size exclusion chromatography) and analysis of solid films (spectroscopy techniques, X-ray scattering methods). To quantify molecular interactions involved, two methods were found to be the most suitable: protein film swelling and solubility. The importance of non-covalent and covalent interactions in protein films can be investigated using different solvents. The research was focused on whey protein, whereas soy protein and wheat gluten were included as further examples of proteins.

Determination Quantification of Molecular Interactions in Protein Films: A Review

PubMed Central

Hammann, Felicia; Schmid, Markus

2014-01-01

Protein based films are nowadays also prepared with the aim of replacing expensive, crude oil-based polymers as environmentally friendly and renewable alternatives. The protein structure determines the ability of protein chains to form intra- and intermolecular bonds, whereas the degree of cross-linking depends on the amino acid composition and molecular weight of the protein, besides the conditions used in film preparation and processing. The functionality varies significantly depending on the type of protein and affects the resulting film quality and properties. This paper reviews the methods used in examination of molecular interactions in protein films and discusses how these intermolecular interactions can be quantified. The qualitative determination methods can be distinguished by structural analysis of solutions (electrophoretic analysis, size exclusion chromatography) and analysis of solid films (spectroscopy techniques, X-ray scattering methods). To quantify molecular interactions involved, two methods were found to be the most suitable: protein film swelling and solubility. The importance of non-covalent and covalent interactions in protein films can be investigated using different solvents. The research was focused on whey protein, whereas soy protein and wheat gluten were included as further examples of proteins. PMID:28788285

Molecular Diffusion Coefficients: Experimental Determination and Demonstration.

ERIC Educational Resources Information Center

Fate, Gwendolyn; Lynn, David G.

1990-01-01

Presented are laboratory methods which allow the demonstration and determination of the diffusion coefficients of compounds ranging in size from water to small proteins. Included are the procedures involving the use of a spectrometer, UV cell, triterated agar, and oxygen diffusion. Results including quantification are described. (CW)

Development and validation of a bioanalytical LC-MS method for the quantification of GHRP-6 in human plasma.

PubMed

Gil, Jeovanis; Cabrales, Ania; Reyes, Osvaldo; Morera, Vivian; Betancourt, Lázaro; Sánchez, Aniel; García, Gerardo; Moya, Galina; Padrón, Gabriel; Besada, Vladimir; González, Luis Javier

2012-02-23

Growth hormone-releasing peptide 6 (GHRP-6, His-(DTrp)-Ala-Trp-(DPhe)-Lys-NH₂, MW=872.44 Da) is a potent growth hormone secretagogue that exhibits a cytoprotective effect, maintaining tissue viability during acute ischemia/reperfusion episodes in different organs like small bowel, liver and kidneys. In the present work a quantitative method to analyze GHRP-6 in human plasma was developed and fully validated following FDA guidelines. The method uses an internal standard (IS) of GHRP-6 with ¹³C-labeled Alanine for quantification. Sample processing includes a precipitation step with cold acetone to remove the most abundant plasma proteins, recovering the GHRP-6 peptide with a high yield. Quantification was achieved by LC-MS in positive full scan mode in a Q-Tof mass spectrometer. The sensitivity of the method was evaluated, establishing the lower limit of quantification at 5 ng/mL and a range for the calibration curve from 5 ng/mL to 50 ng/mL. A dilution integrity test was performed to analyze samples at higher concentration of GHRP-6. The validation process involved five calibration curves and the analysis of quality control samples to determine accuracy and precision. The calibration curves showed R² higher than 0.988. The stability of the analyte and its internal standard (IS) was demonstrated in all conditions the samples would experience in a real time analyses. This method was applied to the quantification of GHRP-6 in plasma from nine healthy volunteers participating in a phase I clinical trial. Copyright © 2011 Elsevier B.V. All rights reserved.

Improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles

NASA Astrophysics Data System (ADS)

Syrvatka, Vasyl J.; Slyvchuk, Yurij I.; Rozgoni, Ivan I.; Gevkan, Ivan I.; Overchuk, Marta O.

2014-02-01

Modern routine enzyme immunoassays for detection and quantification of biomolecules have several disadvantages such as high cost, insufficient sensitivity, complexity and long-term execution. The surface plasmon resonance of silver nanoparticles gives reasons of creating new in the basis of simple, highly sensitive and low cost colorimetric assays that can be applied to the detection of small molecules, DNA, proteins and pollutants. The main aim of the study was the improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles. For this purpose we developed method for synthesis of silver nanoparticles with hyaluronic acid and studied possibility of use these nanoparticles in direct determination of target molecules concentration (in particular proteins) and for improving of enzyme immunoassay. As model we used conventional enzyme immunoassays for determination of progesterone and estradiol concentration. We obtained the possibility to produce silver nanoparticles with hyaluronan homogeneous in size between 10 and 12 nm, soluble and stable in water during long term of storage using modified procedure of silver nanoparticles synthesis. New method allows to obtain silver nanoparticles with strong optical properties at the higher concentrations - 60-90 μg/ml with the peak of absorbance at the wavelength 400 nm. Therefore surface plasmon resonance of silver nanoparticles with hyaluronan and ultraviolet-visible spectroscopy provide an opportunity for rapid determination of target molecules concentration (especial protein). We used silver nanoparticles as enzyme carriers and signal enhancers. Our preliminary data show that silver nanoparticles increased absorbance of samples that allows improving upper limit of determination of estradiol and progesterone concentration.

Studies on fatty acid-binding proteins. The detection and quantification of the protein from rat liver by using a fluorescent fatty acid analogue.

PubMed Central

Wilkinson, T C; Wilton, D C

1986-01-01

Fatty acid-binding protein from rat liver is shown to bind the fluorescent fatty acid probe dansyl undecanoic acid. Binding is accompanied by a shift in the fluorescence emission maximum from 550 nm to 500 nm and a 60-fold fluorescence enhancement at 500 nm. These spectral properties have allowed the use of this probe to detect and quantify microgram amounts of liver fatty acid-binding protein during purification procedures. In conjunction with h.p.l.c. the method allows the rapid estimation of liver fatty acid-binding protein in biological samples. The validity of the method is demonstrated by measuring the concentration of fatty acid-binding protein in livers from control and hypolipidaemic-drug-treated rats. The dramatic diurnal rhythm previously reported for this protein [Dempsey (1984) Curr. Top. Cell. Regul. 24, 63-86] was not observed with this method. Images Fig. 1. PMID:3800946

Electrochemical Aptamer Scaffold Biosensors for Detection of Botulism and Ricin Proteins.

PubMed

Daniel, Jessica; Fetter, Lisa; Jett, Susan; Rowland, Teisha J; Bonham, Andrew J

2017-01-01

Electrochemical DNA (E-DNA) biosensors enable the detection and quantification of a variety of molecular targets, including oligonucleotides, small molecules, heavy metals, antibodies, and proteins. Here we describe the design, electrode preparation and sensor attachment, and voltammetry conditions needed to generate and perform measurements using E-DNA biosensors against two protein targets, the biological toxins ricin and botulinum neurotoxin. This method can be applied to generate E-DNA biosensors for the detection of many other protein targets, with potential advantages over other systems including sensitive detection limits typically in the nanomolar range, real-time monitoring, and reusable biosensors.

Proteomic Analyses of Corneal Tissue Subjected to Alkali Exposure

PubMed Central

Parikh, Toral; Eisner, Natalie; Venugopalan, Praseeda; Yang, Qin; Lam, Byron L.

2011-01-01

Purpose. To determine whether exposure to alkaline chemicals results in predictable changes in corneal protein profile. To determine whether protein profile changes are indicative of severity and duration of alkali exposure. Methods. Enucleated bovine and porcine (n = 59 each) eyes were used for exposure to sodium, ammonium, and calcium hydroxide, respectively. Eyes were subjected to fluorescein staining, 5-bromo-2′-deoxy-uridine (BrdU) labeling. Excised cornea was subjected to protein extraction, spectrophotometric determination of protein amount, dynamic light scattering and SDS-PAGE profiling, mass spectrometric protein identification, and iTRAQ-labeled quantification. Select identified proteins were subjected to Western blot and immunohistochemical analyses. Results. Alkali exposure resulted in lower protein extractability from corneal tissue. Elevated aggregate formation was found with strong alkali exposure (sodium hydroxide>ammonium, calcium hydroxide), even with a short duration of exposure compared with controls. The protein yield after exposure varied as a function of postexposure time. Protein profiles changed because of alkali exposure. Concentration and strength of the alkali affected the profile change significantly. Mass spectrometry identified 15 proteins from different bands with relative quantification. Plexin D1 was identified for the first time in the cornea at a protein level that was further confirmed by Western blot and immunohistochemical analyses. Conclusions. Exposure to alkaline chemicals results in predictable and reproducible changes in corneal protein profile. Stronger alkali, longer durations, or both, of exposure resulted in lower yields and significant protein profile changes compared with controls. PMID:20861482

Quantification of triglyceride content in oleaginous materials using thermo-gravimetry

DOE PAGES

Maddi, Balakrishna; Vadlamani, Agasteswar; Viamajala, Sridhar; ...

2017-10-16

Laboratory analytical methods for quantification of triglyceride content in oleaginous biomass samples, especially microalgae, require toxic chemicals and/or organic solvents and involve multiple steps. We describe a simple triglyceride quantification method that uses thermo-gravimetry. This method is based on the observation that triglycerides undergo near-complete volatilization/degradation over a narrow temperature interval with a derivative weight loss peak at 420 °C when heated in an inert atmosphere. Degradation of the other constituents of oleaginous biomass (protein and carbohydrates) is largely complete after prolonged exposure of samples at 320 °C. Based on these observations, the triglyceride content of oleaginous biomass was estimatedmore » by using the following two-step process. In Step 1, samples were heated to 320 °C and kept isothermal at this temperature for 15 min. In Step 2, samples were heated from 320 °C to 420 °C and then kept isothermal at 420 °C for 15 min. The results show that mass loss in step 2 correlated well with triglyceride content estimates obtained from conventional techniques for diverse microalgae and oilseed samples.« less

Quantification of triglyceride content in oleaginous materials using thermo-gravimetry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maddi, Balakrishna; Vadlamani, Agasteswar; Viamajala, Sridhar

Laboratory analytical methods for quantification of triglyceride content in oleaginous biomass samples, especially microalgae, require toxic chemicals and/or organic solvents and involve multiple steps. We describe a simple triglyceride quantification method that uses thermo-gravimetry. This method is based on the observation that triglycerides undergo near-complete volatilization/degradation over a narrow temperature interval with a derivative weight loss peak at 420 °C when heated in an inert atmosphere. Degradation of the other constituents of oleaginous biomass (protein and carbohydrates) is largely complete after prolonged exposure of samples at 320 °C. Based on these observations, the triglyceride content of oleaginous biomass was estimatedmore » by using the following two-step process. In Step 1, samples were heated to 320 °C and kept isothermal at this temperature for 15 min. In Step 2, samples were heated from 320 °C to 420 °C and then kept isothermal at 420 °C for 15 min. The results show that mass loss in step 2 correlated well with triglyceride content estimates obtained from conventional techniques for diverse microalgae and oilseed samples.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Webb-Robertson, Bobbie-Jo M.; Matzke, Melissa M.; Datta, Susmita

As the capability of mass spectrometry-based proteomics has matured, tens of thousands of peptides can be measured simultaneously, which has the benefit of offering a systems view of protein expression. However, a major challenge is that with an increase in throughput, protein quantification estimation from the native measured peptides has become a computational task. A limitation to existing computationally-driven protein quantification methods is that most ignore protein variation, such as alternate splicing of the RNA transcript and post-translational modifications or other possible proteoforms, which will affect a significant fraction of the proteome. The consequence of this assumption is that statisticalmore » inference at the protein level, and consequently downstream analyses, such as network and pathway modeling, have only limited power for biomarker discovery. Here, we describe a Bayesian model (BP-Quant) that uses statistically derived peptides signatures to identify peptides that are outside the dominant pattern, or the existence of multiple over-expressed patterns to improve relative protein abundance estimates. It is a research-driven approach that utilizes the objectives of the experiment, defined in the context of a standard statistical hypothesis, to identify a set of peptides exhibiting similar statistical behavior relating to a protein. This approach infers that changes in relative protein abundance can be used as a surrogate for changes in function, without necessarily taking into account the effect of differential post-translational modifications, processing, or splicing in altering protein function. We verify the approach using a dilution study from mouse plasma samples and demonstrate that BP-Quant achieves similar accuracy as the current state-of-the-art methods at proteoform identification with significantly better specificity. BP-Quant is available as a MatLab ® and R packages at https://github.com/PNNL-Comp-Mass-Spec/BP-Quant.« less

Detection of protein modifications and counterfeit protein pharmaceuticals using isotope tags for relative and absolute quantification and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry: studies of insulins.

PubMed

Ye, Hongping; Hill, John; Kauffman, John; Gryniewicz, Connie; Han, Xianlin

2008-08-15

Isotope tags for relative and absolute quantification (iTRAQ) reagent coupled with matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometric analysis has been evaluated as both a qualitative and quantitative method for the detection of modifications to active pharmaceutical ingredients derived from recombinant DNA technologies and as a method to detect counterfeit drug products. Five types of insulin (human, bovine, porcine, Lispro, and Lantus) were used as model products in the study because of their minor variations in amino acid sequence. Several experiments were conducted in which each insulin variant was separately digested with Glu-C, and the digestate was labeled with one of four different iTRAQ reagents. All digestates were then combined for desalting and MALDI-TOF/TOF mass spectrometric analysis. When the digestion procedure was optimized, the insulin sequence coverage was 100%. Five different types of insulin were readily differentiated, including human insulin (P28K29) and Lispro insulin (K28P29), which differ only by the interchange of two contiguous residues. Moreover, quantitative analyses show that the results obtained from the iTRAQ method agree well with those determined by other conventional methods. Collectively, the iTRAQ method can be used as a qualitative and quantitative technique for the detection of protein modification and counterfeiting.

Current trends in quantitative proteomics - an update.

PubMed

Li, H; Han, J; Pan, J; Liu, T; Parker, C E; Borchers, C H

2017-05-01

Proteins can provide insights into biological processes at the functional level, so they are very promising biomarker candidates. The quantification of proteins in biological samples has been routinely used for the diagnosis of diseases and monitoring the treatment. Although large-scale protein quantification in complex samples is still a challenging task, a great amount of effort has been made to advance the technologies that enable quantitative proteomics. Seven years ago, in 2009, we wrote an article about the current trends in quantitative proteomics. In writing this current paper, we realized that, today, we have an even wider selection of potential tools for quantitative proteomics. These tools include new derivatization reagents, novel sampling formats, new types of analyzers and scanning techniques, and recently developed software to assist in assay development and data analysis. In this review article, we will discuss these innovative methods, and their current and potential applications in proteomics. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Quantitative Analysis of Staphylococcal Enterotoxins A and B in Food Matrices Using Ultra High-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS).

PubMed

Muratovic, Aida Zuberovic; Hagström, Thomas; Rosén, Johan; Granelli, Kristina; Hellenäs, Karl-Erik

2015-09-11

A method that uses mass spectrometry (MS) for identification and quantification of protein toxins, staphylococcal enterotoxins A and B (SEA and SEB), in milk and shrimp is described. The analysis was performed using a tryptic peptide, from each of the toxins, as the target analyte together with the corresponding (13)C-labeled synthetic internal standard peptide. The performance of the method was evaluated by analyzing spiked samples in the quantification range 2.5-30 ng/g (R² = 0.92-0.99). The limit of quantification (LOQ) in milk and the limit of detection (LOD) in shrimp was 2.5 ng/g, for both SEA and SEB toxins. The in-house reproducibility (RSD) was 8%-30% and 5%-41% at different concentrations for milk and shrimp, respectively. The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g. The comparison showed good coherence for the two methods: 2.9 (MS)/1.8 (ELISA) and 3.6 (MS)/3.8 (ELISA) ng/g. The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach. Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis.

Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography–Multiple-Reaction–Monitoring Mass Spectrometry

PubMed Central

Agger, Sean A.; Marney, Luke C.; Hoofnagle, Andrew N.

2011-01-01

BACKGROUND If liquid-chromatography–multiple-reaction–monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). METHODS We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. RESULTS We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay – 36.6; Sx|y = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; Sx|y = 7.9 for apoB). CONCLUSIONS Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability. PMID:20923952

Recommendations for the generation, quantification, storage and handling of peptides used for mass spectrometry-based assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoofnagle, Andrew N.; Whiteaker, Jeffrey R.; Carr, Steven A.

2015-12-30

The Clinical Proteomic Tumor Analysis Consortium (1) (CPTAC) of the National Cancer Institute (NCI) is a comprehensive and coordinated effort to accelerate the understanding of the molecular basis of cancer through the application of robust technologies and workflows for the quantitative measurements of proteins. The Assay Development Working Group of the CPTAC Program aims to foster broad uptake of targeted mass spectrometry-based assays employing isotopically labeled peptides for confident assignment and quantification, including multiple reaction monitoring (MRM; also referred to as Selected Reaction Monitoring), parallel reaction monitoring (PRM), and other targeted methods.

Protein turnover measurement using selected reaction monitoring-mass spectrometry (SRM-MS)

PubMed Central

Holman, Stephen W.; Hammond, Dean E.; Simpson, Deborah M.; Waters, John; Hurst, Jane L.

2016-01-01

Protein turnover represents an important mechanism in the functioning of cells, with deregulated synthesis and degradation of proteins implicated in many diseased states. Therefore, proteomics strategies to measure turnover rates with high confidence are of vital importance to understanding many biological processes. In this study, the more widely used approach of non-targeted precursor ion signal intensity (MS1) quantification is compared with selected reaction monitoring (SRM), a data acquisition strategy that records data for specific peptides, to determine if improved quantitative data would be obtained using a targeted quantification approach. Using mouse liver as a model system, turnover measurement of four tricarboxylic acid cycle proteins was performed using both MS1 and SRM quantification strategies. SRM outperformed MS1 in terms of sensitivity and selectivity of measurement, allowing more confident determination of protein turnover rates. SRM data are acquired using cheaper and more widely available tandem quadrupole mass spectrometers, making the approach accessible to a larger number of researchers than MS1 quantification, which is best performed on high mass resolution instruments. SRM acquisition is ideally suited to focused studies where the turnover of tens of proteins is measured, making it applicable in determining the dynamics of proteins complexes and complete metabolic pathways. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644981

Quantification of protein expression in cells and cellular subcompartments on immunohistochemical sections using a computer supported image analysis system.

PubMed

Braun, Martin; Kirsten, Robert; Rupp, Niels J; Moch, Holger; Fend, Falko; Wernert, Nicolas; Kristiansen, Glen; Perner, Sven

2013-05-01

Quantification of protein expression based on immunohistochemistry (IHC) is an important step for translational research and clinical routine. Several manual ('eyeballing') scoring systems are used in order to semi-quantify protein expression based on chromogenic intensities and distribution patterns. However, manual scoring systems are time-consuming and subject to significant intra- and interobserver variability. The aim of our study was to explore, whether new image analysis software proves to be sufficient as an alternative tool to quantify protein expression. For IHC experiments, one nucleus specific marker (i.e., ERG antibody), one cytoplasmic specific marker (i.e., SLC45A3 antibody), and one marker expressed in both compartments (i.e., TMPRSS2 antibody) were chosen. Stainings were applied on TMAs, containing tumor material of 630 prostate cancer patients. A pathologist visually quantified all IHC stainings in a blinded manner, applying a four-step scoring system. For digital quantification, image analysis software (Tissue Studio v.2.1, Definiens AG, Munich, Germany) was applied to obtain a continuous spectrum of average staining intensity. For each of the three antibodies we found a strong correlation of the manual protein expression score and the score of the image analysis software. Spearman's rank correlation coefficient was 0.94, 0.92, and 0.90 for ERG, SLC45A3, and TMPRSS2, respectively (p⟨0.01). Our data suggest that the image analysis software Tissue Studio is a powerful tool for quantification of protein expression in IHC stainings. Further, since the digital analysis is precise and reproducible, computer supported protein quantification might help to overcome intra- and interobserver variability and increase objectivity of IHC based protein assessment.

Quantification of proteins by flow cytometry: Quantification of human hepatic transporter P-gp and OATP1B1 using flow cytometry and mass spectrometry.

PubMed

Hogg, Karen; Thomas, Jerry; Ashford, David; Cartwright, Jared; Coldwell, Ruth; Weston, Daniel J; Pillmoor, John; Surry, Dominic; O'Toole, Peter

2015-07-01

Flow cytometry is a powerful tool for the quantitation of fluorescence and is proven to be able to correlate the fluorescence intensity to the number of protein on cells surface. Mass spectroscopy can also be used to determine the number of proteins per cell. Here we have developed two methods, using flow cytometry and mass spectroscopy to quantify number of transporters in human cells. These two approaches were then used to analyse the same samples so that a direct comparison could be made. Transporters have a major impact on the behaviour of a diverse number of drugs in human systems. While active uptake studies by transmembrane protein transporters using model substrates are routinely undertaken in human cell lines and hepatocytes as part of drug discovery and development, the interpretation of these results is currently limited by the inability to quantify the number of transporters present in the test samples. Here we provide a flow cytometric method for accurate quantification of transporter levels both on the cell surface and within the cell, and compare this to a quantitative mass spectrometric approach. Two transporters were selected for the study: OATP1B1 (also known as SLCO1B1, LST-1, OATP-C, OATP2) due to its important role in hepatic drug uptake and elimination; P-gp (also known as P-glycoprotein, MDR1, ABCB1) as a well characterised system and due to its potential impact on oral bioavailability, biliary and renal clearance, and brain penetration of drugs that are substrates for this transporter. In all cases the mass spectrometric method gave higher levels than the flow cytometry method. However, the two methods showed very similar trends in the relative ratios of both transporters in the hepatocyte samples investigated. The P-gp antibody allowed quantitative discrimination between externally facing transporters located in the cytoplasmic membrane and the total number of transporters on and in the cell. The proportion of externally facing transporter varied considerably in the four hepatocyte samples analysed, ranging from only 6% to 35% of intact and viable cells. The sample with only 6% externally facing transporter was further analysed by confocal microscopy which qualitatively confirmed the low level of transporter in the membrane and the large internal population. Here we prove that flow cytometry is an important tool for future protein analysis as it can not only quantify the number of proteins that a cell express but also identify the number of proteins on the surface and it is easy to apply for routine assays. Copyright © 2015 Elsevier Inc. All rights reserved.

Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions

PubMed Central

Weber, Daniela; Davies, Michael J.; Grune, Tilman

2015-01-01

Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples. PMID:26141921

Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions.

PubMed

Weber, Daniela; Davies, Michael J; Grune, Tilman

2015-08-01

Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples. © 2015 Published by Elsevier Ltd.

PCR technology for screening and quantification of genetically modified organisms (GMOs).

PubMed

Holst-Jensen, Arne; Rønning, Sissel B; Løvseth, Astrid; Berdal, Knut G

2003-04-01

Although PCR technology has obvious limitations, the potentially high degree of sensitivity and specificity explains why it has been the first choice of most analytical laboratories interested in detection of genetically modified (GM) organisms (GMOs) and derived materials. Because the products that laboratories receive for analysis are often processed and refined, the quality and quantity of target analyte (e.g. protein or DNA) frequently challenges the sensitivity of any detection method. Among the currently available methods, PCR methods are generally accepted as the most sensitive and reliable methods for detection of GM-derived material in routine applications. The choice of target sequence motif is the single most important factor controlling the specificity of the PCR method. The target sequence is normally a part of the modified gene construct, for example a promoter, a terminator, a gene, or a junction between two of these elements. However, the elements may originate from wildtype organisms, they may be present in more than one GMO, and their copy number may also vary from one GMO to another. They may even be combined in a similar way in more than one GMO. Thus, the choice of method should fit the purpose. Recent developments include event-specific methods, particularly useful for identification and quantification of GM content. Thresholds for labelling are now in place in many countries including those in the European Union. The success of the labelling schemes is dependent upon the efficiency with which GM-derived material can be detected. We will present an overview of currently available PCR methods for screening and quantification of GM-derived DNA, and discuss their applicability and limitations. In addition, we will discuss some of the major challenges related to determination of the limits of detection (LOD) and quantification (LOQ), and to validation of methods.

Investigation of Ion Transmission Effects on Intact Protein Quantification in a Triple Quadrupole Mass Spectrometer

NASA Astrophysics Data System (ADS)

Wang, Evelyn H.; Appulage, Dananjaya Kalu; McAllister, Erin A.; Schug, Kevin A.

2017-09-01

Recently, direct intact protein quantitation using triple quadrupole mass spectrometry (QqQ-MS) and multiple reaction monitoring (MRM) was demonstrated (J. Am. Soc. Mass Spectrom. 27, 886-896 (2016)). Even though QqQ-MS is known to provide extraordinary detection sensitivity for quantitative analysis, we found that intact proteins exhibited a less than 5% ion transmission from the first quadrupole to the third quadrupole mass analyzer in the presence of zero collision energy (ZCE). With the goal to enhance intact protein quantitation sensitivity, ion scattering effects, proton transfer effects, and mass filter resolution widths were examined for their contributions to the lost signal. Protein standards myoglobin and ubiquitin along with small molecules reserpine and vancomycin were analyzed together with various collision induced dissociation (CID) gases (N2, He, and Ar) at different gas pressures. Mass resolution settings played a significant role in reducing ion transmission signal. By narrowing the mass resolution window by 0.35 m/z on each side, roughly 75%-90% of the ion signal was lost. The multiply charged proteins experienced additional proton transfer effects, corresponding to 10-fold signal reduction. A study of increased sensitivity of the method was also conducted with various MRM summation techniques. Although the degree of enhancement was analyte-dependent, an up to 17-fold increase in sensitivity was observed for ubiquitin using a summation of multiple MRM transitions. Biological matrix, human urine, and equine plasma were spiked with proteins to demonstrate the specificity of the method. This study provides additional insight into optimizing the use and sensitivity of QqQ-MS for intact protein quantification. [Figure not available: see fulltext.

High-performance liquid chromatography as a technique to determine protein adsorption onto hydrophilic/hydrophobic surfaces.

PubMed

Huang, Tongtong; Anselme, Karine; Sarrailh, Segolene; Ponche, Arnaud

2016-01-30

The purpose of this study is to evaluate the potential of simple high performance liquid chromatography (HPLC) setup for quantification of adsorbed proteins on various type of plane substrates with limited area (<3 cm(2)). Protein quantification was investigated with a liquid chromatography chain equipped with a size exclusion column or a reversed-phase column. By evaluating the validation of the method according to guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), all the results obtained by HPLC were reliable. By simple adsorption test at the contact of hydrophilic (glass) and hydrophobic (polydimethylsiloxane: PDMS) surfaces, kinetics of adsorption were determined and amounts of adsorbed bovine serum albumin, myoglobin and lysozyme were obtained: as expected for each protein, the amount adsorbed at the plateau on glass (between 0.15 μg/cm(2) and 0.4 μg/cm(2)) is lower than for hydrophobic PDMS surfaces (between 0.45 μg/cm(2) and 0.8 μg/cm(2)). These results were consistent with bicinchoninic acid protein determination. According to ICH guidelines, both Reversed Phase and Size Exclusion HPLC can be validated for quantification of adsorbed protein. However, we consider the size exclusion approach more interesting in this field because additional informations can be obtained for aggregative proteins. Indeed, monomer, dimer and oligomer of bovine serum albumin (BSA) were observed in the chromatogram. On increasing the temperature, we found a decrease of peak intensity of bovine serum albumin as well as the fraction of dimer and oligomer after contact with PDMS and glass surface. As the surface can act as a denaturation parameter, these informations can have a huge impact on the elucidation of the interfacial behavior of protein and in particular for aggregation processes in pharmaceutical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Ariadne's Thread: A Robust Software Solution Leading to Automated Absolute and Relative Quantification of SRM Data.

PubMed

Nasso, Sara; Goetze, Sandra; Martens, Lennart

2015-09-04

Selected reaction monitoring (SRM) MS is a highly selective and sensitive technique to quantify protein abundances in complex biological samples. To enhance the pace of SRM large studies, a validated, robust method to fully automate absolute quantification and to substitute for interactive evaluation would be valuable. To address this demand, we present Ariadne, a Matlab software. To quantify monitored targets, Ariadne exploits metadata imported from the transition lists, and targets can be filtered according to mProphet output. Signal processing and statistical learning approaches are combined to compute peptide quantifications. To robustly estimate absolute abundances, the external calibration curve method is applied, ensuring linearity over the measured dynamic range. Ariadne was benchmarked against mProphet and Skyline by comparing its quantification performance on three different dilution series, featuring either noisy/smooth traces without background or smooth traces with complex background. Results, evaluated as efficiency, linearity, accuracy, and precision of quantification, showed that Ariadne's performance is independent of data smoothness and complex background presence and that Ariadne outperforms mProphet on the noisier data set and improved 2-fold Skyline's accuracy and precision for the lowest abundant dilution with complex background. Remarkably, Ariadne could statistically distinguish from each other all different abundances, discriminating dilutions as low as 0.1 and 0.2 fmol. These results suggest that Ariadne offers reliable and automated analysis of large-scale SRM differential expression studies.

Improved catalyzed reporter deposition, iCARD.

PubMed

Lohse, Jesper; Petersen, Kenneth Heesche; Woller, Nina Claire; Pedersen, Hans Christian; Skladtchikova, Galina; Jørgensen, Rikke Malene

2014-06-18

Novel reporters have been synthesized with extended hydrophilic linkers that in combination with polymerizing cross-linkers result in very efficient reporter deposition. By utilizing antibodies to stain HER2 proteins in a cell line model it is demonstrated that the method is highly specific and sensitive with virtually no background. The detection of HER2 proteins in tissue was used to visualize individual antigens as small dots visible in a microscope. Image analysis-assisted counting of fluorescent or colored dots allowed assessment of relative protein levels in tissue. Taken together, we have developed novel reporters that improve the CARD method allowing highly sensitive in situ detection of proteins in tissue. Our findings suggest that in situ protein quantification in biological samples can be performed by object recognition and enumeration of dots, rather than intensity-based fluorescent or colorimetric assays.

A new high-performance thin layer chromatography-based assay of detergents and surfactants commonly used in membrane protein studies.

PubMed

Barret, Laurie-Anne; Polidori, Ange; Bonneté, Françoise; Bernard-Savary, Pierre; Jungas, Colette

2013-03-15

The hydrophobic nature of membrane proteins (MPs) necessitates the use of detergents for their extraction, solubilization and purification. Because the concentration of amphiphiles is crucial in the crystallization process, detergent quantification is essential to routine analysis. Here we describe a quantitative high-performance thin-layer chromatography (HPTLC) method we developed for the detection of small quantities of detergent bound to solubilized MPs. After optimization of aqueous deposit conditions, we show that most detergents widely used in membrane protein crystallography display distinctive mobilities in a mixture of dichloromethane, methanol and acetic acid 32:7.6:0.4 (v/v/v). Migration and derivatization conditions were optimized with n-dodecyl-β-D-maltoside (DDM), the most popular detergent for membrane protein crystallization. A linear calibration curve very well fits our data from 0.1 to 1.6 μg of DDM in water with a limit of detection of 0.05 μg. This limit of detection is the best achieved to date for a routine detergent assay, being not modified by the addition of NaCl, commonly used in protein buffers. With these chromatographic conditions, no prior treatment is required to assess the quantities of detergent bound to purified MPs, thus enabling the quantification of close structure detergents via a single procedure. This HPTLC method, which is fast and requires low sample volume, is fully suitable for routine measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparative Evaluation of Small Molecular Additives and Their Effects on Peptide/Protein Identification.

PubMed

Gao, Jing; Zhong, Shaoyun; Zhou, Yanting; He, Han; Peng, Shuying; Zhu, Zhenyun; Liu, Xing; Zheng, Jing; Xu, Bin; Zhou, Hu

2017-06-06

Detergents and salts are widely used in lysis buffers to enhance protein extraction from biological samples, facilitating in-depth proteomic analysis. However, these detergents and salt additives must be efficiently removed from the digested samples prior to LC-MS/MS analysis to obtain high-quality mass spectra. Although filter-aided sample preparation (FASP), acetone precipitation (AP), followed by in-solution digestion, and strong cation exchange-based centrifugal proteomic reactors (CPRs) are commonly used for proteomic sample processing, little is known about their efficiencies at removing detergents and salt additives. In this study, we (i) developed an integrative workflow for the quantification of small molecular additives in proteomic samples, developing a multiple reaction monitoring (MRM)-based LC-MS approach for the quantification of six additives (i.e., Tris, urea, CHAPS, SDS, SDC, and Triton X-100) and (ii) systematically evaluated the relationships between the level of additive remaining in samples following sample processing and the number of peptides/proteins identified by mass spectrometry. Although FASP outperformed the other two methods, the results were complementary in terms of peptide/protein identification, as well as the GRAVY index and amino acid distributions. This is the first systematic and quantitative study of the effect of detergents and salt additives on protein identification. This MRM-based approach can be used for an unbiased evaluation of the performance of new sample preparation methods. Data are available via ProteomeXchange under identifier PXD005405.

A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays.

PubMed

Hampf, Mathias; Gossen, Manfred

2006-09-01

We established a quantitative reporter gene protocol, the P/Rluc assay system, allowing the sequential measurement of Photinus and Renilla luciferase activities from the same extract. Other than comparable commercial reporter assay systems and their noncommercial counterparts, the P/Rluc assay system was formulated under the aspect of full compatibility with standard methods for protein assays. This feature greatly expands the range of applications for assay systems quantifying the expression of multiple luciferase reporters.

Assays for Determination of Protein Concentration.

PubMed

Olson, Bradley J S C

2016-06-01

Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Development and validation of MRM methods to quantify protein isoforms of polyphenol oxidase in loquat fruits.

PubMed

Martínez-Márquez, Ascensión; Morante-Carriel, Jaime; Sellés-Marchart, Susana; Martínez-Esteso, María José; Pineda-Lucas, José Luis; Luque, Ignacio; Bru-Martínez, Roque

2013-12-06

Multiple reaction monitoring (MRM) is emerging as a promising technique for the detection and quantification of protein biomarkers in complex biological samples. Compared to Western blotting or enzyme assays, its high sensitivity, specificity, accuracy, assay speed, and sample throughput represent a clear advantage for being the approach of choice for the analysis of proteins. MRM assays are capable of detecting and quantifying proteolytic peptides differing in mass unique to particular proteins, that is, proteotypic peptides, through which different protein isoforms can be distinguished. We have focused on polyphenol oxidase (PPO), a plant conspicuous enzyme encoded by a multigenic family in loquat (Eriobotrya japonica Lindl.) and other related species. PPO is responsible for both the protection of plants from biotic stress as a feeding deterrent for herbivore insects and the enzymatic browning of fruits and vegetables. The latter makes fruit more attractive to seed dispersal agents but is also a major cause of important economic losses in agriculture and food industry. An adequate management of PPO at plant breeding level would maximize the benefits and minimize the disadvantages of this enzyme, but it would require a precise knowledge of the biological role played by each isoform in the plant. Thus, for the functional study of the PPOs, we have cloned and overexpressed fragments of three PPO isoforms from loquat to develop MRM-based methods for the quantification of each isoform. The method was developed using an ion trap instrument and validated in a QQQ instrument. It resulted in the selection of at least two peptides for each isoform that can be monitored by at least three transitions. A combination of SDS-PAGE and MRM lead to detect two out of three monitored isoforms in different gel bands corresponding to different processing stages of PPO. The method was applied to determine the amount of the PPO2 isoform in protein extracts from fruit samples using external calibrants.

Systematic Errors in Peptide and Protein Identification and Quantification by Modified Peptides*

PubMed Central

Bogdanow, Boris; Zauber, Henrik; Selbach, Matthias

2016-01-01

The principle of shotgun proteomics is to use peptide mass spectra in order to identify corresponding sequences in a protein database. The quality of peptide and protein identification and quantification critically depends on the sensitivity and specificity of this assignment process. Many peptides in proteomic samples carry biochemical modifications, and a large fraction of unassigned spectra arise from modified peptides. Spectra derived from modified peptides can erroneously be assigned to wrong amino acid sequences. However, the impact of this problem on proteomic data has not yet been investigated systematically. Here we use combinations of different database searches to show that modified peptides can be responsible for 20–50% of false positive identifications in deep proteomic data sets. These false positive hits are particularly problematic as they have significantly higher scores and higher intensities than other false positive matches. Furthermore, these wrong peptide assignments lead to hundreds of false protein identifications and systematic biases in protein quantification. We devise a “cleaned search” strategy to address this problem and show that this considerably improves the sensitivity and specificity of proteomic data. In summary, we show that modified peptides cause systematic errors in peptide and protein identification and quantification and should therefore be considered to further improve the quality of proteomic data annotation. PMID:27215553

Membrane protease degradomics: proteomic identification and quantification of cell surface protease substrates.

PubMed

Butler, Georgina S; Dean, Richard A; Smith, Derek; Overall, Christopher M

2009-01-01

The modification of cell surface proteins by plasma membrane and soluble proteases is important for physiological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying protease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control conditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching.

To elute or not to elute in immunocapture bottom-up LC-MS.

PubMed

Levernæs, Maren Christin Stillesby; Broughton, Marianne Nordlund; Reubsaet, Léon; Halvorsen, Trine Grønhaug

2017-06-15

Immunocapture-based bottom-up LC-MS is a promising technique for the quantification of low abundant proteins. Magnetic immunocapture beads provide efficient enrichment from complex samples through the highly specific interaction between the target protein and its antibody. In this article, we have performed the first thorough comparison between digestion of proteins while bound to antibody coated beads versus after elution from the beads. Two previously validated immunocapture based MS methods for the quantification of pro-gastrin releasing peptide (ProGRP) and human chorionic gonadotropin (hCG) were used as model systems. The tryptic peptide generation was shown to be protein dependent and influenced by protein folding and accessibility towards trypsin both on-beads and in the eluate. The elution of proteins bound to the beads was also shown to be incomplete. In addition, the on-beads digestion suffered from non-specific binding of the trypsin generated peptides. A combination of on-beads digestion and elution may be applied to improve both the quantitative (peak area of the signature peptides) and qualitative yield (number of missed cleavages, total number of identified peptides, coverage, signal intensity and number of zero missed cleavage peptides) of the target proteins. The quantitative yield of signature peptides was shown to be reproducible in all procedures tested. Copyright © 2017 Elsevier B.V. All rights reserved.

Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Nonprotein Thiol Imaging and Quantification in Mitochondria.

PubMed

Wang, Shenggang; Yin, Huihui; Huang, Yue; Guan, Xiangming

2018-06-11

Cellular thiols are divided into two major categories: nonprotein thiols (NPSH) and protein thiols (PSH). Thiols are unevenly distributed inside the cell and compartmentalized in subcellular structures. Most of our knowledge on functions/dysfunctions of cellular/subcellular thiols is based on the quantification of cellular/subcellular thiols through homogenization of cellular/subcellular structures followed by a thiol quantification method. We would like to report a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide {7,7'-thiobis( N-rhodamine-benzo[c][1,2,5]oxadiazole-4-sulfonamide) (TBROS)} that can effectively image and quantify live cell NPSH in mitochondria through fluorescence intensity. Limited methods are available for imaging thiols in mitochondria in live cells especially in a quantitative manner. The thiol specificity of TBROS was demonstrated by its ability to react with thiols and inability to react with biologically relevant nucleophilic functional groups other than thiols. TBROS, with minimal fluorescence, formed strong fluorescent thiol adducts (λ ex = 550 nm, λ em = 580 nm) when reacting with NPSH confirming its fluorogenicity. TBROS failed to react with PSH from bovine serum albumin and cell homogenate proteins. The high mitochondrial thiol selectivity of TBROS was achieved by its mitochondria targeting structure and its higher reaction rate with NPSH at mitochondrial pH. Imaging of mitochondrial NPSH in live cells was confirmed by two colocalization methods and use of a thiol-depleting reagent. TBROS effectively imaged NPSH changes in a quantitative manner in mitochondria in live cells. The reagent will be a useful tool in exploring physiological and pathological roles of mitochondrial thiols.

Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

NASA Astrophysics Data System (ADS)

Samanta, Sudipta; Mukherjee, Sanchita

2017-10-01

The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings

PubMed Central

Lowry, Troy W.; Hariri, Hanaa; Prommapan, Plengchart; Kusi-Appiah, Aubrey; Vafai, Nicholas; Bienkiewicz, Ewa A.; Van Winkle, David H.; Stagg, Scott M.

2016-01-01

The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid–protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (KD) and kinetics (kon and koff). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached. PMID:26649649

Identification and Quantification of Preterm Birth Biomarkers in Human Cervicovaginal Fluid by Liquid Chromatography/Tandem Mass Spectrometry

PubMed Central

Shah, Sumit J.; Yu, Kenneth H.; Sangar, Vineet; Parry, Samuel I.; Blair, Ian A.

2009-01-01

Spontaneous preterm birth (PTB) before 37 completed weeks of gestation resulting from preterm labor (PTL) is a leading contributor of perinatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could alleviate this health issue; however, conventional methods such as obstetric history and clinical risk factors, uterine activity monitoring, biochemical markers, and cervical sonography for screening women at risk for PTB have proven unsuccessful in lowering the rate of PTB. Cervicovaginal fluid (CVF) might prove to be a useful, readily available biological fluid for identifying diagnostic PTB biomarkers. Human columnar epithelial endocervical-1 (End1) and vaginal (Vk2) cell secretomes were employed to generate a stable isotope labeled proteome (SILAP) standard to facilitate characterization and relative quantification of proteins present in CVF. The SILAP standard was prepared using stable isotope labeling by amino acids in cell culture (SILAC) of End1 and Vk2 through seven passages. The labeled secreted proteins from both cell lines were combined and characterized by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). 1211 proteins were identified in the End1-Vk2 SILAP standard, with 236 proteins being consistently identified in each of the replicates analyzed. Individual proteins were found to contain < 0.5 % of the endogenous unlabeled forms. Identified proteins were screened to provide a set of fifteen candidates that have either previously been identified as potential PTB biomarkers or could be linked mechanistically to PTB. Stable isotope dilution LC-multiple reaction monitoring (MRM/MS) assays were then developed for conducting relative quantification of the fifteen candidate biomarkers in human CVF samples from term and PTB cases. Three proteins were significantly elevated in PTB cases (desmoplakin isoform 1, stratifin, and thrombospondin 1 precursor), providing a foundation for further validation in larger patient cohorts. PMID:19271751

Identification and quantification of preterm birth biomarkers in human cervicovaginal fluid by liquid chromatography/tandem mass spectrometry.

PubMed

Shah, Sumit J; Yu, Kenneth H; Sangar, Vineet; Parry, Samuel I; Blair, Ian A

2009-05-01

Spontaneous preterm birth (PTB) before 37 completed weeks of gestation resulting from preterm labor (PTL) is a leading contributor of perinatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could alleviate this health issue; however, conventional methods such as obstetric history and clinical risk factors, uterine activity monitoring, biochemical markers, and cervical sonography for screening women at risk for PTB have proven unsuccessful in lowering the rate of PTB. Cervicovaginal fluid (CVF) might prove to be a useful, readily available biological fluid for identifying diagnostic PTB biomarkers. Human columnar epithelial endocervical-1 (End1) and vaginal (Vk2) cell secretomes were employed to generate a stable isotope labeled proteome (SILAP) standard to facilitate characterization and relative quantification of proteins present in CVF. The SILAP standard was prepared using stable isotope labeling by amino acids in cell culture (SILAC) of End1 and Vk2 through seven passages. The labeled secreted proteins from both cell lines were combined and characterized by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). In total, 1211 proteins were identified in the End1-Vk2 SILAP standard, with 236 proteins being consistently identified in each of the replicates analyzed. Individual proteins were found to contain <0.5% of the endogenous unlabeled forms. Identified proteins were screened to provide a set of 15 candidates that have either previously been identified as potential PTB biomarkers or could be linked mechanistically to PTB. Stable isotope dilution LC-multiple reaction monitoring (MRM/MS) assays were then developed for conducting relative quantification of the 15 candidate biomarkers in human CVF samples from term and PTB cases. Three proteins were significantly elevated in PTB cases (desmoplakin isoform 1, stratifin, and thrombospondin 1 precursor), providing a foundation for further validation in larger patient cohorts.

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.

PubMed

Yang, Yang; Guan, Xiangming

2017-05-01

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY's reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY's reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY's reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY's reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP.

Ultraviolet, Visible, and Fluorescence Spectroscopy

NASA Astrophysics Data System (ADS)

Penner, Michael H.

Spectroscopy in the ultraviolet-visible (UV-Vis) range is one of the most commonly encountered laboratory techniques in food analysis. Diverse examples, such as the quantification of macrocomponents (total carbohydrate by the phenol-sulfuric acid method), quantification of microcomponents, (thiamin by the thiochrome fluorometric procedure), estimates of rancidity (lipid oxidation status by the thiobarbituric acid test), and surveillance testing (enzyme-linked immunoassays), are presented in this text. In each of these cases, the analytical signal for which the assay is based is either the emission or absorption of radiation in the UV-Vis range. This signal may be inherent in the analyte, such as the absorbance of radiation in the visible range by pigments, or a result of a chemical reaction involving the analyte, such as the colorimetric copper-based Lowry method for the analysis of soluble protein.

A new optical method for a fast and simple detection of ephedrine

NASA Astrophysics Data System (ADS)

Varriale, Antonio; Staiano, Maria; Strianese, Maria; Marzullo, Vincenzo; Ruggiero, Giuseppe; Secchi, Alberto; Dispenza, Massimiliano; Fiorello, Anna Maria; D'Auria, Sabato

2011-11-01

In this work we describe the synthesis of a new ephedrine derivative with a carbon linker featuring an amino reactive group, and its conjugation to the glutamine binding protein (GlnBP) from E. coli as a carrier protein for the production of polyclonal antibodies in rabbits against ephedrine. Proof-of-principle results that an efficient SPR-based indirect competitive immunoassay for the detection and quantification of ephedrine are presented. The detection limit of this assay was found to be about 33ng/ml.

Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side-by-side.

PubMed

Linscheid, Michael W

2018-03-30

To understand biological processes, not only reliable identification, but quantification of constituents in biological processes play a pivotal role. This is especially true for the proteome: protein quantification must follow protein identification, since sometimes minute changes in abundance tell the real tale. To obtain quantitative data, many sophisticated strategies using electrospray and MALDI mass spectrometry (MS) have been developed in recent years. All of them have advantages and limitations. Several years ago, we started to work on strategies, which are principally capable to overcome some of these limits. The fundamental idea is to use elemental signals as a measure for quantities. We began by replacing the radioactive 32 P with the "cold" natural 31 P to quantify modified nucleotides and phosphorylated peptides and proteins and later used tagging strategies for quantification of proteins more generally. To do this, we introduced Inductively Coupled Plasma Mass Spectrometry (ICP-MS) into the bioanalytical workflows, allowing not only reliable and sensitive detection but also quantification based on isotope dilution absolute measurements using poly-isotopic elements. The detection capability of ICP-MS becomes particularly attractive with heavy metals. The covalently bound proteins tags developed in our group are based on the well-known DOTA chelate complex (1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid) carrying ions of lanthanoides as metal core. In this review, I will outline the development of this mutual assistance between molecular and elemental mass spectrometry and discuss the scope and limitations particularly of peptide and protein quantification. The lanthanoide tags provide low detection limits, but offer multiplexing capabilities due to the number of very similar lanthanoides and their isotopes. With isotope dilution comes previously unknown accuracy. Separation techniques such as electrophoresis and HPLC were used and just slightly adapted workflows, already in use for quantification in bioanalysis. Imaging mass spectrometry (MSI) with MALDI and laser ablation ICP-MS complemented the range of application in recent years. © 2018 Wiley Periodicals, Inc.

Robust high-throughput batch screening method in 384-well format with optical in-line resin quantification.

PubMed

Kittelmann, Jörg; Ottens, Marcel; Hubbuch, Jürgen

2015-04-15

High-throughput batch screening technologies have become an important tool in downstream process development. Although continuative miniaturization saves time and sample consumption, there is yet no screening process described in the 384-well microplate format. Several processes are established in the 96-well dimension to investigate protein-adsorbent interactions, utilizing between 6.8 and 50 μL resin per well. However, as sample consumption scales with resin volumes and throughput scales with experiments per microplate, they are limited in costs and saved time. In this work, a new method for in-well resin quantification by optical means, applicable in the 384-well format, and resin volumes as small as 0.1 μL is introduced. A HTS batch isotherm process is described, utilizing this new method in combination with optical sample volume quantification for screening of isotherm parameters in 384-well microplates. Results are qualified by confidence bounds determined by bootstrap analysis and a comprehensive Monte Carlo study of error propagation. This new approach opens the door to a variety of screening processes in the 384-well format on HTS stations, higher quality screening data and an increase in throughput. Copyright © 2015 Elsevier B.V. All rights reserved.

Automated Protein Biomarker Analysis: on-line extraction of clinical samples by Molecularly Imprinted Polymers

NASA Astrophysics Data System (ADS)

Rossetti, Cecilia; Świtnicka-Plak, Magdalena A.; Grønhaug Halvorsen, Trine; Cormack, Peter A. G.; Sellergren, Börje; Reubsaet, Léon

2017-03-01

Robust biomarker quantification is essential for the accurate diagnosis of diseases and is of great value in cancer management. In this paper, an innovative diagnostic platform is presented which provides automated molecularly imprinted solid-phase extraction (MISPE) followed by liquid chromatography-mass spectrometry (LC-MS) for biomarker determination using ProGastrin Releasing Peptide (ProGRP), a highly sensitive biomarker for Small Cell Lung Cancer, as a model. Molecularly imprinted polymer microspheres were synthesized by precipitation polymerization and analytical optimization of the most promising material led to the development of an automated quantification method for ProGRP. The method enabled analysis of patient serum samples with elevated ProGRP levels. Particularly low sample volumes were permitted using the automated extraction within a method which was time-efficient, thereby demonstrating the potential of such a strategy in a clinical setting.

[Evaluation study on a new method of dietary assessment with instant photography applied in urban pregnant women in Nanjing city].

PubMed

Jiang, Tingting; Dai, Yongmei; Miao, Miao; Zhang, Yue; Song, Chenglin; Wang, Zhixu

2015-07-01

To evaluate the usefulness and efficiency of a novel dietary method among urban pregnant women. Sixty one pregnant women were recruited from the ward and provided with a meal accurately weighed before cooking. The meal was photographed from three different angles before and after eating. The subjects were also interviewed for 24 h dietary recall by the investigators. Food weighting, image quantification and 24 h dietary recall were conducted by investigators from three different groups, and the messages were isolated from each other. Food consumption was analyzed on bases of classification and total summation. Nutrient intake from the meal was calculated for each subject. The data obtained from the dietary recall and the image quantification were compared with the actual values. Correlation and regression analyses were carried out on values between weight method and image quantification as well as dietary recall. Total twenty three kinds of food including rice, vegetables, fish, meats and soy bean curd were included in the experimental meal for the study. Compared with data from 24 h dietary recall (r = 0.413, P < 0.05), food weight estimated by image quantification (r = 0.778, P < 0.05, n = 308) were more correlated with weighed data, and show more concentrated linear distribution. Absolute difference distribution between image quantification and weight method of all food was 77.23 ± 56.02 (P < 0.05, n = 61), which was much small than the difference (172.77 ± 115.18) between 24 h recall and weight method. Values of almost all nutrients, including energy, protein, fat, carbohydrate, vitamin A, vitamin C, calcium, iron and zine calculated based on food weight from image quantification were more close to those of weighed data compared with 24 h dietary recall (P < 0.01). The results found by the Bland Altman analysis showed that the majority of the measurements for nutrient intake, were scattered along the mean difference line and close to the equality line (difference = 0). The plots show fairly good agreement between estimated and actual food consumption. It indicate that the differences (including the outliers) were random and did not exhibit any systematic bias, being consistent over different levels of mean food amount. On the other hand, the questionnaire showed that fifty six pregnant women considered the image quantification was less time-consuming and burdened than 24 h recall. Fifty eight of them would like to use image quantification to know their dietary status. The novel method which called instant photography (image quantification) for dietary assessment is more effective than conventional 24 h dietary recall and it also can obtain food intake values close to weighed data.

Fluorogenic Ag+–Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining

PubMed Central

Xie, Sheng; Wong, Alex Y. H.; Kwok, Ryan T. K.; Li, Ying; Su, Huifang; Lam, Jacky W. Y.

2018-01-01

Abstract Silver staining, which exploits the special bioaffinity and the chromogenic reduction of silver ions, is an indispensable visualization method in biology. It is a most popular method for in‐gel protein detection. However, it is limited by run‐to‐run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) analysis; limitations that are largely attributed to the tricky chromogenic visualization. Herein, we reported a novel water‐soluble fluorogenic Ag+ probe, the sensing mechanism of which is based on an aggregation‐induced emission (AIE) process driven by tetrazolate‐Ag+ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains. PMID:29575702

Compressive Force Spectroscopy: From Living Cells to Single Proteins.

PubMed

Wang, Jiabin; Liu, Meijun; Shen, Yi; Sun, Jielin; Shao, Zhifeng; Czajkowsky, Daniel Mark

2018-03-23

One of the most successful applications of atomic force microscopy (AFM) in biology involves monitoring the effect of force on single biological molecules, often referred to as force spectroscopy. Such studies generally entail the application of pulling forces of different magnitudes and velocities upon individual molecules to resolve individualistic unfolding/separation pathways and the quantification of the force-dependent rate constants. However, a less recognized variation of this method, the application of compressive force, actually pre-dates many of these "tensile" force spectroscopic studies. Further, beyond being limited to the study of single molecules, these compressive force spectroscopic investigations have spanned samples as large as living cells to smaller, multi-molecular complexes such as viruses down to single protein molecules. Correspondingly, these studies have enabled the detailed characterization of individual cell states, subtle differences between seemingly identical viral structures, as well as the quantification of rate constants of functionally important, structural transitions in single proteins. Here, we briefly review some of the recent achievements that have been obtained with compressive force spectroscopy using AFM and highlight exciting areas of its future development.

Ultrasonic-based membrane aided sample preparation of urine proteomes.

PubMed

Jesus, Jemmyson Romário; Santos, Hugo M; López-Fernández, H; Lodeiro, Carlos; Arruda, Marco Aurélio Zezzi; Capelo, J L

2018-02-01

A new ultrafast ultrasonic-based method for shotgun proteomics as well as label-free protein quantification in urine samples is developed. The method first separates the urine proteins using nitrocellulose-based membranes and then proteins are in-membrane digested using trypsin. The enzymatic digestion process is accelerated from overnight to four minutes using a sonoreactor ultrasonic device. Overall, the sample treatment pipeline comprising protein separation, digestion and identification is done in just 3h. The process is assessed using urine of healthy volunteers. The method shows that male can be differentiated from female using the protein content of urine in a fast, easy and straightforward way. 232 and 226 proteins are identified in urine of male and female, respectively. From this, 162 are common to both genders, whilst 70 are unique to male and 64 to female. From the 162 common proteins, 13 are present at levels statistically different (p < 0.05). The method matches the analytical minimalism concept as outlined by Halls, as each stage of this analysis is evaluated to minimize the time, cost, sample requirement, reagent consumption, energy requirements and production of waste products. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein identification and quantification from riverbank grape, Vitis riparia: Comparing SDS-PAGE and FASP-GPF techniques for shotgun proteomic analysis.

PubMed

George, Iniga S; Fennell, Anne Y; Haynes, Paul A

2015-09-01

Protein sample preparation optimisation is critical for establishing reproducible high throughput proteomic analysis. In this study, two different fractionation sample preparation techniques (in-gel digestion and in-solution digestion) for shotgun proteomics were used to quantitatively compare proteins identified in Vitis riparia leaf samples. The total number of proteins and peptides identified were compared between filter aided sample preparation (FASP) coupled with gas phase fractionation (GPF) and SDS-PAGE methods. There was a 24% increase in the total number of reproducibly identified proteins when FASP-GPF was used. FASP-GPF is more reproducible, less expensive and a better method than SDS-PAGE for shotgun proteomics of grapevine samples as it significantly increases protein identification across biological replicates. Total peptide and protein information from the two fractionation techniques is available in PRIDE with the identifier PXD001399 (http://proteomecentral.proteomexchange.org/dataset/PXD001399). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acoustic technology for high-performance disruption and extraction of plant proteins.

PubMed

Toorchi, Mahmoud; Nouri, Mohammad-Zaman; Tsumura, Makoto; Komatsu, Setsuko

2008-07-01

Acoustic technology shows the capability of protein pellet homogenization from different tissue samples of soybean and rice in a manner comparable to the ordinary mortar/pestle method and far better than the vortex/ultrasonic method with respect to the resolution of the protein pattern through two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). With acoustic technology, noncontact tissue disruption and protein pellet homogenization can be carried out in a computer-controlled manner, which ultimately increases the efficiency of the process for a large number of samples. A lysis buffer termed the T-buffer containing TBP, thiourea, and CHAPS yields an excellent result for the 2D-PAGE separation of soybean plasma membrane proteins followed by the 2D-PAGE separation of crude protein of soybean and rice tissues. For this technology, the T-buffer is preferred because protein quantification is possible by eliminating the interfering compound 2-mercaptoethanol and because of the high reproducibility of 2D-PAGE separation.

Engineering of filamentous bacteriophage for protein sensing

NASA Astrophysics Data System (ADS)

Brasino, Michael

Methods of high throughput, sensitive and cost effective quantification of proteins enables personalized medicine by allowing healthcare professionals to better monitor patient condition and response to treatment. My doctoral research has attempted to advance these methods through the use of filamentous bacteriophage (phage). These bacterial viruses are particularly amenable to both genetic and chemical engineering and can be produced efficiently in large amounts. Here, I discuss several strategies for modifying phage for use in protein sensing assays. These include the expression of bio-orthogonal conjugation handles on the phage coat, the incorporation of specific recognition sequences within the phage genome, and the creation of antibody-phage conjugates via a photo-crosslinking non-canonical amino acid. The physical and chemical characterization of these engineered phage and the results of their use in modified protein sensing assays will be presented.

Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples.

PubMed

Abbas, Ioana M; Hoffmann, Holger; Montes-Bayón, María; Weller, Michael G

2018-06-01

Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the "sticky" character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5-40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials. Graphical abstract Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).

Relative quantification of biomarkers using mixed-isotope labeling coupled with MS

PubMed Central

Chapman, Heidi M; Schutt, Katherine L; Dieter, Emily M; Lamos, Shane M

2013-01-01

The identification and quantification of important biomarkers is a critical first step in the elucidation of biological systems. Biomarkers take many forms as cellular responses to stimuli and can be manifested during transcription, translation, and/or metabolic processing. Increasingly, researchers have relied upon mixed-isotope labeling (MIL) coupled with MS to perform relative quantification of biomarkers between two or more biological samples. MIL effectively tags biomarkers of interest for ease of identification and quantification within the mass spectrometer by using isotopic labels that introduce a heavy and light form of the tag. In addition to MIL coupled with MS, a number of other approaches have been used to quantify biomarkers including protein gel staining, enzymatic labeling, metabolic labeling, and several label-free approaches that generate quantitative data from the MS signal response. This review focuses on MIL techniques coupled with MS for the quantification of protein and small-molecule biomarkers. PMID:23157360

Integrated protocol for reliable and fast quantification and documentation of electrophoresis gels.

PubMed

Rehbein, Peter; Schwalbe, Harald

2015-06-01

Quantitative analysis of electrophoresis gels is an important part in molecular cloning, as well as in protein expression and purification. Parallel quantifications in yield and purity can be most conveniently obtained from densitometric analysis. This communication reports a comprehensive, reliable and simple protocol for gel quantification and documentation, applicable for single samples and with special features for protein expression screens. As major component of the protocol, the fully annotated code of a proprietary open source computer program for semi-automatic densitometric quantification of digitized electrophoresis gels is disclosed. The program ("GelQuant") is implemented for the C-based macro-language of the widespread integrated development environment of IGOR Pro. Copyright © 2014 Elsevier Inc. All rights reserved.

A liquid chromatography/tandem mass spectrometry assay for the analysis of atomoxetine in human plasma and in vitro cellular samples

PubMed Central

Appel, David I.; Brinda, Bryan; Markowitz, John S.; Newcorn, Jeffrey H.; Zhu, Hao-Jie

2012-01-01

A simple, rapid and sensitive method for quantification of atomoxetine by liquid chromatography- tandem mass spectrometry (LC-MS/MS) was developed. This assay represents the first LC-MS/MS quantification method for atomoxetine utilizing electrospray ionization. Deuterated atomoxetine (d3-atomoxetine) was adopted as the internal standard. Direct protein precipitation was utilized for sample preparation. This method was validated for both human plasma and in vitro cellular samples. The lower limit of quantification was 3 ng/ml and 10 nM for human plasma and cellular samples, respectively. The calibration curves were linear within the ranges of 3 ng/ml to 900 ng/ml and 10 nM to 10 μM for human plasma and cellular samples, respectively (r2 > 0.999). The intra- and inter-day assay accuracy and precision were evaluated using quality control samples at 3 different concentrations in both human plasma and cellular lysate. Sample run stability, assay selectivity, matrix effect, and recovery were also successfully demonstrated. The present assay is superior to previously published LC-MS and LC-MS/MS methods in terms of sensitivity or the simplicity of sample preparation. This assay is applicable to the analysis of atomoxetine in both human plasma and in vitro cellular samples. PMID:22275222

Clinical applications of MS-based protein quantification.

PubMed

Sabbagh, Bassel; Mindt, Sonani; Neumaier, Michael; Findeisen, Peter

2016-04-01

Mass spectrometry-based assays are increasingly important in clinical laboratory medicine and nowadays are already commonly used in several areas of routine diagnostics. These include therapeutic drug monitoring, toxicology, endocrinology, pediatrics, and microbiology. Accordingly, some of the most common analyses are therapeutic drug monitoring of immunosuppressants, vitamin D, steroids, newborn screening, and bacterial identification. However, MS-based quantification of peptides and proteins for routine diagnostic use is rather rare up to now despite excellent analytical specificity and good sensitivity. Here, we want to give an overview over current fit-for-purpose assays for MS-based protein quantification. Advantages as well as challenges of this approach will be discussed with focus on feasibility for routine diagnostic use. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic Methods of Detection and Quantification of Protein Toxins.

PubMed

Duracova, Miloslava; Klimentova, Jana; Fucikova, Alena; Dresler, Jiri

2018-02-28

Biological toxins are a heterogeneous group of compounds that share commonalities with biological and chemical agents. Among them, protein toxins represent a considerable, diverse set. They cover a broad range of molecular weights from less than 1000 Da to more than 150 kDa. This review aims to compare conventional detection methods of protein toxins such as in vitro bioassays with proteomic methods, including immunoassays and mass spectrometry-based techniques and their combination. Special emphasis is given to toxins falling into a group of selected agents, according to the Centers for Disease Control and Prevention, such as Staphylococcal enterotoxins , Bacillus anthracis toxins, Clostridium botulinum toxins, Clostridium perfringens epsilon toxin, ricin from Ricinus communis , Abrin from Abrus precatorius or control of trade in dual-use items in the European Union, including lesser known protein toxins such as Viscumin from Viscum album . The analysis of protein toxins and monitoring for biological threats, i.e., the deliberate spread of infectious microorganisms or toxins through water, food, or the air, requires rapid and reliable methods for the early identification of these agents.

Proteomic Methods of Detection and Quantification of Protein Toxins

PubMed Central

Klimentova, Jana; Fucikova, Alena

2018-01-01

Biological toxins are a heterogeneous group of compounds that share commonalities with biological and chemical agents. Among them, protein toxins represent a considerable, diverse set. They cover a broad range of molecular weights from less than 1000 Da to more than 150 kDa. This review aims to compare conventional detection methods of protein toxins such as in vitro bioassays with proteomic methods, including immunoassays and mass spectrometry-based techniques and their combination. Special emphasis is given to toxins falling into a group of selected agents, according to the Centers for Disease Control and Prevention, such as Staphylococcal enterotoxins, Bacillus anthracis toxins, Clostridium botulinum toxins, Clostridium perfringens epsilon toxin, ricin from Ricinus communis, Abrin from Abrus precatorius or control of trade in dual-use items in the European Union, including lesser known protein toxins such as Viscumin from Viscum album. The analysis of protein toxins and monitoring for biological threats, i.e., the deliberate spread of infectious microorganisms or toxins through water, food, or the air, requires rapid and reliable methods for the early identification of these agents. PMID:29495560

Quantification of the Triazole Antifungal Compounds Voriconazole and Posaconazole in Human Serum or Plasma Using Liquid Chromatography Electrospray Tandem Mass Spectrometry (HPLC-ESI-MS/MS).

PubMed

Molinelli, Alejandro R; Rose, Charles H

2016-01-01

Voriconazole and posaconazole are triazole antifungal compounds used in the treatment of fungal infections. Therapeutic drug monitoring of both compounds is recommended in order to guide drug dosing to achieve optimal blood concentrations. In this chapter we describe an HPLC-ESI-MS/MS method for the quantification of both compounds in human plasma or serum following a simple specimen preparation procedure. Specimen preparation consists of protein precipitation using methanol and acetonitrile followed by a cleanup step that involves filtration through a cellulose acetate membrane. The specimen is then injected into an HPLC-ESI-MS/MS equipped with a C18 column and separated over an acetonitrile gradient. Quantification of the drugs in the specimen is achieved by comparing the response of the unknown specimen to that of the calibrators in the standard curve using multiple reaction monitoring.

In situ, rapid, and temporally resolved measurements of cellulase adsorption onto lignocellulosic substrates by UV-vis spectrophotometry

Treesearch

Hao Liu; J. Y. Zhu; X. S. Chai

2011-01-01

This study demonstrated two in situ UV-vis spectrophotometric methods for rapid and temporally resolved measurements of cellulase adsorption onto cellulosic and lignocellulosic substrates during enzymatic hydrolysis. The cellulase protein absorption peak at 280 nm was used for quantification. The spectral interferences from light scattering by small fibers (fines) and...

Breast density quantification with cone-beam CT: A post-mortem study

PubMed Central

Johnson, Travis; Ding, Huanjun; Le, Huy Q.; Ducote, Justin L.; Molloi, Sabee

2014-01-01

Forty post-mortem breasts were imaged with a flat-panel based cone-beam x-ray CT system at 50 kVp. The feasibility of breast density quantification has been investigated using standard histogram thresholding and an automatic segmentation method based on the fuzzy c-means algorithm (FCM). The breasts were chemically decomposed into water, lipid, and protein immediately after image acquisition was completed. The percent fibroglandular volume (%FGV) from chemical analysis was used as the gold standard for breast density comparison. Both image-based segmentation techniques showed good precision in breast density quantification with high linear coefficients between the right and left breast of each pair. When comparing with the gold standard using %FGV from chemical analysis, Pearson’s r-values were estimated to be 0.983 and 0.968 for the FCM clustering and the histogram thresholding techniques, respectively. The standard error of the estimate (SEE) was also reduced from 3.92% to 2.45% by applying the automatic clustering technique. The results of the postmortem study suggested that breast tissue can be characterized in terms of water, lipid and protein contents with high accuracy by using chemical analysis, which offers a gold standard for breast density studies comparing different techniques. In the investigated image segmentation techniques, the FCM algorithm had high precision and accuracy in breast density quantification. In comparison to conventional histogram thresholding, it was more efficient and reduced inter-observer variation. PMID:24254317

A Statistics-based Platform for Quantitative N-terminome Analysis and Identification of Protease Cleavage Products*

PubMed Central

auf dem Keller, Ulrich; Prudova, Anna; Gioia, Magda; Butler, Georgina S.; Overall, Christopher M.

2010-01-01

Terminal amine isotopic labeling of substrates (TAILS), our recently introduced platform for quantitative N-terminome analysis, enables wide dynamic range identification of original mature protein N-termini and protease cleavage products. Modifying TAILS by use of isobaric tag for relative and absolute quantification (iTRAQ)-like labels for quantification together with a robust statistical classifier derived from experimental protease cleavage data, we report reliable and statistically valid identification of proteolytic events in complex biological systems in MS2 mode. The statistical classifier is supported by a novel parameter evaluating ion intensity-dependent quantification confidences of single peptide quantifications, the quantification confidence factor (QCF). Furthermore, the isoform assignment score (IAS) is introduced, a new scoring system for the evaluation of single peptide-to-protein assignments based on high confidence protein identifications in the same sample prior to negative selection enrichment of N-terminal peptides. By these approaches, we identified and validated, in addition to known substrates, low abundance novel bioactive MMP-2 targets including the plasminogen receptor S100A10 (p11) and the proinflammatory cytokine proEMAP/p43 that were previously undescribed. PMID:20305283

Analytical platform evaluation for quantification of ERG in prostate cancer using protein and mRNA detection methods

DOE PAGES

He, Jintang; Schepmoes, Athena A.; Shi, Tujin; ...

2015-01-01

Background: The established methods for detecting prostate cancer (CaP) are based on tests using PSA (blood), PCA3 (urine), and AMACR (tissue) as biomarkers in patient samples. The demonstration of ERG oncoprotein overexpression due to gene fusion in CaP has thus provided ERG as an additional biomarker. Based on this, we hypothesized that ERG protein quantification methods can be of use in the diagnosis of prostate cancer. Methods: Therefore, an antibody-free assay for ERG3 protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERGmore » negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment). Results: Recombinant ERG3 protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was around approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine. The sensitivity of the in-house enzyme-linked immunosorbent assay (ELISA) was similar to the PRISM-SRM assay, with detection of 30 pg of purified recombinant ERG3 protein and 10,000 VCaP cells. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 100 VCaP cells, in comparison to NanoString methodologies which detected ERG from 10,000 cells. Conclusions: Based on this data, we propose that the detection of both ERG transcriptional products with RNA-based assays, as well as protein products of ERG using PRISM-SRM assays, may be of clinical value in developing diagnostics and prognostics assays for prostate cancer given their sensitivity, specificity, and reproducibility.« less

Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.

PubMed

Branigan, Emma; Pliotas, Christos; Hagelueken, Gregor; Naismith, James H

2013-11-01

Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.

Quantification of SAA1 and SAA2 in lung cancer plasma using the isotype-specific PRM assays.

PubMed

Kim, Yeoun Jin; Gallien, Sebastien; El-Khoury, Victoria; Goswami, Panchali; Sertamo, Katriina; Schlesser, Marc; Berchem, Guy; Domon, Bruno

2015-09-01

The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross-reactivity. On the other hand, when mass spectrometry (MS)-based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype-specific (SAA1α,  SAA 1β,  SAA1γ,  SAA2α,  SAA2β), protein-specific (SAA1 or SAA2), and pan SAA (SAA1 and SAA2) were chosen to differentiate SAAs in lung cancer plasma samples using a panel of PRM assays. The measurement of specific isotypes, leveraging the analytical performance of PRM, allowed to quantify the allelic variants of both target proteins. The isotypes detected were corroborated with the genetic information obtained from the same samples. The combination of SAA2α and SAA2β assays representing the total SAA2 concentration demonstrated a superior analytical outcome than the previously used assay on the common peptide when applied to the detection of lung cancer. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MStern Blotting-High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates.

PubMed

Berger, Sebastian T; Ahmed, Saima; Muntel, Jan; Cuevas Polo, Nerea; Bachur, Richard; Kentsis, Alex; Steen, Judith; Steen, Hanno

2015-10-01

We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. In summary, our new method maintains the strengths of FASP and simultaneously overcomes one of the major limitations of FASP without compromising protein identification and quantification. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

MStern Blotting–High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates*

PubMed Central

Berger, Sebastian T.; Ahmed, Saima; Muntel, Jan; Cuevas Polo, Nerea; Bachur, Richard; Kentsis, Alex; Steen, Judith; Steen, Hanno

2015-01-01

We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. In summary, our new method maintains the strengths of FASP and simultaneously overcomes one of the major limitations of FASP without compromising protein identification and quantification. PMID:26223766

Clinical review: improving the measurement of serum thyroglobulin with mass spectrometry.

PubMed

Hoofnagle, Andrew N; Roth, Mara Y

2013-04-01

Serum thyroglobulin (Tg) measurements are central to the management of patients treated for differentiated thyroid carcinoma. For decades, Tg measurements have relied on methods that are subject to interference by commonly found substances in human serum and plasma, such as Tg autoantibodies. As a result, many patients need additional imaging studies to rule out cancer persistence or recurrence that could be avoided with more sensitive and specific testing methods. The aims of this review are to: 1) briefly review the interferences common to Tg immunoassays; 2) introduce readers to liquid chromatography-tandem mass spectrometry as a method for quantifying proteins in human serum/plasma; and 3) discuss the potential benefits and limitations of the method in the quantification of serum Tg. Mass spectrometric methods have traditionally lacked the sensitivity, robustness, and throughput to be useful clinical assays. These methods failed to meet the necessary clinical benchmarks due to the nature of the mass spectrometry workflow and instrumentation. Over the past few years, there have been major advances in reagents, automation, and instrumentation for the quantification of proteins using mass spectrometry. More recently, methods using mass spectrometry to detect and quantify Tg have been developed and are of sufficient quality to be used in the management of patients. Novel serum Tg assays that use mass spectrometry may avoid the issue of autoantibody interference and other problems with currently available immunoassays for Tg. Prospective studies are needed to fully understand the potential benefits of novel Tg assays to patients and care providers.

Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring*

PubMed Central

Lawless, Craig; Holman, Stephen W.; Brownridge, Philip; Lanthaler, Karin; Harman, Victoria M.; Watkins, Rachel; Hammond, Dean E.; Miller, Rebecca L.; Sims, Paul F. G.; Grant, Christopher M.; Eyers, Claire E.; Beynon, Robert J.

2016-01-01

Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800 Saccharomyces cerevisiae proteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ∼100 million molecules-per-cell, a median of ∼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a “gold-standard” reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies. PMID:26750110

Evaluation of Normalization Methods on GeLC-MS/MS Label-Free Spectral Counting Data to Correct for Variation during Proteomic Workflows

NASA Astrophysics Data System (ADS)

Gokce, Emine; Shuford, Christopher M.; Franck, William L.; Dean, Ralph A.; Muddiman, David C.

2011-12-01

Normalization of spectral counts (SpCs) in label-free shotgun proteomic approaches is important to achieve reliable relative quantification. Three different SpC normalization methods, total spectral count (TSpC) normalization, normalized spectral abundance factor (NSAF) normalization, and normalization to selected proteins (NSP) were evaluated based on their ability to correct for day-to-day variation between gel-based sample preparation and chromatographic performance. Three spectral counting data sets obtained from the same biological conidia sample of the rice blast fungus Magnaporthe oryzae were analyzed by 1D gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Equine myoglobin and chicken ovalbumin were spiked into the protein extracts prior to 1D-SDS- PAGE as internal protein standards for NSP. The correlation between SpCs of the same proteins across the different data sets was investigated. We report that TSpC normalization and NSAF normalization yielded almost ideal slopes of unity for normalized SpC versus average normalized SpC plots, while NSP did not afford effective corrections of the unnormalized data. Furthermore, when utilizing TSpC normalization prior to relative protein quantification, t-testing and fold-change revealed the cutoff limits for determining real biological change to be a function of the absolute number of SpCs. For instance, we observed the variance decreased as the number of SpCs increased, which resulted in a higher propensity for detecting statistically significant, yet artificial, change for highly abundant proteins. Thus, we suggest applying higher confidence level and lower fold-change cutoffs for proteins with higher SpCs, rather than using a single criterion for the entire data set. By choosing appropriate cutoff values to maintain a constant false positive rate across different protein levels (i.e., SpC levels), it is expected this will reduce the overall false negative rate, particularly for proteins with higher SpCs.

A new automated turbidimetric immunoassay for the measurement of canine C-reactive protein.

PubMed

Piñeiro, Matilde; Pato, Raquel; Soler, Lourdes; Peña, Raquel; García, Natalia; Torrente, Carlos; Saco, Yolanda; Lampreave, Fermín; Bassols, Anna; Canalias, Francesca

2018-03-01

In dogs, as in humans, C-reactive protein (CRP) is a major acute phase protein that is rapidly and prominently increased after exposure to inflammatory stimuli. CRP measurements are used in the diagnosis and monitoring of infectious and inflammatory diseases. The study aim was to develop and validate a turbidimetric immunoassay for the quantification of canine CRP (cCRP), using canine-specific reagents and standards. A particle-enhanced turbidimetric immunoassay was developed. The assay was set up in a fully automated analyzer, and studies of imprecision, limits of linearity, limits of detection, prozone effects, and interferences were carried out. The new method was compared with 2 other commercially available automated immunoassays for cCRP: one turbidimetric immunoassay (Gentian CRP) and one point-of-care assay based on magnetic permeability (Life Assays CRP). The within-run and between-day imprecision were <1.7% and 4.2%, respectively. The assay quantified CRP proportionally in an analytic range up to 150 mg/L, with a prozone effect appearing at cCRP concentrations >320 mg/L. No interference from hemoglobin (20 g/L), triglycerides (10 g/L), or bilirubin (150 mg/L) was detected. Good agreement was observed between the results obtained with the new method and the Gentian cCRP turbidimetric immunoassay. The new turbidimetric immunoassay (Turbovet canine CRP, Acuvet Biotech) is a rapid, robust, precise, and accurate method for the quantification of cCRP. The method can be easily set up in automated analyzers, providing a suitable tool for routine clinical use. © 2018 American Society for Veterinary Clinical Pathology.

PEGylated human serum albumin (HSA) nanoparticles: preparation, characterization and quantification of the PEGylation extent

NASA Astrophysics Data System (ADS)

Fahrländer, E.; Schelhaas, S.; Jacobs, A. H.; Langer, K.

2015-04-01

Modification with poly(ethylene glycol) (PEG) is a widely used method for the prolongation of plasma half-life of colloidal carrier systems such as nanoparticles prepared from human serum albumin (HSA). However, the quantification of the PEGylation extent is still challenging. Moreover, the influence of different PEG derivatives, which are commonly used for nanoparticle conjugation, has not been investigated so far. The objective of the present study is to develop a method for the quantification of PEG and to monitor the influence of diverse PEG reagents on the amount of PEG linked to the surface of HSA nanoparticles. A size exclusion chromatography method with refractive index detection was established which enabled the quantification of unreacted PEG in the supernatant. The achieved results were confirmed using a fluorescent PEG derivative, which was detected by photometry and fluorimetry. Additionally, PEGylated HSA nanoparticles were enzymatically digested and the linked amount of fluorescently active PEG was directly determined. All the analytical methods confirmed that under optimized PEGylation conditions a PEGylation efficiency of up to 0.5 mg PEG per mg nanoparticle could be achieved. Model calculations made a ‘brush’ conformation of the PEG chains on the particle surface very likely. By incubating the nanoparticles with fetal bovine serum the reduced adsorption of serum proteins on PEGylated HSA nanoparticles compared to non-PEGylated HSA nanoparticles was demonstrated using sodium dodecylsulfate polyacrylamide gel electrophoresis. Finally, the positive effect of PEGylation on plasma half-life was demonstrated in an in vivo study in mice. Compared to unmodified nanoparticles the PEGylation led to a four times larger plasma half-life.

Quantification of protein interaction kinetics in a micro droplet

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, L. L.; College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044; Wang, S. P., E-mail: shaopeng.wang@asu.edu, E-mail: njtao@asu.edu

Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the averagemore » SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.« less

Quantification of protein interaction kinetics in a micro droplet

NASA Astrophysics Data System (ADS)

Yin, L. L.; Wang, S. P.; Shan, X. N.; Zhang, S. T.; Tao, N. J.

2015-11-01

Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

Absolute protein quantification of clinically relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based targeted proteomics.

PubMed

Gröer, C; Busch, D; Patrzyk, M; Beyer, K; Busemann, A; Heidecke, C D; Drozdzik, M; Siegmund, W; Oswald, S

2014-11-01

Cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGT) are major determinants in the pharmacokinetics of most drugs on the market. To investigate their impact on intestinal and hepatic drug metabolism, we developed and validated quantification methods for nine CYP (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and four UGT enzymes (UGT1A1, UGT1A3, UGT2B7 and UGT2B15) that have been shown to be of clinical relevance in human drug metabolism. Protein quantification was performed by targeted proteomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based determination of enzyme specific peptides after tryptic digestion using in each case stable isotope labelled peptides as internal standard. The chromatography of the respective peptides was performed with gradient elution using a reversed phase (C18) column (Ascentis(®) Express Peptide ES-C18, 100mm×2.1mm, 2.7μm) and 0.1% formic acid (FA) as well as acetonitrile with 0.1% FA as mobile phases at a flow rate of 300μl/min. The MS/MS detection of all peptides was done simultaneously with a scheduled multiple reaction monitoring (MRM) method in the positive mode by monitoring in each case three mass transitions per proteospecific peptide and the internal standard. The assays were validated according to current bioanalytical guidelines with respect to specificity, linearity (0.25-50nM), within-day and between-day accuracy and precision, digestion efficiency as well as stability. Finally, the developed method was successfully applied to determine the CYP and UGT protein amount in human liver and intestinal microsomes. The method was shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to quantify clinically relevant human CYP and UGT enzymes. Copyright © 2014 Elsevier B.V. All rights reserved.

Highly sensitive simultaneous quantification of estrogenic tamoxifen metabolites and steroid hormones by LC-MS/MS.

PubMed

Johänning, Janina; Heinkele, Georg; Precht, Jana C; Brauch, Hiltrud; Eichelbaum, Michel; Schwab, Matthias; Schroth, Werner; Mürdter, Thomas E

2015-09-01

Tamoxifen is a mainstay in the treatment of estrogen receptor-positive breast cancer and is metabolized to more than 30 different compounds. Little is known about in vivo concentrations of estrogenic metabolites E-metabolite E, Z-metabolite E, and bisphenol and their relevance for tamoxifen efficacy. Therefore, we developed a highly sensitive HPLC-ESI-MS/MS quantification method for tamoxifen metabolites bisphenol, E-metabolite E, and Z-metabolite E as well as for the sex steroid hormones estradiol, estrone, testosterone, androstenedione, and progesterone. Plasma samples were subjected to protein precipitation followed by solid phase extraction. Upon derivatization with 3-[(N-succinimide-1-yl)oxycarbonyl]-1-methylpyridinium iodide, all analytes were separated on a sub-2-μm column with a gradient of acetonitrile in water with 0.1 % of formic acid. Analytes were detected on a triple-quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Our method demonstrated high sensitivity, accuracy, and precision. The lower limits of quantification were 12, 8, and 25 pM for bisphenol, E-metabolite E, and Z-metabolite E, respectively, and 4 pM for estradiol and estrogen, 50 pM for testosterone and androstenedione, and 25 pM for progesterone. The method was applied to plasma samples of postmenopausal patients taken at baseline and under tamoxifen therapy. Graphical Abstract Sample preparation and derivatization for highly sensitive quantification of estrogenic tamoxifen metabolites and steroid hormones by HPLC-MS/MS.

Electrophoresis Gel Quantification with a Flatbed Scanner and Versatile Lighting from a Screen Scavenged from a Liquid Crystal Display (LCD) Monitor

ERIC Educational Resources Information Center

Yeung, Brendan; Ng, Tuck Wah; Tan, Han Yen; Liew, Oi Wah

2012-01-01

The use of different types of stains in the quantification of proteins separated on gels using electrophoresis offers the capability of deriving good outcomes in terms of linear dynamic range, sensitivity, and compatibility with specific proteins. An inexpensive, simple, and versatile lighting system based on liquid crystal display backlighting is…

A brief review of other notable protein blotting methods.

PubMed

Kurien, Biji T; Scofield, R Hal

2009-01-01

A plethora of methods have been used for transferring proteins from the gel to the membrane. These include centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low and high molecular weight proteins, blotting of Coomassie Brilliant Blue (CBB)-stained proteins from polyacrylamide gels to transparencies, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before MALDI tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

Fast and selective determination of total protein in milk powder via titration of moving reaction boundary electrophoresis.

PubMed

Guo, Cheng-ye; Wang, Hou-yu; Liu, Xiao-ping; Fan, Liu-yin; Zhang, Lei; Cao, Cheng-xi

2013-05-01

In this paper, moving reaction boundary titration (MRBT) was developed for rapid and accurate quantification of total protein in infant milk powder, from the concept of moving reaction boundary (MRB) electrophoresis. In the method, the MRB was formed by the hydroxide ions and the acidic residues of milk proteins immobilized via cross-linked polyacrylamide gel (PAG), an acid-base indicator was used to denote the boundary motion. As a proof of concept, we chose five brands of infant milk powders to study the feasibility of MRBT method. The calibration curve of MRB velocity versus logarithmic total protein content of infant milk powder sample was established based on the visual signal of MRB motion as a function of logarithmic milk protein content. Weak influence of nonprotein nitrogen (NPN) reagents (e.g., melamine and urea) on MRBT method was observed, due to the fact that MRB was formed with hydroxide ions and the acidic residues of captured milk proteins, rather than the alkaline residues or the NPN reagents added. The total protein contents in infant milk powder samples detected via the MRBT method were in good agreement with those achieved by the classic Kjeldahl method. In addition, the developed method had much faster measuring speed compared with the Kjeldahl method. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An efficient method for native protein purification in the selected range from prostate cancer tissue digests

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahmad, Rumana; Nicora, Carrie D.; Shukla, Anil K.

Prostate cancer (CP) cells differ from their normal counterpart in gene expression. Genes encoding secreted or extracellular proteins with increased expression in CP may serve as potential biomarkers. For their detection and quantification, assays based on monoclonal antibodies are best suited for development in a clinical setting. One approach to obtain antibodies is to use recombinant proteins as immunogen. However, the synthesis of recombinant protein for each identified candidate is time-consuming and expensive. It is also not practical to generate high quality antibodies to all identified candidates individually. Furthermore, non-native forms (e.g., recombinant) of proteins may not always lead tomore » useful antibodies. Our approach was to purify a subset of proteins from CP tissue specimens for use as immunogen.« less

Characterization of Medium Conditioned by Irradiated Cells Using Proteome-Wide, High-Throughput Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Springer, David L.; Ahram, Mamoun; Adkins, Joshua N.

Shedding, the release of cell surface proteins by regulated proteolysis, is a general cellular response to injury and is responsible for generating numerous bioactive molecules including growth factors and cytokines. The purpose of our work is to determine whether low doses of low-linear energy transfer (LET) radiation induce shedding of bioactive molecules. Using a mass spectrometry-based global proteomics method, we tested this hypothesis by analyzing for shed proteins in medium from irradiated human mammary epithelial cells (HMEC). Several hundred proteins were identified, including transforming growth factor beta (TGFB); however, no changes in protein abundances attributable to radiation exposure, based onmore » immunoblotting methods, were observed. These results demonstrate that our proteomic-based approach has the sensitivity to identify the kinds of proteins believed to be released after low-dose radiation exposure but that improvements in mass spectrometry-based protein quantification will be required to detect the small changes in abundance associated with this type of insult.« less

Antibodies as means for selective mass spectrometry.

PubMed

Boström, Tove; Takanen, Jenny Ottosson; Hober, Sophia

2016-05-15

For protein analysis of biological samples, two major strategies are used today; mass spectrometry (MS) and antibody-based methods. Each strategy offers advantages and drawbacks. However, combining the two using an immunoenrichment step with MS analysis brings together the benefits of each method resulting in increased sensitivity, faster analysis and possibility of higher degrees of multiplexing. The immunoenrichment can be performed either on protein or peptide level and quantification standards can be added in order to enable determination of the absolute protein concentration in the sample. The combination of immunoenrichment and MS holds great promise for the future in both proteomics and clinical diagnostics. This review describes different setups of immunoenrichment coupled to mass spectrometry and how these can be utilized in various applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantification of Free Phenytoin by Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS).

PubMed

Peat, Judy; Frazee, Clint; Garg, Uttam

2016-01-01

Phenytoin (diphenylhydantoin) is an anticonvulsant drug that has been used for decades for the treatment of many types of seizures. The drug is highly protein bound and measurement of free-active form of the drug is warranted particularly in patients with conditions that can affect drug protein binding. Here, we describe a LC/MS/MS method for the measurement of free phenytoin. Free drug is separated by ultrafiltration of serum or plasma. Ultrafiltrate is treated with acetonitrile containing internal standard phenytoin d-10 to precipitate proteins. The mixture is centrifuged and supernatant is injected onto LC-MS-MS, and analyzed using multiple reaction monitoring. This method is linear from 0.1 to 4.0 μg/mL and does not demonstrate any significant ion suppression or enhancement.

Relative quantification of N(epsilon)-(Carboxymethyl)lysine, imidazolone A, and the Amadori product in glycated lysozyme by MALDI-TOF mass spectrometry.

PubMed

Kislinger, Thomas; Humeny, Andreas; Peich, Carlo C; Zhang, Xiaohong; Niwa, Toshimitsu; Pischetsrieder, Monika; Becker, Cord-Michael

2003-01-01

The nonenzymatic glycation of proteins by reducing sugars, also known as the Maillard reaction, has received increasing recognition from nutritional science and medical research. In this study, we applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to perform relative and simultaneous quantification of the Amadori product, which is an early glycation product, and of N(epsilon)-(carboxymethyl)lysine and imidazolone A, two important advanced glycation end products. Therefore, native lysozyme was incubated with d-glucose for increasing periods of time (1, 4, 8, and 16 weeks) in phosphate-buffered saline pH 7.8 at 50 degrees C. After enzymatic digestion with endoproteinase Glu-C, the N-terminal peptide fragment (m/z 838; amino acid sequence KVFGRCE) and the C-terminal peptide fragment (m/z 1202; amino acid sequence VQAWIRGCRL) were used for relative quantification of the three Maillard products. Amadori product, N(epsilon)-(carboxymethyl)lysine, and imidazolone A were the main glycation products formed under these conditions. Their formation was dependent on glucose concentration and reaction time. The kinetics were similar to those obtained by competitive ELISA, an established method for quantification of N(epsilon)-(carboxymethyl)lysine and imidazolone A. Inhibition experiments showed that coincubation with N(alpha)-acetylargine suppressed formation of imidazolone A but not of the Amadori product or N(epsilon)-(carboxymethyl)lysine. The presence of N(alpha)-acetyllysine resulted in the inhibition of lysine modifications but in higher concentrations of imidazolone A. o-Phenylenediamine decreased the yield of the Amadori product and completely inhibited the formation of N(epsilon)-(carboxymethyl)lysine and imidazolone A. MALDI-TOF-MS proved to be a new analytical tool for the simultaneous, relative quantification of specific products of the Maillard reaction. For the first time, kinetic data of defined products on specific sites of glycated protein could be measured. This characterizes MALDI-TOF-MS as a valuable method for monitoring the Maillard reaction in the course of food processing.

Quantification of asymmetric microtubule nucleation at sub-cellular structures

PubMed Central

Zhu, Xiaodong; Kaverina, Irina

2012-01-01

Cell polarization is important for multiple physiological processes. In polarized cells, microtubules (MTs) are organized into a spatially polarized array. Generally, in non-differentiated cells, it is assumed that MTs are symmetrically nucleated exclusively from centrosome (microtubule organizing center, MTOC) and then reorganized into the asymmetric array. We have recently identified the Golgi complex as an additional MTOC that asymmetrically nucleates MTs toward one side of the cell. Methods used for alternative MTOC identification include microtubule re-growth after complete drug-induced depolymerization and tracking of growing microtubules using fluorescence labeled MT +TIP binding proteins in living cells. These approaches can be used for quantification of MT nucleation sites at diverse sub-cellular structures. PMID:21773933

Other notable protein blotting methods: a brief review.

PubMed

Kurien, Biji T; Scofield, R Hal

2015-01-01

Proteins have been transferred from the gel to the membrane by a variety of methods. These include vacuum blotting, centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low- and high-molecular-weight proteins, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before matrix-assisted laser desorption/ionization tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

Development and characterization of novel 8-plex DiLeu isobaric labels for quantitative proteomics and peptidomics

PubMed Central

Frost, Dustin C.; Greer, Tyler; Xiang, Feng; Liang, Zhidan; Li, Lingjun

2015-01-01

Rationale Relative quantification of proteins via their enzymatically digested peptide products determines disease biomarker candidate lists in discovery studies. Isobaric label-based strategies using TMT and iTRAQ allow for up to 10 samples to be multiplexed in one experiment, but their expense limits their use. The demand for cost-effective tagging reagents capable of multiplexing many samples led us to develop an 8-plex version of our isobaric labeling reagent, DiLeu. Methods The original 4-plex DiLeu reagent was extended to an 8-plex set by coupling isotopic variants of dimethylated leucine to an alanine balance group designed to offset the increasing mass of the label’s reporter group. Tryptic peptides from a single protein digest, a protein mixture digest, and Saccharomyces cerevisiae lysate digest were labeled with 8-plex DiLeu and analyzed via nanoLC-MS2 on a Q-Exactive Orbitrap mass spectrometer. Characteristics of 8-plex DiLeu-labeled peptides, including quantitative accuracy and fragmentation, were examined. Results An 8-plex set of DiLeu reagents with 1 Da-spaced reporters was synthesized at a yield of 36%. The average cost to label eight 100 μg peptide samples was calculated to be approximately $15. Normalized collision energy tests on the Q-Exactive revealed that a higher-energy collisional dissociation value of 27 generated the optimum number of high-quality spectral matches. Relative quantification of DiLeu-labeled peptides yielded normalized median ratios accurate to within 12% of their expected values. Conclusions Cost-effective 8-plex DiLeu reagents can be synthesized and applied to relative peptide and protein quantification. These labels increase the multiplexing capacity of our previous 4-plex implementation without requiring high-resolution instrumentation to resolve reporter ion signals. PMID:25981542

A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.

PubMed

Chen, WeiYue; Avezov, Edward; Schlachter, Simon C; Gielen, Fabrice; Laine, Romain F; Harding, Heather P; Hollfelder, Florian; Ron, David; Kaminski, Clemens F

2015-03-10

FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Validated method for quantification of genetically modified organisms in samples of maize flour.

PubMed

Kunert, Renate; Gach, Johannes S; Vorauer-Uhl, Karola; Engel, Edwin; Katinger, Hermann

2006-02-08

Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material is the prerequisite for detection of 1% or 0.5% genetically modified ingredients in food products or raw materials thereof. Compared to ELISA detection of expressed proteins, real-time PCR (RT-PCR) amplification has easier sample preparation and detection limits are lower. Of the different methods of DNA preparation CTAB method with high flexibility in starting material and generation of sufficient DNA with relevant quality was chosen. Previous RT-PCR data generated with the SYBR green detection method showed that the method is highly sensitive to sample matrices and genomic DNA content influencing the interpretation of results. Therefore, this paper describes a real-time DNA quantification based on the TaqMan probe method, indicating high accuracy and sensitivity with detection limits of lower than 18 copies per sample applicable and comparable to highly purified plasmid standards as well as complex matrices of genomic DNA samples. The results were evaluated with ValiData for homology of variance, linearity, accuracy of the standard curve, and standard deviation.

Identification of Phosphorylated Proteins on a Global Scale.

PubMed

Iliuk, Anton

2018-05-31

Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) has enabled researchers to analyze complex biological samples with unprecedented depth. It facilitates the identification and quantification of modifications within thousands of proteins in a single large-scale proteomic experiment. Analysis of phosphorylation, one of the most common and important post-translational modifications, has particularly benefited from such progress in the field. Here, detailed protocols are provided for a few well-regarded, common sample preparation methods for an effective phosphoproteomic experiment. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Gel-based methods in redox proteomics.

PubMed

Charles, Rebecca; Jayawardhana, Tamani; Eaton, Philip

2014-02-01

The key to understanding the full significance of oxidants in health and disease is the development of tools and methods that allow the study of proteins that sense and transduce changes in cellular redox. Oxidant-reactive deprotonated thiols commonly operate as redox sensors in proteins and a variety of methods have been developed that allow us to monitor their oxidative modification. This outline review specifically focuses on gel-based methods used to detect, quantify and identify protein thiol oxidative modifications. The techniques we discuss fall into one of two broad categories. Firstly, methods that allow oxidation of thiols in specific proteins or the global cellular pool to be monitored are discussed. These typically utilise thiol-labelling reagents that add a reporter moiety (e.g. affinity tag, fluorophore, chromophore), in which loss of labelling signifies oxidation. Secondly, we outline methods that allow specific thiol oxidation states of proteins (e.g. S-sulfenylation, S-nitrosylation, S-thionylation and interprotein disulfide bond formation) to be investigated. A variety of different gel-based methods for identifying thiol proteins that are sensitive to oxidative modifications have been developed. These methods can aid the detection and quantification of thiol redox state, as well as identifying the sensor protein. By understanding how cellular redox is sensed and transduced to a functional effect by protein thiol redox sensors, this will help us better appreciate the role of oxidants in health and disease. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Copyright © 2013 Elsevier B.V. All rights reserved.

Comprehensive Analysis of Protein Modifications by Top-down Mass Spectrometry

PubMed Central

Zhang, Han; Ge, Ying

2012-01-01

Mass spectrometry (MS)-based proteomics is playing an increasingly important role in cardiovascular research. Proteomics includes not only identification and quantification of proteins, but also the characterization of protein modifications such as post-translational modifications and sequence variants. The conventional bottom-up approach, involving proteolytic digestion of proteins into small peptides prior to MS analysis, is routinely used for protein identification and quantification with high throughput and automation. Nevertheless, it has limitations in the analysis of protein modifications mainly due to the partial sequence coverage and loss of connections among modifications on disparate portions of a protein. An alternative approach, top-down MS, has emerged as a powerful tool for the analysis of protein modifications. The top-down approach analyzes whole proteins directly, providing a “bird’s eye” view of all existing modifications. Subsequently, each modified protein form can be isolated and fragmented in the mass spectrometer to locate the modification site. The incorporation of the non-ergodic dissociation methods such as electron capture dissociation (ECD) greatly enhances the top-down capabilities. ECD is especially useful for mapping labile post-translational modifications which are well-preserved during the ECD fragmentation process. Top-down MS with ECD has been successfully applied to cardiovascular research with the unique advantages in unraveling the molecular complexity, quantifying modified protein forms, complete mapping of modifications with full sequence coverage, discovering unexpected modifications, and identifying and quantifying positional isomers and determining the order of multiple modifications. Nevertheless, top-down MS still needs to overcome some technical challenges to realize its full potential. Herein, we reviewed the advantages and challenges of top-down methodology with a focus on its application in cardiovascular research. PMID:22187450

Biomarkers in Cerebrospinal Fluid: Analysis of Cell-Free Circulating Mitochondrial DNA by Digital PCR.

PubMed

Podlesniy, Petar; Trullas, Ramon

2018-01-01

Cerebrospinal fluid (CSF) contains molecules directly linked with brain function because it permeates brain tissue. The analysis of protein biomarkers in CSF is currently recommended for the diagnosis of neurodegenerative disorders, but the clinical sensitivity and specificity are still being investigated. A major drawback is that most of the currently used biomarkers of neurodegenerative diseases are proteins that are found at very low concentrations in CSF and need to be measured by immunoassays that provide relative values, which sometimes are difficult to reproduce between laboratories. In contrast, the recent availability of digital PCR platforms allows the absolute quantification of nucleic acids at single-molecule resolution, but their presence in CSF has not been characterized. CSF contains cell-free mitochondrial DNA (mtDNA) and changes in the concentration of this nucleic acid are linked to neurodegeneration. Here we describe a method to measure the concentration of cell-free circulating mtDNA directly in unpurified CSF using droplet digital PCR with either hydrolysis probes or fluorescent DNA-binding dye methods. This protocol allows the detection and absolute quantification of mtDNA content in the CSF with high analytical sensitivity, specificity, and accuracy.

Imaging and quantification of trans-membrane protein diffusion in living bacteria.

PubMed

Oswald, Felix; L M Bank, Ernst; Bollen, Yves J M; Peterman, Erwin J G

2014-07-07

The cytoplasmic membrane forms the barrier between any cell's interior and the outside world. It contains many proteins that enable essential processes such as the transmission of signals, the uptake of nutrients, and cell division. In the case of prokaryotes, which do not contain intracellular membranes, the cytoplasmic membrane also contains proteins for respiration and protein folding. Mutual interactions and specific localization of these proteins depend on two-dimensional diffusion driven by thermal fluctuations. The experimental investigation of membrane-protein diffusion in bacteria is challenging due to their small size, only a few times larger than the resolution of an optical microscope. Here, we review fluorescence microscopy-based methods to study diffusion of membrane proteins in living bacteria. The main focus is on data-analysis tools to extract diffusion coefficients from single-particle tracking data obtained by single-molecule fluorescence microscopy. We introduce a novel approach, IPODD (inverse projection of displacement distributions), to obtain diffusion coefficients from the usually obtained 2-D projected diffusion trajectories of the highly 3-D curved bacterial membrane. This method provides, in contrast to traditional mean-squared-displacement methods, correct diffusion coefficients and allows unravelling of heterogeneously diffusing populations.

High-sensitivity MALDI-TOF MS quantification of anthrax lethal toxin for diagnostics and evaluation of medical countermeasures.

PubMed

Boyer, Anne E; Gallegos-Candela, Maribel; Quinn, Conrad P; Woolfitt, Adrian R; Brumlow, Judith O; Isbell, Katherine; Hoffmaster, Alex R; Lins, Renato C; Barr, John R

2015-04-01

Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (<4 h total time to detection), sensitive at 0.033 ng/mL LTx in plasma for the fast analysis (0.0075 ng/mL LTx in plasma for an 18 h reaction), precise (6.3-9.9% coefficient of variation), and accurate (0.1-12.7%error; n ≥ 25). Diagnostic sensitivity was 100% (n = 27 animal/clinical cases). Diagnostic specificity was 100% (n = 141). LTx was detected post-antibiotic treatment in 6/6 treated rhesus macaques and 3/3 clinical cases of inhalation anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics.

Absolute quantification of Dehalococcoides proteins: enzyme bioindicators of chlorinated ethene dehalorespiration.

PubMed

Werner, Jeffrey J; Ptak, A Celeste; Rahm, Brian G; Zhang, Sheng; Richardson, Ruth E

2009-10-01

The quantification of trace proteins in complex environmental samples and mixed microbial communities would be a valuable monitoring tool in countless applications, including the bioremediation of groundwater contaminated with chlorinated solvents. Measuring the concentrations of specific proteins provides unique information about the activity and physiological state of organisms in a sample. We developed sensitive (< 5 fmol), selective bioindicator assays for the absolute quantification of select proteins used by Dehalococcoides spp. when reducing carbon atoms in the common pollutants trichloroethene (TCE) and tetrachloroethene (PCE). From complex whole-sample digests of two different dechlorinating mixed communities, we monitored the chromatographic peaks of selected tryptic peptides chosen to represent 19 specific Dehalococcoides proteins. This was accomplished using multiple-reaction monitoring (MRM) assays using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS), which provided the selectivity, sensitivity and reproducibility required to quantify Dehalococcoides proteins in complex samples. We observed reproducible peak areas (average CV = 0.14 over 4 days, n = 3) and linear responses in standard curves (n = 5, R(2) > 0.98) using synthetic peptide standards spiked into a background matrix of sediment peptides. We detected and quantified TCE reductive dehalogenase (TceA) at 7.6 +/- 1.7 x 10(3) proteins cell(-1) in the KB1 bioaugmentation culture, previously thought to be lacking TceA. Fragmentation data from MS/MS shotgun proteomics experiments were helpful in developing the MRM targets. Similar shotgun proteomics data are emerging in labs around the world for many environmentally relevant microbial proteins, and these data are a valuable resource for the future development of MRM assays. We expect targeted peptide quantification in environmental samples to be a useful tool in environmental monitoring.

Application of Quality by Design Approach to Bioanalysis: Development of a Method for Elvitegravir Quantification in Human Plasma.

PubMed

Baldelli, Sara; Marrubini, Giorgio; Cattaneo, Dario; Clementi, Emilio; Cerea, Matteo

2017-10-01

The application of Quality by Design (QbD) principles in clinical laboratories can help to develop an analytical method through a systematic approach, providing a significant advance over the traditional heuristic and empirical methodology. In this work, we applied for the first time the QbD concept in the development of a method for drug quantification in human plasma using elvitegravir as the test molecule. The goal of the study was to develop a fast and inexpensive quantification method, with precision and accuracy as requested by the European Medicines Agency guidelines on bioanalytical method validation. The method was divided into operative units, and for each unit critical variables affecting the results were identified. A risk analysis was performed to select critical process parameters that should be introduced in the design of experiments (DoEs). Different DoEs were used depending on the phase of advancement of the study. Protein precipitation and high-performance liquid chromatography-tandem mass spectrometry were selected as the techniques to be investigated. For every operative unit (sample preparation, chromatographic conditions, and detector settings), a model based on factors affecting the responses was developed and optimized. The obtained method was validated and clinically applied with success. To the best of our knowledge, this is the first investigation thoroughly addressing the application of QbD to the analysis of a drug in a biological matrix applied in a clinical laboratory. The extensive optimization process generated a robust method compliant with its intended use. The performance of the method is continuously monitored using control charts.

Novel method to detect microRNAs using chip-based QuantStudio 3D digital PCR.

PubMed

Conte, Davide; Verri, Carla; Borzi, Cristina; Suatoni, Paola; Pastorino, Ugo; Sozzi, Gabriella; Fortunato, Orazio

2015-10-23

Research efforts for the management of cancer, in particular for lung cancer, are directed to identify new strategies for its early detection. MicroRNAs (miRNAs) are a new promising class of circulating biomarkers for cancer detection, but lack of consensus on data normalization methods has affected the diagnostic potential of circulating miRNAs. There is a growing interest in techniques that allow an absolute quantification of miRNAs which could be useful for early diagnosis. Recently, digital PCR, mainly based on droplets generation, emerged as an affordable technology for precise and absolute quantification of nucleic acids. In this work, we described a new interesting approach for profiling circulating miRNAs in plasma samples using a chip-based platform, the QuantStudio 3D digital PCR. The proposed method was validated using synthethic oligonucleotide at serial dilutions in plasma samples of lung cancer patients and in lung tissues and cell lines. Given its reproducibility and reliability, our approach could be potentially applied for the identification and quantification of miRNAs in other biological samples such as circulating exosomes or protein complexes. As chip-digital PCR becomes more established, it would be a robust tool for quantitative assessment of miRNA copy number for diagnosis of lung cancer and other diseases.

Quantification of sulphur amino acids by ultra-high performance liquid chromatography in aquatic invertebrates.

PubMed

Thera, Jennifer C; Kidd, Karen A; Dodge-Lynch, M Elaine; Bertolo, Robert F

2017-12-15

We examined the performance of an ultra-high performance liquid chromatography method to quantify protein-bound sulphur amino acids in zooplankton. Both cysteic acid and methionine sulfone were linear from 5 to 250 pmol (r 2  = 0.99), with a method detection limit of 13 pmol and 9 pmol, respectively. Although there was no matrix effect on linearity, adjacent peaks and co-eluting noise from the invertebrate proteins increased the detection limits when compared to common standards. Overall, performance characteristics were reproducible and accurate, and provide a means for quantifying sulphur amino acids in aquatic invertebrates, an understudied group. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of processing on the detectability of peanut protein by ELISA.

PubMed

Iqbal, Amjad; Ateeq, Nadia

2013-12-01

Chicken IgY was used for the detection and quantification of peanut proteins by indirect competitive ELISA. The method was optimized by using a checker board approach to determine the optimal concentration of coating antigen, primary antibody and secondary antibody. Peanut protein could be detected in foods down to levels of 10 ppm. The effect of physical (heat treatment at 80 °C and 100 °C) and chemical (acid, alkali and reducing sugar) treatments on the IgY binding of peanut proteins was investigated. The optimized assay was relatively sensitive for the roasted peanut proteins. However, the binding ability of chicken IgYs to peanut proteins was found to be significantly altered by denaturation and hydrolysis of proteins. It was also observed that the effect of Millard chemistry on the detectability of peanut protein was less pronounced at high temperatures than at low temperatures. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mass spectrometric approaches for the identification and quantification of reactive carbonyl species protein adducts.

PubMed

Colzani, Mara; Aldini, Giancarlo; Carini, Marina

2013-10-30

Our current knowledge of the occurrence of proteins covalently modified by reactive carbonyl species (RCS) generated by lipid peroxidation indicates their involvement as pathogenic factors associated with several chronic degenerative diseases. Proteomics and mass spectrometry (MS) in the last decade have played a fundamental role in this context, allowing the demonstration of the formation of RCS-protein adducts in vitro and in vivo under different experimental conditions. In conjunction with functional and computational studies, MS has been widely applied in vitro to study the stoichiometry of the protein-RCS adduct formation, and, by identifying the site(s) of modification, to elucidate the molecular mechanisms of protein carbonylation and the physiologic impact of such modification on protein function. This review will provide an update of the MS methods commonly used in detecting and characterizing protein modification by RCS generated by lipid peroxidation, among which 4-hydroxy-trans-2-nonenal and acrolein represent the most studied and cytotoxic compounds. Research in this field, employing state-of-the-art MS, is rapidly and continuously evolving, owing also to the development of suitable derivatization and enrichment procedures enabling the improve MS detectability of RCS-protein adducts in complex biological matrices. By considering the emerging role of RCS in several human diseases, unequivocal analytical approaches by MS are needed to provide levels of intermediate diagnostic biomarkers for human diseases. This review focuses also on the different MS-based approaches so far developed for RCS-protein adduct quantification. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

Global Proteomic Analysis of Human Liver Microsomes: Rapid Characterization and Quantification of Hepatic Drug-Metabolizing Enzymes.

PubMed

Achour, Brahim; Al Feteisi, Hajar; Lanucara, Francesco; Rostami-Hodjegan, Amin; Barber, Jill

2017-06-01

Many genetic and environmental factors lead to interindividual variations in the metabolism and transport of drugs, profoundly affecting efficacy and toxicity. Precision dosing, that is, targeting drug dose to a well characterized subpopulation, is dependent on quantitative models of the profiles of drug-metabolizing enzymes (DMEs) and transporters within that subpopulation, informed by quantitative proteomics. We report the first use of ion mobility-mass spectrometry for this purpose, allowing rapid, robust, label-free quantification of human liver microsomal (HLM) proteins from distinct individuals. Approximately 1000 proteins were identified and quantified in four samples, including an average of 70 DMEs. Technical and biological variabilities were distinguishable, with technical variability accounting for about 10% of total variability. The biological variation between patients was clearly identified, with samples showing a range of expression profiles for cytochrome P450 and uridine 5'-diphosphoglucuronosyltransferase enzymes. Our results showed excellent agreement with previous data from targeted methods. The label-free method, however, allowed a fuller characterization of the in vitro system, showing, for the first time, that HLMs are significantly heterogeneous. Further, the traditional units of measurement of DMEs (pmol mg -1 HLM protein) are shown to introduce error arising from variability in unrelated, highly abundant proteins. Simulations of this variability suggest that up to 1.7-fold variation in apparent CYP3A4 abundance is artifactual, as are background positive correlations of up to 0.2 (Spearman correlation coefficient) between the abundances of DMEs. We suggest that protein concentrations used in pharmacokinetic predictions and scaling to in vivo clinical situations (physiologically based pharmacokinetics and in vitro-in vivo extrapolation) should be referenced instead to tissue mass. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences*

PubMed Central

Hessling, Bernd; Büttner, Knut; Hecker, Michael; Becher, Dörte

2013-01-01

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LC–electrospray ionization workflows. Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis. Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF- and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments. PMID:23788530

Global relative quantification with liquid chromatography-matrix-assisted laser desorption ionization time-of-flight (LC-MALDI-TOF)--cross-validation with LTQ-Orbitrap proves reliability and reveals complementary ionization preferences.

PubMed

Hessling, Bernd; Büttner, Knut; Hecker, Michael; Becher, Dörte

2013-10-01

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LC-electrospray ionization workflows. Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis. Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF- and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments.

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

PubMed

de la Calle, Maria B; Devesa, Vicenta; Fiamegos, Yiannis; Vélez, Dinoraz

2017-09-01

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to inorganic arsenic through diet, information about distribution of arsenic species in various food types must be generated. A method, previously validated in a collaborative trial, has been applied to determine inorganic arsenic in a wide variety of food matrices, covering grains, mushrooms and food of marine origin (31 samples in total). The method is based on detection by flow injection-hydride generation-atomic absorption spectrometry of the iAs selectively extracted into chloroform after digestion of the proteins with concentrated HCl. The method is characterized by a limit of quantification of 10 µg/kg dry weight, which allowed quantification of inorganic arsenic in a large amount of food matrices. Information is provided about performance scores given to results obtained with this method and which were reported by different laboratories in several proficiency tests. The percentage of satisfactory results obtained with the discussed method is higher than that of the results obtained with other analytical approaches.

Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells

NASA Astrophysics Data System (ADS)

Okumus, Burak; Landgraf, Dirk; Lai, Ghee Chuan; Bakhsi, Somenath; Arias-Castro, Juan Carlos; Yildiz, Sadik; Huh, Dann; Fernandez-Lopez, Raul; Peterson, Celeste N.; Toprak, Erdal; El Karoui, Meriem; Paulsson, Johan

2016-05-01

Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3-4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting.

Food for thought: Selecting the right enzyme for the digestion of gluten.

PubMed

Colgrave, Michelle L; Byrne, Keren; Howitt, Crispin A

2017-11-01

Gluten describes a complex mixture of proteins found in wheat, rye, barley and oats that pose a health risk to people affected by conditions such as coeliac disease and non-coeliac gluten sensitivity. Complete digestion of gluten proteins is of critical importance during quantitative analysis. To this end, chymotrypsin was investigated for its ability to efficiently and reproducibly digest specific classes of gluten in barley. Using proteomics a chymotryptic peptide marker panel was elucidated and subjected to relative quantification using LC-MRM-MS. Thorough investigation of peptide markers revealed robust and reproducible quantification with CVs <15% was possible, however a greater proportion of non-specific cleavage variants were observed relative to trypsin. The selected peptide markers were assessed to ensure their efficient liberation from their parent proteins. While trypsin remains the preferred enzyme for quantification of the avenin-like A proteins, the B-, D- and γ-hordeins, chymotrypsin was the enzyme of choice for the C-hordeins. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

PubMed

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

2014-11-27

In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

Multiplex single-molecule interaction profiling of DNA barcoded proteins

PubMed Central

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

2014-01-01

In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

A reliable and rapid tool for plasma quantification of 18 psychotropic drugs by ESI tandem mass spectrometry.

PubMed

Vecchione, Gennaro; Casetta, Bruno; Chiapparino, Antonella; Bertolino, Alessandro; Tomaiuolo, Michela; Cappucci, Filomena; Gatta, Raffaella; Margaglione, Maurizio; Grandone, Elvira

2012-01-01

A simple liquid chromatographic tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous analysis of 17 basic and one acid psychotropic drugs in human plasma. The method relies on a protein precipitation step for sample preparation and offers high sensitivity, wide linearity without interferences from endogenous matrix components. Chromatography was run on a reversed-phase column with an acetonitrile-H₂O mixture. The quantification of target compounds was performed in multiple reaction monitoring (MRM) and by switching the ionization polarity within the analytical run. A further sensitivity increase was obtained by implementing the functionality "scheduled multiple reaction monitoring" (sMRM) offered by the recent version of the software package managing the instrument. The overall injection interval was less than 5.5 min. Regression coefficients of the calibration curves and limits of quantification (LOQ) showed a good coverage of over-therapeutic, therapeutic and sub-therapeutic ranges. Recovery rates, measured as percentage of recovery of spiked plasma samples, were ≥ 94%. Precision and accuracy data have been satisfactory for a therapeutic drug monitoring (TDM) service as for managing plasma samples from patients receiving psycho-pharmacological treatment. Copyright © 2012 Elsevier B.V. All rights reserved.

High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy.

PubMed

Bravo-San Pedro, J M; Pietrocola, F; Sica, V; Izzo, V; Sauvat, A; Kepp, O; Maiuri, M C; Kroemer, G; Galluzzi, L

2017-01-01

Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy. In physiological conditions, cells transfected temporarily or stably with a GFP-LC3-encoding construct exhibit a diffuse green fluorescence over the cytoplasm and nucleus. Conversely, in response to macroautophagy-promoting stimuli, the GFP-LC3 signal becomes punctate and often (but not always) predominantly cytoplasmic. The accumulation of GFP-LC3 in cytoplasmic dots, however, also ensues the blockage of any of the steps that ensure the degradation of mature autophagosomes, calling for the implementation of strategies that accurately discriminate between an increase in autophagic flux and an arrest in autophagic degradation. Various cell lines have been engineered to stably express GFP-LC3, which-combined with the appropriate controls of flux, high-throughput imaging stations, and automated image analysis-offer a relatively straightforward tool to screen large chemical or biological libraries for inducers or inhibitors of autophagy. Here, we describe a simple and robust method for the high-throughput quantification of GFP-LC3 + dots by automated fluorescence microscopy. © 2017 Elsevier Inc. All rights reserved.

Image Processing for Bioluminescence Resonance Energy Transfer Measurement-BRET-Analyzer.

PubMed

Chastagnier, Yan; Moutin, Enora; Hemonnot, Anne-Laure; Perroy, Julie

2017-01-01

A growing number of tools now allow live recordings of various signaling pathways and protein-protein interaction dynamics in time and space by ratiometric measurements, such as Bioluminescence Resonance Energy Transfer (BRET) Imaging. Accurate and reproducible analysis of ratiometric measurements has thus become mandatory to interpret quantitative imaging. In order to fulfill this necessity, we have developed an open source toolset for Fiji- BRET-Analyzer -allowing a systematic analysis, from image processing to ratio quantification. We share this open source solution and a step-by-step tutorial at https://github.com/ychastagnier/BRET-Analyzer. This toolset proposes (1) image background subtraction, (2) image alignment over time, (3) a composite thresholding method of the image used as the denominator of the ratio to refine the precise limits of the sample, (4) pixel by pixel division of the images and efficient distribution of the ratio intensity on a pseudocolor scale, and (5) quantification of the ratio mean intensity and standard variation among pixels in chosen areas. In addition to systematize the analysis process, we show that the BRET-Analyzer allows proper reconstitution and quantification of the ratiometric image in time and space, even from heterogeneous subcellular volumes. Indeed, analyzing twice the same images, we demonstrate that compared to standard analysis BRET-Analyzer precisely define the luminescent specimen limits, enlightening proficient strengths from small and big ensembles over time. For example, we followed and quantified, in live, scaffold proteins interaction dynamics in neuronal sub-cellular compartments including dendritic spines, for half an hour. In conclusion, BRET-Analyzer provides a complete, versatile and efficient toolset for automated reproducible and meaningful image ratio analysis.

Defining the consequences of genetic variation on a proteome–wide scale

PubMed Central

Chick, Joel M.; Munger, Steven C.; Simecek, Petr; Huttlin, Edward L.; Choi, Kwangbom; Gatti, Daniel M.; Raghupathy, Narayanan; Svenson, Karen L.; Churchill, Gary A.; Gygi, Steven P.

2016-01-01

Genetic variation modulates protein expression through both transcriptional and post-transcriptional mechanisms. To characterize the consequences of natural genetic diversity on the proteome, here we combine a multiplexed, mass spectrometry-based method for protein quantification with an emerging outbred mouse model containing extensive genetic variation from eight inbred founder strains. By measuring genome-wide transcript and protein expression in livers from 192 Diversity outbred mice, we identify 2,866 protein quantitative trait loci (pQTL) with twice as many local as distant genetic variants. These data support distinct transcriptional and post-transcriptional models underlying the observed pQTL effects. Using a sensitive approach to mediation analysis, we often identified a second protein or transcript as the causal mediator of distant pQTL. Our analysis reveals an extensive network of direct protein–protein interactions. Finally, we show that local genotype can provide accurate predictions of protein abundance in an independent cohort of collaborative cross mice. PMID:27309819

Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics

PubMed Central

Malmström, Erik; Kilsgård, Ola; Hauri, Simon; Smeds, Emanuel; Herwald, Heiko; Malmström, Lars; Malmström, Johan

2016-01-01

The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics. PMID:26732734

Bioluminescent Antibodies for Point‐of‐Care Diagnostics

PubMed Central

Xue, Lin; Yu, Qiuliyang; Griss, Rudolf; Schena, Alberto

2017-01-01

Abstract We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no‐wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP‐tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔR max>500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera. PMID:28510347

GoIFISH: a system for the quantification of single cell heterogeneity from IFISH images.

PubMed

Trinh, Anne; Rye, Inga H; Almendro, Vanessa; Helland, Aslaug; Russnes, Hege G; Markowetz, Florian

2014-08-26

Molecular analysis has revealed extensive intra-tumor heterogeneity in human cancer samples, but cannot identify cell-to-cell variations within the tissue microenvironment. In contrast, in situ analysis can identify genetic aberrations in phenotypically defined cell subpopulations while preserving tissue-context specificity. GoIFISHGoIFISH is a widely applicable, user-friendly system tailored for the objective and semi-automated visualization, detection and quantification of genomic alterations and protein expression obtained from fluorescence in situ analysis. In a sample set of HER2-positive breast cancers GoIFISHGoIFISH is highly robust in visual analysis and its accuracy compares favorably to other leading image analysis methods. GoIFISHGoIFISH is freely available at www.sourceforge.net/projects/goifish/.

Protein, enzyme and carbohydrate quantification using smartphone through colorimetric digitization technique.

PubMed

Dutta, Sibasish; Saikia, Gunjan Prasad; Sarma, Dhruva Jyoti; Gupta, Kuldeep; Das, Priyanka; Nath, Pabitra

2017-05-01

In this paper the utilization of smartphone as a detection platform for colorimetric quantification of biological macromolecules has been demonstrated. Using V-channel of HSV color space, the quantification of BSA protein, catalase enzyme and carbohydrate (using D-glucose) have been successfully investigated. A custom designed android application has been developed for estimating the total concentration of biological macromolecules. The results have been compared with that of a standard spectrophotometer which is generally used for colorimetric quantification in laboratory settings by measuring its absorbance at a specific wavelength. The results obtained with the designed sensor is found to be similar when compared with the spectrophotometer data. The designed sensor is low cost, robust and we envision that it could promote diverse fields of bio-analytical investigations. Schematic illustration of the smartphone sensing mechanism for colorimetric analysis of biomolecular samples. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A specific endogenous reference for genetically modified common bean (Phaseolus vulgaris L.) DNA quantification by real-time PCR targeting lectin gene.

PubMed

Venturelli, Gustavo L; Brod, Fábio C A; Rossi, Gabriela B; Zimmermann, Naíra F; Oliveira, Jaison P; Faria, Josias C; Arisi, Ana C M

2014-11-01

The Embrapa 5.1 genetically modified (GM) common bean was approved for commercialization in Brazil. Methods for the quantification of this new genetically modified organism (GMO) are necessary. The development of a suitable endogenous reference is essential for GMO quantification by real-time PCR. Based on this, a new taxon-specific endogenous reference quantification assay was developed for Phaseolus vulgaris L. Three genes encoding common bean proteins (phaseolin, arcelin, and lectin) were selected as candidates for endogenous reference. Primers targeting these candidate genes were designed and the detection was evaluated using the SYBR Green chemistry. The assay targeting lectin gene showed higher specificity than the remaining assays, and a hydrolysis probe was then designed. This assay showed high specificity for 50 common bean samples from two gene pools, Andean and Mesoamerican. For GM common bean varieties, the results were similar to those obtained for non-GM isogenic varieties with PCR efficiency values ranging from 92 to 101 %. Moreover, this assay presented a limit of detection of ten haploid genome copies. The primers and probe developed in this work are suitable to detect and quantify either GM or non-GM common bean.

Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

PubMed Central

Boogaard, Ivette; Smith, Melanie; Pulli, Kristiina; Szynol, Agnieszka; Albertus, Faywell; Lamers, Marieke B. A. C.; Dijkstra, Sipke; Kordt, Daniel; Reindl, Wolfgang; Herrmann, Frank; McAllister, George; Fischer, David F.; Munoz-Sanjuan, Ignacio

2014-01-01

The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples. PMID:24816435

EvaGreen real-time PCR protocol for specific 'Candidatus Phytoplasma mali' detection and quantification in insects.

PubMed

Monti, Monia; Martini, Marta; Tedeschi, Rosemarie

2013-01-01

In this paper the validation and implementation of a Real-time PCR protocol based on ribosomal protein genes has been carried out for sensitive and specific quantification of 'Candidatus (Ca.) Phytoplasma mali' (apple proliferation phytoplasma, APP) in insects. The method combines the use of EvaGreen(®) dye as chemistry detection system and the specific primer pair rpAP15f-mod/rpAP15r3, which amplifies a fragment of 238 bp of the ribosomal protein rplV (rpl22) gene of APP. Primers specificity was demonstrated by running in the same Real-time PCR 'Ca. Phytoplasma mali' samples with phytoplasmas belonging to the same group (16SrX) as 'Ca. Phytoplasma pyri' and 'Ca. Phytoplasma prunorum', and also phytoplasmas from different groups, as 'Ca. Phytoplasma phoenicium' (16SrIX) and Flavescence dorée phytoplasma (16SrV). 'Ca. Phytoplasma mali' titre in insects was quantified using a specific approach, which relates the concentration of the phytoplasma to insect 18S rDNA. Absolute quantification of APP and insect 18S rDNA were calculated using standard curves prepared from serial dilutions of plasmids containing rplV-rpsC and a portion of 18S rDNA genes, respectively. APP titre in insects was expressed as genome units (GU) of phytoplasma per picogram (pg) of individual insect 18S rDNA. 'Ca. Phytoplasma mali' concentration in examined samples (Cacopsylla melanoneura overwintered adults) ranged from 5.94 × 10(2) to 2.51 × 10(4) GU/pg of insect 18S rDNA. Repeatability and reproducibility of the method were also evaluated by calculation of the coefficient of variation (CV%) of GU of phytoplasma and pg of 18S rDNA fragment for both assays. CV less than 14% and 9% (for reproducibility test) and less than 10 and 11% (for repeatability test) were obtained for phytoplasma and insect qPCR assays, respectively. Sensitivity of the method was also evaluated, in comparison with conventional 16S rDNA-based nested-PCR procedure. The method described has been demonstrated reliable, sensitive and specific for the quantification of 'Ca. Phytoplasma mali' in insects. The possibility to study the trend of phytoplasma titre in the vectors will allow a deepen investigation on the epidemiology of the disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

Compositional Analysis of Asymmetric and Symmetric Dimethylated H3R2 Using Liquid Chromatography-Tandem Mass Spectrometry-Based Targeted Proteomics.

PubMed

Xu, Qingqing; Xu, Feifei; Liu, Liang; Chen, Yun

2016-09-06

Protein arginine methylation is one of the common post-translational modifications in cellular processes. To date, two isomeric forms of dimethylated arginine have been identified: asymmetric N(G),N(G)-dimethylarginine (aDMA), and symmetric N(G),N'(G)-dimethylarginine (sDMA). Evidence indicated that these isomers can coexist and have different or even opposite functions, with aDMA and sDMA forms of arginine 2 on histone H3 (i.e., H3R2me2a and H3R2me2s) being an example. Thus, specific detection and quantification of each isomeric form is important. Current methods are capable of predicting and detecting thousands of methylarginine sites in proteins, whereas differentiation and stoichiometric measurement of dimethylated protein isomers are still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics has emerged as a promising technique for site-specific quantification of protein methylation using enzymatic peptides as surrogates of target proteins. However, it should be pointed out that a routine targeted proteomics strategy cannot easily distinguish sDMA- and aDMA-containing surrogate peptides due to their common nature. The estimated amount should be considered as the sum of both arginine dimethylated isomers. In this study, compositional analysis based on a linear algebra algorithm as an add-on to targeted proteomics was employed to quantify H3R2me2a and H3R2me2s (i.e., surrogate peptides of AR(me2a)TK(me1/2)QT and AR(me2s)TK(me1/2)QT). To achieve this simultaneous quantification, a targeted proteomics assay was developed and validated for each isomer first. With the slope and intercept of their calibration curves for each multiple reaction monitoring (MRM) transition, linear algebraic equations were derived. Using a series of mock mixtures consisting of isomers in varying concentrations, the reliability of the method was confirmed. Finally, the H3R2 dimethylation status was analyzed in normal MCF-10A cells, parental drug-sensitive MCF-7/WT cancer cells, and drug-resistant MCF-7/ADR cancer cells. Dimethylated H3R2 was also monitored in MCF-7/WT cells with the treatment of doxorubicin (DOX) for confirmation.

Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples.

PubMed

Al Ali, Ahmad; Touboul, David; Le Caër, Jean-Pierre; Schmitz-Afonso, Isabelle; Flinois, Jean-Pierre; Marchetti, Catherine; De Waziers, Isabelle; Brunelle, Alain; Laprévote, Olivier; Beaune, Philippe

2014-08-01

Cytochromes P450 (CYPs) play critical roles in oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing a simple method to quantify six CYPs (CYP3A4, CYP3A5, CYP1A2, CYP2D6, CYP2C9, and CYP2J2) in various biological samples without isotopic labeling. The biological matrix was spiked with the standard peptides prior to the digestion step to realize a label-free quantification by mass spectrometry. The method was validated and applied to quantify these six isoforms in both human liver microsomes and mitochondria, but also in recombinant expression systems such as baculosomes and the HepG2 cell line. The results showed intra-assay and interassay accuracy and precision within 16 % and 5 %, respectively, at the low quality control level, and demonstrated the advantages of the method in terms of reproducibility and cost.

Short communication: separation and quantification of caseins and casein macropeptide using ion-exchange chromatography.

PubMed

Holland, B; Rahimi Yazdi, S; Ion Titapiccolo, G; Corredig, M

2010-03-01

The aim of this work was to improve an existing method to separate and quantify the 4 major caseins from milk samples (i.e., containing whey proteins) using ion-exchange chromatography. The separation process was carried out using a mini-preparative cation exchange column (1 or 5mL of column volume), using urea acetate as elution buffer at pH 3.5 with a NaCl gradient. All 4 major caseins were separated, and the purity of each peak was assessed using sodium dodecyl sulfate-PAGE. Purified casein fractions were also added to raw milk to confirm their elution volumes. The quantification was carried out using purified caseins in buffer as well as added directly to fresh skim milk. This method can also be employed to determine the decrease in kappa-casein and the release of the casein-macropeptide during enzymatic hydrolysis using rennet. In this case, the main advantage of using this method is the lack of organic solvents compared with the conventional method for separation of macropeptide (using reversed phase HPLC).

Quantitative Assessment of In-solution Digestion Efficiency Identifies Optimal Protocols for Unbiased Protein Analysis*

PubMed Central

León, Ileana R.; Schwämmle, Veit; Jensen, Ole N.; Sprenger, Richard R.

2013-01-01

The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods. PMID:23792921

STEM VQ Method, Using Scanning Transmission Electron Microscopy (STEM) for Accurate Virus Quantification

DTIC Science & Technology

2017-02-02

Corresponding Author Abstract Accurate virus quantification is sought, but a perfect method still eludes the scientific community. Electron...unlimited. UNCLASSIFIED 2 provides morphology data and counts all viral particles, including partial or noninfectious particles; however, EM methods ...consistent, reproducible virus quantification method called Scanning Transmission Electron Microscopy – Virus Quantification (STEM-VQ) which simplifies

A peptide-retrieval strategy enables significant improvement of quantitative performance without compromising confidence of identification.

PubMed

Tu, Chengjian; Shen, Shichen; Sheng, Quanhu; Shyr, Yu; Qu, Jun

2017-01-30

Reliable quantification of low-abundance proteins in complex proteomes is challenging largely owing to the limited number of spectra/peptides identified. In this study we developed a straightforward method to improve the quantitative accuracy and precision of proteins by strategically retrieving the less confident peptides that were previously filtered out using the standard target-decoy search strategy. The filtered-out MS/MS spectra matched to confidently-identified proteins were recovered, and the peptide-spectrum-match FDR were re-calculated and controlled at a confident level of FDR≤1%, while protein FDR maintained at ~1%. We evaluated the performance of this strategy in both spectral count- and ion current-based methods. >60% increase of total quantified spectra/peptides was respectively achieved for analyzing a spike-in sample set and a public dataset from CPTAC. Incorporating the peptide retrieval strategy significantly improved the quantitative accuracy and precision, especially for low-abundance proteins (e.g. one-hit proteins). Moreover, the capacity of confidently discovering significantly-altered proteins was also enhanced substantially, as demonstrated with two spike-in datasets. In summary, improved quantitative performance was achieved by this peptide recovery strategy without compromising confidence of protein identification, which can be readily implemented in a broad range of quantitative proteomics techniques including label-free or labeling approaches. We hypothesize that more quantifiable spectra and peptides in a protein, even including less confident peptides, could help reduce variations and improve protein quantification. Hence the peptide retrieval strategy was developed and evaluated in two spike-in sample sets with different LC-MS/MS variations using both MS1- and MS2-based quantitative approach. The list of confidently identified proteins using the standard target-decoy search strategy was fixed and more spectra/peptides with less confidence matched to confident proteins were retrieved. However, the total peptide-spectrum-match false discovery rate (PSM FDR) after retrieval analysis was still controlled at a confident level of FDR≤1%. As expected, the penalty for occasionally incorporating incorrect peptide identifications is negligible by comparison with the improvements in quantitative performance. More quantifiable peptides, lower missing value rate, better quantitative accuracy and precision were significantly achieved for the same protein identifications by this simple strategy. This strategy is theoretically applicable for any quantitative approaches in proteomics and thereby provides more quantitative information, especially on low-abundance proteins. Published by Elsevier B.V.

Quantification of Protein Biomarker Using SERS Nano-Stress Sensing with Peak Intensity Ratiometry

NASA Astrophysics Data System (ADS)

Goh, Douglas; Kong, Kien Voon; Jayakumar, Perumal; Gong, Tianxun; Dinish, U. S.; Olivo, Malini

We report a surface enhanced Raman spectroscopy (SERS) ratiometry method based on peak intensity coupled in a nano-stress sensing platform to detect and quantify biological molecules. Herein, we employed an antibody-conjugated p-aminothiophenol (ATP) functionalized on a bimetallic-film-over-nanosphere (BMFON) substrate as a sensitive SERS platform to detect human haptoglobin (Hp) protein, which is an acute phase protein and a biomarker for various cancers. Correlation between change in the ATP spectral characteristics and concentration of Hp protein was established by examining the peak intensity ratio at 1572cm-1 and 1592cm-1 that reflects the degree of stress experienced by the aromatic ring of ATP during Hp protein-antibody interaction. Development of this platform shows the potential in developing a low-cost and sensitive SERS sensor for the pre-screening of various biomarkers.

Quantification of anti-nutritional factors and their correlations with protein and oil in soybeans.

PubMed

Bueno, Rafael D; Borges, Leandro L; God, Pedro I V Good; Piovesan, Newton D; Teixeira, Arlindo I; Cruz, Cosme Damião; Barros, Everaldo G DE

2018-01-01

Soybeans contain about 30% carbohydrate, mainly consisting of non-starch polysaccharides (NSP) and oligosaccharides. NSP are not hydrolyzed in the gastrointestinal tract of monogastric animals. These NSP negatively affect the development of these animals, especially the soluble fraction. This work aimed to establish a method to quantify NSP in soybeans, using high performance liquid chromatography (HPLC), and to estimate correlations between NSP, oligosaccharides, protein and oil. Sucrose, raffinose + stachyose, soluble and insoluble NSP contents were determined by HPLC. Oil and protein contents were determined by near-infrared spectroscopy (NIRS). The soluble PNAs content showed no significant correlation with protein, oil, sucrose and raffinose + stachyose contents, but oligosaccharides showed a negative correlation with protein content. These findings open up the possibility of developing cultivars with low soluble NSP content, aiming to develop feed for monogastric animals.

Simultaneous differentiation and quantification of ricin and agglutinin by an antibody-sandwich surface plasmon resonance sensor.

PubMed

Stern, Daniel; Pauly, Diana; Zydek, Martin; Müller, Christian; Avondet, Marc A; Worbs, Sylvia; Lisdat, Fred; Dorner, Martin B; Dorner, Brigitte G

2016-04-15

Ricin is one of the most toxic plant toxins known. Its accessibility and relative ease of preparation makes it a potential agent for criminal or bio-terrorist attacks. Detection of ricin from unknown samples requires differentiation of ricin from the highly homologous Ricinus communis agglutinin which is currently not feasible using immunological methods. Here we have developed a simple and sensitive surface plasmon resonance (SPR) sensing system for rapid differentiation between ricin and agglutinin done in real time. Both lectins were quantified in a sandwich immunoassay-like setting by capturing with a cross-reactive antibody (R109) binding to both proteins while differentiating by injection of a ricin-specific antibody (R18) in a subsequent enhancement step. The SPR-assay was reproducible and sensitive for different R. communis cultivars, showing no false positive results when other lectins were tested. Quantification and differentiation of both molecules was also demonstrated from a crude castor bean extract and complex matrices. For the first time, we have demonstrated how the closely related lectins can be discerned and quantified in a single assay based on immunological methods. This novel approach delivers crucial information regarding the composition, purity, concentration, and toxicity of suspicious samples containing ricin in less than 30 minutes. Furthermore, we show how enhancement injections during SPR-measurements can be used to determine the ratio of two related proteins independently of the actual protein concentration by comparing normalized enhancement response levels. Copyright © 2015 Elsevier B.V. All rights reserved.

High-throughput analysis of sub-visible mAb aggregate particles using automated fluorescence microscopy imaging.

PubMed

Paul, Albert Jesuran; Bickel, Fabian; Röhm, Martina; Hospach, Lisa; Halder, Bettina; Rettich, Nina; Handrick, René; Herold, Eva Maria; Kiefer, Hans; Hesse, Friedemann

2017-07-01

Aggregation of therapeutic proteins is a major concern as aggregates lower the yield and can impact the efficacy of the drug as well as the patient's safety. It can occur in all production stages; thus, it is essential to perform a detailed analysis for protein aggregates. Several methods such as size exclusion high-performance liquid chromatography (SE-HPLC), light scattering, turbidity, light obscuration, and microscopy-based approaches are used to analyze aggregates. None of these methods allows determination of all types of higher molecular weight (HMW) species due to a limited size range. Furthermore, quantification and specification of different HMW species are often not possible. Moreover, automation is a perspective challenge coming up with automated robotic laboratory systems. Hence, there is a need for a fast, high-throughput-compatible method, which can detect a broad size range and enable quantification and classification. We describe a novel approach for the detection of aggregates in the size range 1 to 1000 μm combining fluorescent dyes for protein aggregate labelling and automated fluorescence microscope imaging (aFMI). After appropriate selection of the dye and method optimization, our method enabled us to detect various types of HMW species of monoclonal antibodies (mAbs). Using 10 μmol L -1 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (Bis-ANS) in combination with aFMI allowed the analysis of mAb aggregates induced by different stresses occurring during downstream processing, storage, and administration. Validation of our results was performed by SE-HPLC, UV-Vis spectroscopy, and dynamic light scattering. With this new approach, we could not only reliably detect different HMW species but also quantify and classify them in an automated approach. Our method achieves high-throughput requirements and the selection of various fluorescent dyes enables a broad range of applications.

Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes.

PubMed

Wang, Guanghui; Wu, Wells W; Zeng, Weihua; Chou, Chung-Lin; Shen, Rong-Fong

2006-05-01

A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable-isotope labeling (e.g., ICAT, 18O- or 15N-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier-resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative approach that allows global quantification of differentially expressed proteins in complex proteomes. The utility of this method to biomarker discovery is likely to synergize with future improvements in the detecting sensitivity of mass spectrometers.

Application of Targeted Mass Spectrometry for the Quantification of Sirtuins in the Central Nervous System

NASA Astrophysics Data System (ADS)

Jayasena, T.; Poljak, A.; Braidy, N.; Zhong, L.; Rowlands, B.; Muenchhoff, J.; Grant, R.; Smythe, G.; Teo, C.; Raftery, M.; Sachdev, P.

2016-10-01

Sirtuin proteins have a variety of intracellular targets, thereby regulating multiple biological pathways including neurodegeneration. However, relatively little is currently known about the role or expression of the 7 mammalian sirtuins in the central nervous system. Western blotting, PCR and ELISA are the main techniques currently used to measure sirtuin levels. To achieve sufficient sensitivity and selectivity in a multiplex-format, a targeted mass spectrometric assay was developed and validated for the quantification of all seven mammalian sirtuins (SIRT1-7). Quantification of all peptides was by multiple reaction monitoring (MRM) using three mass transitions per protein-specific peptide, two specific peptides for each sirtuin and a stable isotope labelled internal standard. The assay was applied to a variety of samples including cultured brain cells, mammalian brain tissue, CSF and plasma. All sirtuin peptides were detected in the human brain, with SIRT2 being the most abundant. Sirtuins were also detected in human CSF and plasma, and guinea pig and mouse tissues. In conclusion, we have successfully applied MRM mass spectrometry for the detection and quantification of sirtuin proteins in the central nervous system, paving the way for more quantitative and functional studies.

Quantification of genetically modified soya using strong anion exchange chromatography and time-of-flight mass spectrometry.

PubMed

Chang, Po-Chih; Reddy, P Muralidhar; Ho, Yen-Peng

2014-09-01

Stable-isotope dimethyl labeling was applied to the quantification of genetically modified (GM) soya. The herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) was labeled using a dimethyl labeling reagent, formaldehyde-H2 or -D2. The identification and quantification of CP4 EPSPS was performed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The CP4 EPSPS protein was separated from high abundance proteins using strong anion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the tryptic peptides from the samples and reference were labeled with formaldehyde-H2 and formaldehyde-D2, respectively. The two labeled pools were mixed and analyzed using MALDI-MS. The data showed a good correlation between the peak ratio of the H- and D-labeled peptides and the GM soya percentages at 0.5, 1, 3, and 5 %, with R (2) of 0.99. The labeling reagents are readily available. The labeling experiments and the detection procedures are simple. The approach is useful for the quantification of GM soya at a level as low as 0.5 %.

ICPD-a new peak detection algorithm for LC/MS.

PubMed

Zhang, Jianqiu; Haskins, William

2010-12-01

The identification and quantification of proteins using label-free Liquid Chromatography/Mass Spectrometry (LC/MS) play crucial roles in biological and biomedical research. Increasing evidence has shown that biomarkers are often low abundance proteins. However, LC/MS systems are subject to considerable noise and sample variability, whose statistical characteristics are still elusive, making computational identification of low abundance proteins extremely challenging. As a result, the inability of identifying low abundance proteins in a proteomic study is the main bottleneck in protein biomarker discovery. In this paper, we propose a new peak detection method called Information Combining Peak Detection (ICPD ) for high resolution LC/MS. In LC/MS, peptides elute during a certain time period and as a result, peptide isotope patterns are registered in multiple MS scans. The key feature of the new algorithm is that the observed isotope patterns registered in multiple scans are combined together for estimating the likelihood of the peptide existence. An isotope pattern matching score based on the likelihood probability is provided and utilized for peak detection. The performance of the new algorithm is evaluated based on protein standards with 48 known proteins. The evaluation shows better peak detection accuracy for low abundance proteins than other LC/MS peak detection methods.

Semi-automated quantification and neuroanatomical mapping of heterogeneous cell populations.

PubMed

Mendez, Oscar A; Potter, Colin J; Valdez, Michael; Bello, Thomas; Trouard, Theodore P; Koshy, Anita A

2018-07-15

Our group studies the interactions between cells of the brain and the neurotropic parasite Toxoplasma gondii. Using an in vivo system that allows us to permanently mark and identify brain cells injected with Toxoplasma protein, we have identified that Toxoplasma-injected neurons (TINs) are heterogeneously distributed throughout the brain. Unfortunately, standard methods to quantify and map heterogeneous cell populations onto a reference brain atlas are time consuming and prone to user bias. We developed a novel MATLAB-based semi-automated quantification and mapping program to allow the rapid and consistent mapping of heterogeneously distributed cells on to the Allen Institute Mouse Brain Atlas. The system uses two-threshold background subtraction to identify and quantify cells of interest. We demonstrate that we reliably quantify and neuroanatomically localize TINs with low intra- or inter-observer variability. In a follow up experiment, we show that specific regions of the mouse brain are enriched with TINs. The procedure we use takes advantage of simple immunohistochemistry labeling techniques, use of a standard microscope with a motorized stage, and low cost computing that can be readily obtained at a research institute. To our knowledge there is no other program that uses such readily available techniques and equipment for mapping heterogeneous populations of cells across the whole mouse brain. The quantification method described here allows reliable visualization, quantification, and mapping of heterogeneous cell populations in immunolabeled sections across whole mouse brains. Copyright © 2018 Elsevier B.V. All rights reserved.

Mass Spectrometry Method to Measure Membrane Proteins in Dried Blood Spots for the Detection of Blood Doping Practices in Sport.

PubMed

Cox, Holly D; Eichner, Daniel

2017-09-19

The dried blood spot (DBS) matrix has significant utility for applications in the field where venous blood collection and timely shipment of labile blood samples is difficult. Unfortunately, protein measurement in DBS is hindered by high abundance proteins and matrix interference that increases with hematocrit. We developed a DBS method to enrich for membrane proteins and remove soluble proteins and matrix interference. Following a wash in a series of buffers, the membrane proteins are digested with trypsin and quantitated by parallel reaction monitoring mass spectrometry methods. The DBS method was applied to the quantification of four cell-specific cluster of differentiation (CD) proteins used to count cells by flow cytometry, band 3 (CD233), CD71, CD45, and CD41. We demonstrate that the DBS method counts low abundance cell types such as immature reticulocytes as well as high abundance cell types such as red blood cells, white blood cells, and platelets. When tested in 82 individuals, counts obtained by the DBS method demonstrated good agreement with flow cytometry and automated hematology analyzers. Importantly, the method allows longitudinal monitoring of CD protein concentration and calculation of interindividual variation which is difficult by other methods. Interindividual variation of band 3 and CD45 was low, 6 and 8%, respectively, while variation of CD41 and CD71 was higher, 18 and 78%, respectively. Longitudinal measurement of CD71 concentration in DBS over an 8-week period demonstrated intraindividual variation 17.1-38.7%. Thus, the method may allow stable longitudinal measurement of blood parameters currently monitored to detect blood doping practices.

Evaluation of capillary zone electrophoresis for the determination of protein composition in therapeutic immunoglobulins and human albumins.

PubMed

Christians, Stefan; van Treel, Nadine Denise; Bieniara, Gabriele; Eulig-Wien, Annika; Hanschmann, Kay-Martin; Giess, Siegfried

2016-07-01

Capillary zone electrophoresis (CZE) provides an alternative means of separating native proteins on the basis of their inherent electrophoretic mobilities. The major advantage of CZE is the quantification by UV detection, circumventing the drawbacks of staining and densitometry in the case of gel electrophoresis methods. The data of this validation study showed that CZE is a reliable assay for the determination of protein composition in therapeutic preparations of human albumin and human polyclonal immunoglobulins. Data obtained by CZE are in line with "historical" data obtained by the compendial method, provided that peak integration is performed without time correction. The focus here was to establish a rapid and reliable test to substitute the current gel based zone electrophoresis techniques for the control of protein composition of human immunoglobulins or albumins in the European Pharmacopoeia. We believe that the more advanced and modern CZE method described here is a very good alternative to the procedures currently described in the relevant monographs. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Development of an Enhanced Metaproteomic Approach for Deepening the Microbiome Characterization of the Human Infant Gut

PubMed Central

2015-01-01

The establishment of early life microbiota in the human infant gut is highly variable and plays a crucial role in host nutrient availability/uptake and maturation of immunity. Although high-performance mass spectrometry (MS)-based metaproteomics is a powerful method for the functional characterization of complex microbial communities, the acquisition of comprehensive metaproteomic information in human fecal samples is inhibited by the presence of abundant human proteins. To alleviate this restriction, we have designed a novel metaproteomic strategy based on double filtering (DF) the raw samples, a method that fractionates microbial from human cells to enhance microbial protein identification and characterization in complex fecal samples from healthy premature infants. This method dramatically improved the overall depth of infant gut proteome measurement, with an increase in the number of identified low-abundance proteins and a greater than 2-fold improvement in microbial protein identification and quantification. This enhancement of proteome measurement depth enabled a more extensive microbiome comparison between infants by not only increasing the confidence of identified microbial functional categories but also revealing previously undetected categories. PMID:25350865

Systematic analysis of protein turnover in primary cells.

PubMed

Mathieson, Toby; Franken, Holger; Kosinski, Jan; Kurzawa, Nils; Zinn, Nico; Sweetman, Gavain; Poeckel, Daniel; Ratnu, Vikram S; Schramm, Maike; Becher, Isabelle; Steidel, Michael; Noh, Kyung-Min; Bergamini, Giovanna; Beck, Martin; Bantscheff, Marcus; Savitski, Mikhail M

2018-02-15

A better understanding of proteostasis in health and disease requires robust methods to determine protein half-lives. Here we improve the precision and accuracy of peptide ion intensity-based quantification, enabling more accurate protein turnover determination in non-dividing cells by dynamic SILAC-based proteomics. This approach allows exact determination of protein half-lives ranging from 10 to >1000 h. We identified 4000-6000 proteins in several non-dividing cell types, corresponding to 9699 unique protein identifications over the entire data set. We observed similar protein half-lives in B-cells, natural killer cells and monocytes, whereas hepatocytes and mouse embryonic neurons show substantial differences. Our data set extends and statistically validates the previous observation that subunits of protein complexes tend to have coherent turnover. Moreover, analysis of different proteasome and nuclear pore complex assemblies suggests that their turnover rate is architecture dependent. These results illustrate that our approach allows investigating protein turnover and its implications in various cell types.

In-situ protein determination to monitor contamination in a centrifugal partition chromatograph.

PubMed

Bouiche, Feriel; Faure, Karine

2017-05-15

Centrifugal partition chromatography (CPC) works with biphasic liquid systems including aqueous two-phase systems. Metallic rotors are able to retain an aqueous stationary phase able to purify proteins. But the adhesion of proteins to solid surface may pose a cross-contamination risk during downstream processes. So it is of utmost importance to ensure the cleanliness of the equipment and detect possible protein contamination in a timely manner. Thereby, a direct method that allows the determination of the effective presence of proteins and the extent of contamination in the metallic CPC rotors was developed. This in-situ method is derived from the Amino Density Estimation by Colorimetric Assay (ADECA) which is based on the affinity of a dye, Coomassie Brillant Blue (CBB), with protonated N + groups of the proteins. In this paper, the ADECA method was developed dynamically, on a 25 mL stainless-steel rotor with various extents of protein contaminations using bovine serum albumin (BSA) as a fouling model. The eluted CBB dye was quantified and found to respond linearly to BSA contamination up to 70 mg injected. Limits of detection and quantification were recorded as 0.9 mg and 3.1 mg, respectively. While the non-specific interactions between the dye and the rotor cannot currently be neglected, this method allows for in situ determination of proteins contamination and should contribute to the development of CPC as a separation tool in protein purification processes. Copyright © 2017 Elsevier Inc. All rights reserved.

When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

NASA Astrophysics Data System (ADS)

Petre, Brînduşa-Alina; Ulrich, Martina; Stumbaum, Mihaela; Bernevic, Bogdan; Moise, Adrian; Döring, Gerd; Przybylski, Michael

2012-11-01

Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar KD values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.

Assessment of Sample Preparation Bias in Mass Spectrometry-Based Proteomics.

PubMed

Klont, Frank; Bras, Linda; Wolters, Justina C; Ongay, Sara; Bischoff, Rainer; Halmos, Gyorgy B; Horvatovich, Péter

2018-04-17

For mass spectrometry-based proteomics, the selected sample preparation strategy is a key determinant for information that will be obtained. However, the corresponding selection is often not based on a fit-for-purpose evaluation. Here we report a comparison of in-gel (IGD), in-solution (ISD), on-filter (OFD), and on-pellet digestion (OPD) workflows on the basis of targeted (QconCAT-multiple reaction monitoring (MRM) method for mitochondrial proteins) and discovery proteomics (data-dependent acquisition, DDA) analyses using three different human head and neck tissues (i.e., nasal polyps, parotid gland, and palatine tonsils). Our study reveals differences between the sample preparation methods, for example, with respect to protein and peptide losses, quantification variability, protocol-induced methionine oxidation, and asparagine/glutamine deamidation as well as identification of cysteine-containing peptides. However, none of the methods performed best for all types of tissues, which argues against the existence of a universal sample preparation method for proteome analysis.

Versatile aptasensor for electrochemical quantification of cell surface glycan and naked-eye tracking glycolytic inhibition in living cells.

PubMed

Zhang, Jing-Jing; Cheng, Fang-Fang; Zheng, Ting-Ting; Zhu, Jun-Jie

2017-03-15

Quantifying the glycan expression status on cell surfaces is of vital importance for insight into the glycan function in biological processes and related diseases. Here we developed a versatile aptasensor for electrochemical quantification of cell surface glycan by taking advantage of the cell-specific aptamer, and the lectin-functionalized gold nanoparticles acting as both a glycan recognition unit and a signal amplification probe. To construct the aptasensor, amine-functionalized mucin 1 protein (MUC1) aptamer was first covalently conjugated to carboxylated-magnetic beads (MBs) using the succinimide coupling (EDC-NHS) method. On the basis of the specific recognition between aptamer and MUC1 protein that overexpressed on the surface of MCF-7 cells, the aptamer conjugated MBs showed a predominant capability for cell capture with high selectivity. Moreover, a lectin-based nanoprobe was designed by noncovalent assembly of concanavalin A (ConA) on gold nanoparticles (AuNPs). This nanoprobe incorporated the abilities of both the specific carbohydrate recognition and the signal amplification based on the gold-promoted reduction of silver ions. By coupling with electrochemical stripping analysis, the proposed sandwich-type cytosensor showed an excellent analytical performance for the ultrasensitive detection of MCF-7 cells and quantification of cell surface glycan. More importantly, taking advantage of Con A-gold nanoprobe catalyzed silver enhancement, the proposed method was further used for naked-eye tracking glycolytic inhibition in living cells. This aptasensor holds great promise as a new point-of-care diagnostic tool for analyzing glycan expression on living cells and further helps cancer diagnosis and treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

Mass spectrometry-based proteomics: basic principles and emerging technologies and directions.

PubMed

Van Riper, Susan K; de Jong, Ebbing P; Carlis, John V; Griffin, Timothy J

2013-01-01

As the main catalytic and structural molecules within living systems, proteins are the most likely biomolecules to be affected by radiation exposure. Proteomics, the comprehensive characterization of proteins within complex biological samples, is therefore a research approach ideally suited to assess the effects of radiation exposure on cells and tissues. For comprehensive characterization of proteomes, an analytical platform capable of quantifying protein abundance, identifying post-translation modifications and revealing members of protein complexes on a system-wide level is necessary. Mass spectrometry (MS), coupled with technologies for sample fractionation and automated data analysis, provides such a versatile and powerful platform. In this chapter we offer a view on the current state of MS-proteomics, and focus on emerging technologies within three areas: (1) New instrumental methods; (2) New computational methods for peptide identification; and (3) Label-free quantification. These emerging technologies should be valuable for researchers seeking to better understand biological effects of radiation on living systems.

Infectious helminth ova in wastewater and sludge: A review on public health issues and current quantification practices.

PubMed

Gyawali, P

2018-02-01

Raw and partially treated wastewater has been widely used to maintain the global water demand. Presence of viable helminth ova and larvae in the wastewater raised significant public health concern especially when used for agriculture and aquaculture. Depending on the prevalence of helminth infections in communities, up to 1.0 × 10 3 ova/larvae can be presented per litre of wastewater and 4 gm (dry weight) of sludge. Multi-barrier approaches including pathogen reduction, risk assessment, and exposure reduction have been suggested by health regulators to minimise the potential health risk. However, with a lack of a sensitive and specific method for the quantitative detection of viable helminth ova from wastewater, an accurate health risk assessment is difficult to achieve. As a result, helminth infections are difficult to control from the communities despite two decades of global effort (mass drug administration). Molecular methods can be more sensitive and specific than currently adapted culture-based and vital stain methods. The molecular methods, however, required more and thorough investigation for its ability with accurate quantification of viable helminth ova/larvae from wastewater and sludge samples. Understanding different cell stages and corresponding gene copy numbers is pivotal for accurate quantification of helminth ova/larvae in wastewater samples. Identifying specific genetic markers including protein, lipid, and metabolites using multiomics approach could be utilized for cheap, rapid, sensitive, specific and point of care detection tools for helminth ova and larva in the wastewater.

Isobaric Quantification of Cerebrospinal Fluid Amyloid-β Peptides in Alzheimer's Disease: C-Terminal Truncation Relates to Early Measures of Neurodegeneration.

PubMed

Rogeberg, Magnus; Almdahl, Ina Selseth; Wettergreen, Marianne; Nilsson, Lars N G; Fladby, Tormod

2015-11-06

The amyloid beta (Aβ) peptide is the main constituent of the plaques characteristic of Alzheimer's disease (AD). Measurement of Aβ1-42 in cerebrospinal fluid (CSF) is a valuable marker in AD research, where low levels indicate AD. Although the use of immunoassays measuring Aβ1-38 and Aβ1-40 in addition to Aβ1-42 has increased, quantitative assays of other Aβ peptides remain rarely explored. We recently discovered novel Aβ peptides in CSF using antibodies recognizing the Aβ mid-domain region. Here we have developed a method using both Aβ N-terminal and mid-domain antibodies for immunoprecipitation in combination with isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of endogenous Aβ peptides in CSF. The developed method was used in a pilot study to produce Aβ peptide profiles from 38 CSF samples. Statistical comparison between CSF samples from 19 AD patients and 19 cognitively healthy controls revealed no significant differences at group level. A significant correlation was found between several larger C-terminally truncated Aβ peptides and protein biomarkers for neuronal damage, particularly prominent in the control group. Comparison of the isobaric quantification with immunoassays measuring Aβ1-38 or Aβ1-40 showed good correlation (r(2) = 0.84 and 0.85, respectively) between the two analysis methods. The developed method could be used to assess disease-modifying therapies directed at Aβ production or degradation.

Bismuth citrate in the quantification of inorganic phosphate and its utility in the determination of membrane-bound phosphatases.

PubMed

Cariani, L; Thomas, L; Brito, J; del Castillo, J R

2004-01-01

This paper describes a rapid and sensitive method to determine inorganic phosphate, even in the presence of labile organic phosphate compounds and large quantities of proteins. The method eliminates the use of sodium arsenite, a highly toxic compound, substituting bismuth citrate for it to stabilize the phosphomolybdic acid complex formed during the interaction of inorganic phosphate and molybdate reduced by ascorbic acid. This method has also been adapted to microplates and has been used to determine the activities of Na/K ATPase and alkaline phosphatase of intestinal basolateral and luminal plasma membranes.

Immunochemical-based method for detection of hazelnut proteins in processed foods.

PubMed

Ben Rejeb, Samuel; Abbott, Michael; Davies, David; Querry, Jessica; Cléroux, Chantal; Streng, Christine; Delahaut, Philippe; Yeung, Jupiter M

2003-01-01

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect hazelnut by using polyclonal antibodies generated against a protein extract of roasted hazelnut. No cross-reactivity was observed in tests against 39 commodities, including many common allergens, tree nuts, and legumes. Hazelnut protein standard solutions at 0.45 ng/mL [inhibition concentration (IC80) of the competitive test] were clearly identified by the ELISA. An extraction and quantification method was developed and optimized for chocolate, cookies, breakfast cereals, and ice cream, major food commodities likely to be cross-contaminated with undeclared hazelnut during food processing. No sample cleanup was required when extracts were diluted 10-fold. Recovery results were generated with blank matrixes spiked at 4 levels from 1 to 10 microg/g hazelnut protein. With the developed extraction and sample handling procedure, hazelnut proteins were recovered at 64-83% from chocolate and at 78-97% from other matrixes. A confirmatory technique was developed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer. The developed methods were applied to a small market survey of chocolate products and allowed the identification of undeclared hazelnut in these products.

Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum).

PubMed

Gao, Wei; Nan, Tiegui; Tan, Guiyu; Zhao, Hongwei; Tan, Weiming; Meng, Fanyun; Li, Zhaohu; Li, Qing X; Wang, Baomin

2015-01-01

The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

Quantification of vitamin B6 vitamers in human cerebrospinal fluid by ultra performance liquid chromatography-tandem mass spectrometry.

PubMed

van der Ham, M; Albersen, M; de Koning, T J; Visser, G; Middendorp, A; Bosma, M; Verhoeven-Duif, N M; de Sain-van der Velden, M G M

2012-01-27

Since vitamin B6 is essential for normal functioning of the central nervous system, there is growing need for sensitive analysis of B6 vitamers in cerebrospinal fluid (CSF). This manuscript describes the development and validation of a rapid, sensitive and accurate method for quantification of the vitamin B6 vitamers pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxic acid (PA), pyridoxal 5'-phosphate (PLP), pyridoxamine 5'-phosphate (PMP) and pyridoxine 5'-phosphate (PNP) in human CSF. The method is based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with a simple sample preparation procedure of protein precipitation using 50 g L(-1) trichloroacetic acid containing stable isotope labeled internal standards: PL-D(3) for PL and PM, PN-(13)C(4) for PN, PA-D(2) for PA and PLP-D(3) for the phosphorylated vitamers. B6 vitamers were separated (Acquity HSS-T3 UPLC column) with a buffer containing acetic acid, heptafluorobutyric acid and acetonitrile. Positive electrospray ionization was used to monitor transitions m/z 168.1→150.1 (PL), 169.1→134.1 (PM), 170.1→134.1 (PN), 184.1→148.1 (PA), 248.1→150.1 (PLP), 249.1→232.1 (PMP) and 250.1→134.1 (PNP). The method was validated at three concentration levels for each B6 vitamer in CSF. Recoveries of the internal standards were between 93% and 96%. Intra- and inter-assay variations were below 20%. Accuracy tests showed deviations from 3% (PN) to 39% (PMP). Limits of quantification were in the range of 0.03-5.37 nM. Poor results were obtained for quantification of PNP. The method was applied to CSF samples of 20 subjects and two patients on pyridoxine supplementation. Using minimal CSF volumes this method is suitable for implementation in a routine diagnostic setting. Copyright © 2011 Elsevier B.V. All rights reserved.

Absolute quantification of prion protein (90-231) using stable isotope-labeled chymotryptic peptide standards in a LC-MRM AQUA workflow.

PubMed

Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M; Pedersen, Joel A; Li, Lingjun

2012-09-01

Substantial evidence indicates that the disease-associated conformer of the prion protein (PrP(TSE)) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrP(TSE) has the ability to convert the conformation of the normal prion protein (PrP(C)) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrP(TSE) at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrP(TSE), the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

Absolute Quantification of Prion Protein (90-231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow

NASA Astrophysics Data System (ADS)

Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M.; Pedersen, Joel A.; Li, Lingjun

2012-09-01

Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

Sensitive and rapid liquid chromatography/tandem mass spectrometric assay for the quantification of piperaquine in human plasma.

PubMed

Singhal, Puran; Gaur, Ashwani; Gautam, Anirudh; Varshney, Brijesh; Paliwal, Jyoti; Batra, Vijay

2007-11-01

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantification of piperaquine, an antimalarial drug, in human plasma using its structural analogue, piperazine bis chloroquinoline as internal standard (IS). The method involved a simple protein precipitation with methanol followed by rapid isocratic elution of analytes with 10mM ammonium acetate buffer/methanol/formic acid/ammonia solution (25/75/0.2/0.15, v/v) on Chromolith SpeedROD RP-18e reversed phase chromatographic column and quantification by mass spectrometry in the multiple reaction monitoring mode (MRM). The precursor to product ion transitions of m/z 535.3-->288.2 and m/z 409.1-->205.2 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 1.0-250.2 ng/mL for piperaquine in plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) in plasma were 0.2 and 1.0 ng/mL, respectively. Acceptable precision and accuracy (+/-20% deviation for LLOQ standard and +/-15% deviation for other standards from the respective nominal concentration) were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for a sample made it possible to achieve a throughput of more than 400 plasma samples analyzed per day. The validated method was successfully applied to analyze human plasma samples from phase-1 clinical studies. The mean pharmacokinetic parameters of piperaquine following 1000 mg oral dose: observed maximum plasma concentration (Cmax), time to maximum plasma concentration (Tmax) and elimination half-life (T1/2) were 46.1 ng/mL, 3.8h and 13 days, respectively.

Pre-analytical effects of blood sampling and handling in quantitative immunoassays for rheumatoid arthritis.

PubMed

Zhao, Xiaoyan; Qureshi, Ferhan; Eastman, P Scott; Manning, William C; Alexander, Claire; Robinson, William H; Hesterberg, Lyndal K

2012-04-30

Variability in pre-analytical blood sampling and handling can significantly impact results obtained in quantitative immunoassays. Understanding the impact of these variables is critical for accurate quantification and validation of biomarker measurements. Particularly, in the design and execution of large clinical trials, even small differences in sample processing and handling can have dramatic effects in analytical reliability, results interpretation, trial management and outcome. The effects of two common blood sampling methods (serum vs. plasma) and two widely-used serum handling methods (on the clot with ambient temperature shipping, "traditional", vs. centrifuged with cold chain shipping, "protocol") on protein and autoantibody concentrations were examined. Matched serum and plasma samples were collected from 32 rheumatoid arthritis (RA) patients representing a wide range of disease activity status. Additionally, a set of matched serum samples with two sample handling methods was collected. One tube was processed per manufacturer's instructions and shipped overnight on cold packs (protocol). The matched tube, without prior centrifugation, was simultaneously shipped overnight at ambient temperatures (traditional). Upon delivery, the traditional tube was centrifuged. All samples were subsequently aliquoted and frozen prior to analysis of protein and autoantibody biomarkers. Median correlation between paired serum and plasma across all autoantibody assays was 0.99 (0.98-1.00) with a median % difference of -3.3 (-7.5 to 6.0). In contrast, observed protein biomarker concentrations were significantly affected by sample types, with median correlation of 0.99 (0.33-1.00) and a median % difference of -10 (-55 to 23). When the two serum collection/handling methods were compared, the median correlation between paired samples for autoantibodies was 0.99 (0.91-1.00) with a median difference of 4%. In contrast, significant increases were observed in protein biomarker concentrations among certain biomarkers in samples processed with the 'traditional' method. Autoantibody quantification appears robust to both sample type (plasma vs. serum) and pre-analytical sample collection/handling methods (protocol vs. traditional). In contrast, for non-antibody protein biomarker concentrations, sample type had a significant impact; plasma samples generally exhibit decreased protein biomarker concentrations relative to serum. Similarly, sample handling significantly impacted the variability of protein biomarker concentrations. When biomarker concentrations are combined algorithmically into a single test score such as a multi-biomarker disease activity test for rheumatoid arthritis (MBDA), changes in protein biomarker concentrations may result in a bias of the score. These results illustrate the importance of characterizing pre-analytical methodology, sample type, sample processing and handling procedures for clinical testing in order to ensure test accuracy. Copyright © 2012 Elsevier B.V. All rights reserved.

Quantification of Anti-Aggregation Activity of Chaperones: A Test-System Based on Dithiothreitol-Induced Aggregation of Bovine Serum Albumin

PubMed Central

Borzova, Vera A.; Markossian, Kira A.; Kara, Dmitriy A.; Chebotareva, Natalia A.; Makeeva, Valentina F.; Poliansky, Nikolay B.; Muranov, Konstantin O.; Kurganov, Boris I.

2013-01-01

The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin allowed us to make a conclusion that a non-linear character of the dependence of v agg on concentration of intact α-crystallin was due to the dynamic mobility of the quaternary structure of α-crystallin and polydispersity of the α-crystallin–target protein complexes. To characterize the anti-aggregation activity of the chemical chaperones (arginine, arginine ethyl ester, arginine amide and proline), the semi-saturation concentration [L]0.5 was used. Among the chemical chaperones studied, arginine ethyl ester and arginine amide reveal the highest anti-aggregation activity ([L]0.5 = 53 and 58 mM, respectively). PMID:24058554

Generic method for the absolute quantification of glutathione S-conjugates: Application to the conjugates of acetaminophen, clozapine and diclofenac.

PubMed

den Braver, Michiel W; Vermeulen, Nico P E; Commandeur, Jan N M

2017-03-01

Modification of cellular macromolecules by reactive drug metabolites is considered to play an important role in the initiation of tissue injury by many drugs. Detection and identification of reactive intermediates is often performed by analyzing the conjugates formed after trapping by glutathione (GSH). Although sensitivity of modern mass spectrometrical methods is extremely high, absolute quantification of GSH-conjugates is critically dependent on the availability of authentic references. Although 1 H NMR is currently the method of choice for quantification of metabolites formed biosynthetically, its intrinsically low sensitivity can be a limiting factor in quantification of GSH-conjugates which generally are formed at low levels. In the present study, a simple but sensitive and generic method for absolute quantification of GSH-conjugates is presented. The method is based on quantitative alkaline hydrolysis of GSH-conjugates and subsequent quantification of glutamic acid and glycine by HPLC after precolumn derivatization with o-phthaldialdehyde/N-acetylcysteine (OPA/NAC). Because of the lower stability of the glycine OPA/NAC-derivate, quantification of the glutamic acid OPA/NAC-derivate appeared most suitable for quantification of GSH-conjugates. The novel method was used to quantify the concentrations of GSH-conjugates of diclofenac, clozapine and acetaminophen and quantification was consistent with 1 H NMR, but with a more than 100-fold lower detection limit for absolute quantification. Copyright © 2017. Published by Elsevier B.V.

Bioluminescent Antibodies for Point-of-Care Diagnostics.

PubMed

Xue, Lin; Yu, Qiuliyang; Griss, Rudolf; Schena, Alberto; Johnsson, Kai

2017-06-12

We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔR max >500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Identification of 7 Proteins in Sera of RA Patients with Potential to Predict ETA/MTX Treatment Response

PubMed Central

Obry, Antoine; Hardouin, Julie; Lequerré, Thierry; Jarnier, Frédérique; Boyer, Olivier; Fardellone, Patrice; Philippe, Peggy; Marcelli, Christian; Loët, Xavier Le; Vittecoq, Olivier; Cosette, Pascal

2015-01-01

Objective: The recent growth of innovating biologics has opened fascinating avenues for the management of patients. In rheumatoid arthritis, many biologics are currently available, the choice of which being mostly determined empirically. Importantly, a given biologic may not be active in a fraction of patients and may even provoke side effects. Here, we conducted a comparative proteomics study in attempt to identify a predictive theranostic signature of non-response in patients with rheumatoid arthritis treated by etanercept/methotrexate combination. Methods: A serum sample was collected prior to treatment exposure from a cohort of 22 patients with active RA. A proteomic “label free” approach was then designed to quantitate protein biomarkers using mass spectrometry. To verify these results, a relative quantification followed by an absolute quantification of interesting protein candidates were performed on a second cohort. The criterion of judgment was the response to etanercept/methotrexate combination according to the EULAR criteria assessed at 6 months of treatment. Results: These investigations led to the identification of a set of 12 biomarkers with capacity to predict treatment response. A targeted quantitative analysis allowed to confirm the potential of 7 proteins from the latter combination on a new cohort of 16 patients. Two highly discriminating proteins, PROS and CO7, were further evaluated by ELISA on this second cohort. By combining the concentration threshold of each protein associated to a right classification (responders vs non-responders), the sensitivity and specificity reached 88.9 % and 100 %, respectively. Conclusion: Prior to methotrexate/etanercept treatment, abundance of several sera proteins, notably PROS and CO7, were associated to response status of RA patients 6 month after treatment initiation. PMID:26379787

Comparison and validation of methods to quantify Cry1Ab toxin from Bacillus thuringiensis for standardization of insect bioassays.

PubMed

Crespo, André L B; Spencer, Terence A; Nekl, Emily; Pusztai-Carey, Marianne; Moar, William J; Siegfried, Blair D

2008-01-01

Standardization of toxin preparations derived from Bacillus thuringiensis (Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer, Ostrinia nubilalis (Hübner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of O. nubilalis. The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC(50) values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.

Analysis of illegal peptide biopharmaceuticals frequently encountered by controlling agencies.

PubMed

Vanhee, Celine; Janvier, Steven; Desmedt, Bart; Moens, Goedele; Deconinck, Eric; De Beer, Jacques O; Courselle, Patricia

2015-09-01

Recent advances in genomics, recombinant expression technologies and peptide synthesis have led to an increased development of protein and peptide therapeutics. Unfortunately this goes hand in hand with a growing market of counterfeit and illegal biopharmaceuticals, including substances that are still under pre-clinical and clinical development. These counterfeit and illegal protein and peptide substances could imply severe health threats as has been demonstrated by numerous case reports. The Belgian Federal Agency for Medicines and Health Products (FAMHP) and customs are striving, together with their global counterparts, to curtail the trafficking and distributions of these substances. At their request, suspected protein and peptide preparations are analysed in our Official Medicines Control Laboratory (OMCL). It stands to reason that a general screening method would be beneficiary in the battle against counterfeit and illegal peptide drugs. In this paper we present such general screening method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the identification of counterfeit and illegal injectable peptide preparations, extended with a subsequent quantification method using ultra-high performance liquid chromatography with diode array detection (UHPLC-DAD). The screening method, taking only 30 min, is able to selectively detect 25 different peptides and incorporates the proposed minimum of five identification points (IP) as has been recommended for sports drug testing applications. The group of peptides represent substances which have already been detected in illegal and counterfeit products seized by different European countries as well as some biopharmaceutical peptides which have not been confiscated yet by the controlling agencies, but are already being used according to the many internet users forums. Additionally, we also show that when applying the same LC gradient, it is also possible to quantify these peptides without the need for derivatization or the use of expensive labelled peptides. This quantification method was successfully validated for a representative subset of 10 different peptides by using the "total error" approach in accordance with the validation requirements of ISO-17025. Copyright © 2015 Elsevier B.V. All rights reserved.

Targeted, Site-specific quantitation of N- and O-glycopeptides using 18O-labeling and product ion based mass spectrometry.

PubMed

Srikanth, Jandhyam; Agalyadevi, Rathinasamy; Babu, Ponnusamy

2017-02-01

The site-specific quantitation of N- and O-glycosylation is vital to understanding the function(s) of different glycans expressed at a given site of a protein under physiological and disease conditions. Most commonly used precursor ion intensity based quantification method is less accurate and other labeled methods are expensive and require enrichment of glycopeptides. Here, we used glycopeptide product (y and Y0) ions and 18 O-labeling of C-terminal carboxyl group as a strategy to obtain quantitative information about fold-change and relative abundance of most of the glycoforms attached to the glycopeptides. As a proof of concept, the accuracy and robustness of this targeted, relative quantification LC-MS method was demonstrated using Rituximab. Furthermore, the N-glycopeptide quantification results were compared with a biosimilar of Rituximab and validated with quantitative data obtained from 2-AB-UHPLC-FL method. We further demonstrated the intensity fold-change and relative abundance of 46 unique N- and O-glycopeptides and aglycopeptides from innovator and biosimilar samples of Etanercept using both the normal-MS and product ion based quantitation. The results showed a very similar site-specific expression of N- and O-glycopeptides between the samples but with subtle differences. Interestingly, we have also been able to quantify macro-heterogeneity of all N- and O-glycopetides of Etanercept. In addition to applications in biotherapeutics, the developed method can also be used for site-specific quantitation of N- and O-glycopeptides and aglycopeptides of glycoproteins with known glycosylation pattern.

Centrifugal ultrafiltration of human serum for improving immunoglobulin A quantification using attenuated total reflectance infrared spectroscopy.

PubMed

Elsohaby, Ibrahim; McClure, J Trenton; Riley, Christopher B; Bryanton, Janet; Bigsby, Kathryn; Shaw, R Anthony

2018-02-20

Attenuated total reflectance infrared (ATR-IR) spectroscopy is a simple, rapid and cost-effective method for the analysis of serum. However, the complex nature of serum remains a limiting factor to the reliability of this method. We investigated the benefits of coupling the centrifugal ultrafiltration with ATR-IR spectroscopy for quantification of human serum IgA concentration. Human serum samples (n = 196) were analyzed for IgA using an immunoturbidimetric assay. ATR-IR spectra were acquired for whole serum samples and for the retentate (residue) reconstituted with saline following 300 kDa centrifugal ultrafiltration. IR-based analytical methods were developed for each of the two spectroscopic datasets, and the accuracy of each of the two methods compared. Analytical methods were based upon partial least squares regression (PLSR) calibration models - one with 5-PLS factors (for whole serum) and the second with 9-PLS factors (for the reconstituted retentate). Comparison of the two sets of IR-based analytical results to reference IgA values revealed improvements in the Pearson correlation coefficient (from 0.66 to 0.76), and the root mean squared error of prediction in IR-based IgA concentrations (from 102 to 79 mg/dL) for the ultrafiltration retentate-based method as compared to the method built upon whole serum spectra. Depleting human serum low molecular weight proteins using a 300 kDa centrifugal filter thus enhances the accuracy IgA quantification by ATR-IR spectroscopy. Further evaluation and optimization of this general approach may ultimately lead to routine analysis of a range of high molecular-weight analytical targets that are otherwise unsuitable for IR-based analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein.

PubMed Central

Requena, J R; Fu, M X; Ahmed, M U; Jenkins, A J; Lyons, T J; Baynes, J W; Thorpe, S R

1997-01-01

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of < 1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo. PMID:9078279

A flow-proteometric platform for analyzing protein concentration (FAP): Proof of concept for quantification of PD-L1 protein in cells and tissues.

PubMed

Chou, Chao-Kai; Huang, Po-Jung; Tsou, Pei-Hsiang; Wei, Yongkun; Lee, Heng-Huan; Wang, Ying-Nai; Liu, Yen-Liang; Shi, Colin; Yeh, Hsin-Chih; Kameoka, Jun; Hung, Mien-Chie

2018-05-29

Protein expression level is critically related to the cell physiological function. However, current methodologies such as Western blot (WB) and Immunohistochemistry (IHC) in analyzing the protein level are rather semi-quantitative and without the information of actual protein concentration. We have developed a microfluidic technique termed a "flow-proteometric platform for analyzing protein concentration (FAP)" that can measure the concentration of a target protein in cells or tissues without the requirement of a calibration standard, e.g., the purified target molecules. To validate our method, we tested a number of control samples with known target protein concentrations and showed that the FAP measurement resulted in concentrations that well matched the actual concentrations in the control samples (coefficient of determination [R 2 ], 0.998), demonstrating a dynamic range of concentrations from 0.13 to 130 pM of a detection for 2 min. We successfully determined a biomarker protein (for predicting the treatment response of cancer immune check-point therapy) PD-L1 concentration in cancer cell lines (HeLa PD-L1 and MDA-MB-231) and breast cancer patient tumor tissues without any prior process of sample purification and standard line construction. Therefore, FAP is a simple, faster, and reliable method to measure the protein concentration in cells and tissues, which can support the conventional methods such as WB and IHC to determine the actual protein level. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

PubMed

Ermolli, M; Prospero, A; Balla, B; Querci, M; Mazzeo, A; Van Den Eede, G

2006-09-01

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

Quantification of proteins in urine samples using targeted mass spectrometry methods.

PubMed

Khristenko, Nina; Domon, Bruno

2015-01-01

Numerous clinical proteomics studies are focused on the development of biomarkers to improve either diagnostics for early disease detection or the monitoring of the response to the treatment. Although, a wealth of biomarker candidates are available, their evaluation and validation in a true clinical setup remains challenging. In biomarkers evaluation studies, a panel of proteins of interest are systematically analyzed in a large cohort of samples. However, in spite of the latest progresses in mass spectrometry, the consistent detection of pertinent proteins in high complex biological samples is still a challenging task. Thus, targeted LC-MS/MS methods are better suited for the systematic analysis of biomarkers rather than shotgun approaches. This chapter describes the workflow used to perform targeted quantitative analyses of proteins in urinary samples. The peptides, as surrogates of the protein of interest, are commonly measured using a triple quadrupole mass spectrometers operated in selected reaction monitoring (SRM) mode. More recently, the advances in targeted LC-MS/MS analysis based on parallel reaction monitoring (PRM) performed on a quadrupole-orbitrap instrument have allowed to increase the specificity and selectivity of the measurements.

Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells

PubMed Central

Okumus, Burak; Landgraf, Dirk; Lai, Ghee Chuan; Bakhsi, Somenath; Arias-Castro, Juan Carlos; Yildiz, Sadik; Huh, Dann; Fernandez-Lopez, Raul; Peterson, Celeste N.; Toprak, Erdal; El Karoui, Meriem; Paulsson, Johan

2016-01-01

Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3–4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting. PMID:27189321

Quantitation of heat-shock proteins in clinical samples using mass spectrometry.

PubMed

Kaur, Punit; Asea, Alexzander

2011-01-01

Mass spectrometry (MS) is a powerful analytical tool for proteomics research and drug and biomarker discovery. MS enables identification and quantification of known and unknown compounds by revealing their structural and chemical properties. Proper sample preparation for MS-based analysis is a critical step in the proteomics workflow because the quality and reproducibility of sample extraction and preparation for downstream analysis significantly impact the separation and identification capabilities of mass spectrometers. The highly expressed proteins represent potential biomarkers that could aid in diagnosis, therapy, or drug development. Because the proteome is so complex, there is no one standard method for preparing protein samples for MS analysis. Protocols differ depending on the type of sample, source, experiment, and method of analysis. Molecular chaperones play significant roles in almost all biological functions due to their capacity for detecting intracellular denatured/unfolded proteins, initiating refolding or denaturation of such malfolded protein sequences and more recently for their role in the extracellular milieu as chaperokines. In this chapter, we describe the latest techniques for quantitating the expression of molecular chaperones in human clinical samples.

Experimental Null Method to Guide the Development of Technical Procedures and to Control False-Positive Discovery in Quantitative Proteomics.

PubMed

Shen, Xiaomeng; Hu, Qiang; Li, Jun; Wang, Jianmin; Qu, Jun

2015-10-02

Comprehensive and accurate evaluation of data quality and false-positive biomarker discovery is critical to direct the method development/optimization for quantitative proteomics, which nonetheless remains challenging largely due to the high complexity and unique features of proteomic data. Here we describe an experimental null (EN) method to address this need. Because the method experimentally measures the null distribution (either technical or biological replicates) using the same proteomic samples, the same procedures and the same batch as the case-vs-contol experiment, it correctly reflects the collective effects of technical variability (e.g., variation/bias in sample preparation, LC-MS analysis, and data processing) and project-specific features (e.g., characteristics of the proteome and biological variation) on the performances of quantitative analysis. To show a proof of concept, we employed the EN method to assess the quantitative accuracy and precision and the ability to quantify subtle ratio changes between groups using different experimental and data-processing approaches and in various cellular and tissue proteomes. It was found that choices of quantitative features, sample size, experimental design, data-processing strategies, and quality of chromatographic separation can profoundly affect quantitative precision and accuracy of label-free quantification. The EN method was also demonstrated as a practical tool to determine the optimal experimental parameters and rational ratio cutoff for reliable protein quantification in specific proteomic experiments, for example, to identify the necessary number of technical/biological replicates per group that affords sufficient power for discovery. Furthermore, we assessed the ability of EN method to estimate levels of false-positives in the discovery of altered proteins, using two concocted sample sets mimicking proteomic profiling using technical and biological replicates, respectively, where the true-positives/negatives are known and span a wide concentration range. It was observed that the EN method correctly reflects the null distribution in a proteomic system and accurately measures false altered proteins discovery rate (FADR). In summary, the EN method provides a straightforward, practical, and accurate alternative to statistics-based approaches for the development and evaluation of proteomic experiments and can be universally adapted to various types of quantitative techniques.

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

PubMed

Krtolica, Ana; Ortiz de Solorzano, Carlos; Lockett, Stephen; Campisi, Judith

2002-10-01

To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies. Copyright 2002 Wiley-Liss, Inc.

MEERCAT: Multiplexed Efficient Cell Free Expression of Recombinant QconCATs For Large Scale Absolute Proteome Quantification*

PubMed Central

Takemori, Nobuaki; Takemori, Ayako; Tanaka, Yuki; Endo, Yaeta; Hurst, Jane L.; Gómez-Baena, Guadalupe; Harman, Victoria M.; Beynon, Robert J.

2017-01-01

A major challenge in proteomics is the absolute accurate quantification of large numbers of proteins. QconCATs, artificial proteins that are concatenations of multiple standard peptides, are well established as an efficient means to generate standards for proteome quantification. Previously, QconCATs have been expressed in bacteria, but we now describe QconCAT expression in a robust, cell-free system. The new expression approach rescues QconCATs that previously were unable to be expressed in bacteria and can reduce the incidence of proteolytic damage to QconCATs. Moreover, it is possible to cosynthesize QconCATs in a highly-multiplexed translation reaction, coexpressing tens or hundreds of QconCATs simultaneously. By obviating bacterial culture and through the gain of high level multiplexing, it is now possible to generate tens of thousands of standard peptides in a matter of weeks, rendering absolute quantification of a complex proteome highly achievable in a reproducible, broadly deployable system. PMID:29055021

Attomole quantitation of protein separations with accelerator mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vogel, J S; Grant, P G; Buccholz, B A

2000-12-15

Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to sub-attomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5% . Micro-proton-induced-xray-emission quantifies elemental abundancesmore » in separated metalloprotein samples to nanogram amounts and is capable of quantifying phosphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.« less

Protein microarray with horseradish peroxidase chemiluminescence for quantification of serum α-fetoprotein.

PubMed

Zhao, Yuanshun; Zhang, Yonghong; Lin, Dongdong; Li, Kang; Yin, Chengzeng; Liu, Xiuhong; Jin, Boxun; Sun, Libo; Liu, Jinhua; Zhang, Aiying; Li, Ning

2015-10-01

To develop and evaluate a protein microarray assay with horseradish peroxidase (HRP) chemiluminescence for quantification of α-fetoprotein (AFP) in serum from patients with hepatocellular carcinoma (HCC). A protein microarray assay for AFP was developed. Serum was collected from patients with HCC and healthy control subjects. AFP was quantified using protein microarray and enzyme-linked immunosorbent assay (ELISA). Serum AFP concentrations determined via protein microarray were positively correlated (r = 0.973) with those determined via ELISA in patients with HCC (n = 60) and healthy control subjects (n = 30). Protein microarray showed 80% sensitivity and 100% specificity for HCC diagnosis. ELISA had 83.3% sensitivity and 100% specificity. Protein microarray effectively distinguished between patients with HCC and healthy control subjects (area under ROC curve 0.974; 95% CI 0.000, 1.000). Protein microarray is a rapid, simple and low-cost alternative to ELISA for detecting AFP in human serum. © The Author(s) 2015.

Evaluating Kinase ATP Uptake and Tyrosine Phosphorylation using Multiplexed Quantification of Chemically Labeled and Post-Translationally Modified Peptides

PubMed Central

Fang, Bin; Hoffman, Melissa A.; Mirza, Abu-Sayeef; Mishall, Katie M.; Li, Jiannong; Peterman, Scott M.; Smalley, Keiran S. M.; Shain, Kenneth H.; Weinberger, Paul M.; Wu, Jie; Rix, Uwe; Haura, Eric B.; Koomen, John M.

2015-01-01

Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease. PMID:25782629

QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics.

PubMed

Choi, Hyungwon; Kim, Sinae; Fermin, Damian; Tsou, Chih-Chiang; Nesvizhskii, Alexey I

2015-11-03

We introduce QPROT, a statistical framework and computational tool for differential protein expression analysis using protein intensity data. QPROT is an extension of the QSPEC suite, originally developed for spectral count data, adapted for the analysis using continuously measured protein-level intensity data. QPROT offers a new intensity normalization procedure and model-based differential expression analysis, both of which account for missing data. Determination of differential expression of each protein is based on the standardized Z-statistic based on the posterior distribution of the log fold change parameter, guided by the false discovery rate estimated by a well-known Empirical Bayes method. We evaluated the classification performance of QPROT using the quantification calibration data from the clinical proteomic technology assessment for cancer (CPTAC) study and a recently published Escherichia coli benchmark dataset, with evaluation of FDR accuracy in the latter. QPROT is a statistical framework with computational software tool for comparative quantitative proteomics analysis. It features various extensions of QSPEC method originally built for spectral count data analysis, including probabilistic treatment of missing values in protein intensity data. With the increasing popularity of label-free quantitative proteomics data, the proposed method and accompanying software suite will be immediately useful for many proteomics laboratories. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Luminol-Based Chemiluminescent Signals: Clinical and Non-clinical Application and Future Uses

PubMed Central

Khan, Parvez; Idrees, Danish; Moxley, Michael A.; Corbett, John A.; Ahmad, Faizan; von Figura, Guido; Sly, William S.; Waheed, Abdul

2015-01-01

Chemiluminescence (CL) is an important method for quantification and analysis of various macromolecules. A wide range of CL agents such as luminol, hydrogen peroxide, fluorescein, dioxetanes and derivatives of oxalate, and acridinium dyes are used according to their biological specificity and utility. This review describes the application of luminol chemiluminescence (LCL) in forensic, biomedical, and clinical sciences. LCL is a very useful detection method due to its selectivity, simplicity, low cost, and high sensitivity. LCL has a dynamic range of applications, including quantification and detection of macro and micromolecules such as proteins, carbohydrates, DNA, and RNA. Luminol-based methods are used in environmental monitoring as biosensors, in the pharmaceutical industry for cellular localization and as biological tracers, and in reporter gene-based assays and several other immunoassays. Here, we also provide information about different compounds that may enhance or inhibit the LCL along with the effect of pH and concentration on LCL. This review covers most of the significant information related to the applications of luminol in different fields. PMID:24752935

Detection of Free and Protein-Bound ortho-Quinones by Near-Infrared Fluorescence.

PubMed

Mazzulli, Joseph R; Burbulla, Lena F; Krainc, Dimitri; Ischiropoulos, Harry

2016-02-16

Aging and oxidative stress are two prominent pathological mechanisms for Parkinson's disease (PD) that are strongly associated with the degeneration of dopamine (DA) neurons in the midbrain. DA and other catechols readily oxidize into highly reactive o-quinone species that are precursors of neuromelanin (NM) pigment and under pathological conditions can modify and damage macromolecules. The role of DA oxidation in PD pathogenesis remains unclear in part due to the lack of appropriate disease models and the absence of a simple method for the quantification of DA-derived oxidants. Here, we describe a rapid, simple, and reproducible method for the quantification of o-quinones in cells and tissues that relies on the near-infrared fluorescent properties of these species. Importantly, we demonstrate that catechol-derived oxidants can be quantified in human neuroblastoma cells and midbrain dopamine neurons derived from induced pluripotent stem cells, providing a novel model to study the downstream actions of o-quinones. This method should facilitate further study of oxidative stress and DA oxidation in PD and related diseases that affect the dopaminergic system.

Determination of lansoprazole in human plasma by rapid resolution liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study on Chinese volunteers.

PubMed

Wu, Guo-Lan; Zhou, Hui-Li; Shentu, Jian-Zhong; He, Qiao-Jun; Yang, Bo

2008-12-15

A simple, sensitive and rapid LC/MS/MS method was developed for the quantification of lansoprazole in human plasma. After a simple sample preparation procedure by one-step protein precipitation with acetonitrile, lansoprazole and the internal standard bicalutamide were chromatographed on a Zorbax SB-C(18) (3.0 mm x 150 mm, 3.5 microm, Agilent) column with the mobile phase consisted of methanol-water (70:30, v/v, containing 5 mM ammonium formate, pH was adjusted to 7.85 by 1% ammonia solution). Detection was performed on a triple quadrupole tandem mass spectrometry by multiple reaction monitoring (MRM) mode via negative eletrospray ionization source (ESI(-)). The lower limit of quantification was 5.5 ng/mL, and the assay exhibited a linear range of 5.5-2200.0 ng/mL. The validated method was successfully applied to investigate the bioequivalence between two kinds of preparation (test vs. reference product) in twenty-eight healthy male Chinese volunteers.

Proteomic Analysis of Sauvignon Blanc Grape Skin, Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins.

PubMed

Tian, Bin; Harrison, Roland; Morton, James; Deb-Choudhury, Santanu

2015-01-01

Thaumatin-like proteins (TLPs) and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR) proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and determination of PR proteins in berries at harvest.

Proteomic Analysis of Sauvignon Blanc Grape Skin, Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins

PubMed Central

Tian, Bin; Harrison, Roland; Morton, James; Deb-Choudhury, Santanu

2015-01-01

Thaumatin-like proteins (TLPs) and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR) proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and determination of PR proteins in berries at harvest. PMID:26076362

Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture.

PubMed

de Wit, C; Fautz, C; Xu, Y

2000-09-01

Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1x10(6) particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3-6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5'-->3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600 particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell-based production system. Copyright 2000 The International Association for Biologicals.

Combining Metabolic ¹⁵N Labeling with Improved Tandem MOAC for Enhanced Probing of the Phosphoproteome.

PubMed

Thomas, Martin; Huck, Nicola; Hoehenwarter, Wolfgang; Conrath, Uwe; Beckers, Gerold J M

2015-01-01

In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool for studying protein phosphorylation because it enables unbiased localization, and site-specific quantification of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy for identifying phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major pitfalls in MS-based phosphoproteomics. This is particularly true for plants in which the presence of secondary metabolites and endogenous compounds, the overabundance of ribulose-1,5-bisphosphate carboxylase and other components of the photosynthetic apparatus, and the concurrent difficulties in protein extraction necessitate two-step phosphoprotein/phosphopeptide enrichment strategies (Nakagami et al., Plant Cell Physiol 53:118-124, 2012).Approaches for label-free peptide quantification are advantageous due to their low cost and experimental simplicity, but they lack precision. These drawbacks can be overcome by metabolic labeling of whole plants with heavy nitrogen ((15)N) which allows combining two samples very early in the phosphoprotein enrichment workflow. This avoids sample-to-sample variation introduced by the analytical procedures and it results in robust relative quantification values that need no further standardization. The integration of (15)N metabolic labeling into tandem metal-oxide affinity chromatography (MOAC) (Hoehenwarter et al., Mol Cell Proteomics 12:369-380, 2013) presents an improved and highly selective approach for the identification and accurate site-specific quantification of low-abundance phosphoproteins that is based on the successive enrichment of light and heavy nitrogen-labeled phosphoproteins and peptides. This improved strategy combines metabolic labeling of whole plants with the stable heavy nitrogen isotope ((15)N), protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, and tryptic digest of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides and quantitative phosphopeptide measurement by liquid chromatography (LC) and high-resolution accurate mass (HR/AM) mass spectrometry (MS). Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and allows probing of the phosphoproteome to unprecedented depth, while (15)N metabolic labeling enables accurate relative quantification of measured peptides and direct comparison between samples.

Identification of cellular MMP substrates using quantitative proteomics: isotope-coded affinity tags (ICAT) and isobaric tags for relative and absolute quantification (iTRAQ).

PubMed

Butler, Georgina S; Dean, Richard A; Morrison, Charlotte J; Overall, Christopher M

2010-01-01

Identification of protease substrates is essential to understand the functional consequences of normal proteolytic processing and dysregulated proteolysis in disease. Quantitative proteomics and mass spectrometry can be used to identify protease substrates in the cellular context. Here we describe the use of two protein labeling techniques, Isotope-Coded Affinity Tags (ICAT and Isobaric Tags for Relative and Absolute Quantification (iTRAQ), which we have used successfully to identify novel matrix metalloproteinase (MMP) substrates in cell culture systems (1-4). ICAT and iTRAQ can label proteins and protease cleavage products of secreted proteins, protein domains shed from the cell membrane or pericellular matrix of protease-transfected cells that have accumulated in conditioned medium, or cell surface proteins in membrane preparations; isotopically distinct labels are used for control cells. Tryptic digestion and tandem mass spectrometry of the generated fragments enable sequencing of differentially labeled but otherwise identical pooled peptides. The isotopic tag, which is unique for each label, identifies the peptides originating from each sample, for instance, protease-transfected or control cells, and comparison of the peak areas enables relative quantification of the peptide in each sample. Thus proteins present in altered amounts between protease-expressing and null cells are implicated as protease substrates and can be further validated as such.

External cavity-quantum cascade laser (EC-QCL) spectroscopy for protein analysis in bovine milk.

PubMed

Kuligowski, Julia; Schwaighofer, Andreas; Alcaráz, Mirta Raquel; Quintás, Guillermo; Mayer, Helmut; Vento, Máximo; Lendl, Bernhard

2017-04-22

The analytical determination of bovine milk proteins is important in food and non-food industrial applications and yet, rather labour-intensive wet-chemical, low-throughput methods have been employed since decades. This work proposes the use of external cavity-quantum cascade laser (EC-QCL) spectroscopy for the simultaneous quantification of the most abundant bovine milk proteins and the total protein content based on the chemical information contained in mid-infrared (IR) spectral features of the amide I band. Mid-IR spectra of protein standard mixtures were used for building partial least squares (PLS) regression models. Protein concentrations in commercial bovine milk samples were calculated after chemometric compensation of the matrix contribution employing science-based calibration (SBC) without sample pre-processing. The use of EC-QCL spectroscopy together with advanced multivariate data analysis allowed the determination of casein, α-lactalbumin, β-lactoglobulin and total protein content within several minutes. Copyright © 2017 Elsevier B.V. All rights reserved.

Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods

PubMed Central

Friedrich, Ralf P; Janko, Christina; Poettler, Marina; Tripal, Philipp; Zaloga, Jan; Cicha, Iwona; Dürr, Stephan; Nowak, Johannes; Odenbach, Stefan; Slabu, Ioana; Liebl, Maik; Trahms, Lutz; Stapf, Marcus; Hilger, Ingrid; Lyer, Stefan; Alexiou, Christoph

2015-01-01

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEONLA) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEONLA with an additional protein corona formed by bovine serum albumin (SEONLA-BSA) and commercially available Rienso® particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products. PMID:26170658

A study of a self diagnostic platform for the detection of A2 biomarker for Leishmania donovani

NASA Astrophysics Data System (ADS)

Roche, Philip J. R.; Cheung, Maurice C.; Najih, Mohamed; McCall, Laura-Isobel; Fakih, Ibrahim; Chodavarapu, Vamsy P.; Ward, Brian; Ndao, Momar; Kirk, Andrew G.

2012-03-01

Visceral leishmaniasis (L.donovani) is a protozoan infection that attacks mononuclear phagocytes and causes the liver and spleen damage that can cause death. The investigation presented is a proof of concept development applying a plasmonic diagnostic platform with simple microfluidic sample delivery and optical readout. An immune-assay method is applied to the quantification of A2 protein, a highly immunogenic biomarker for the pathogen. Quantification of A2 was performed in the ng/ml range, analysis by ELISA suggested that a limit of 0.1ng/ml of A2 is approximate to 1 pathogen per ml and the sensing system shows the potential to deliver a similar level of quantification. Significant reduction in assay complexity as further enzyme linked enhancement is not required when applying a plasmonic methodology to an immunoassay. The basic instrumentation required for a portable device and potential dual optical readout where both plasmonic and photoluminescent response are assessed and investigated including consideration of the application of the device to testing where non-literate communication of results is considered and issues of performance are addressed.

Recommended Immunological Assays to Screen for Ricin-Containing Samples

PubMed Central

Simon, Stéphanie; Worbs, Sylvia; Avondet, Marc-André; Tracz, Dobryan M.; Dano, Julie; Schmidt, Lisa; Volland, Hervé; Dorner, Brigitte G.; Corbett, Cindi R.

2015-01-01

Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC) as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories’ capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120). Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests). Using these immunological methods “dangerous” samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin. PMID:26703725

Recommended Immunological Assays to Screen for Ricin-Containing Samples.

PubMed

Simon, Stéphanie; Worbs, Sylvia; Avondet, Marc-André; Tracz, Dobryan M; Dano, Julie; Schmidt, Lisa; Volland, Hervé; Dorner, Brigitte G; Corbett, Cindi R

2015-11-26

Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC) as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories' capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120). Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests). Using these immunological methods "dangerous" samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin.

A novel quantification-driven proteomic strategy identifies an endogenous peptide of pleiotrophin as a new biomarker of Alzheimer's disease.

PubMed

Skillbäck, Tobias; Mattsson, Niklas; Hansson, Karl; Mirgorodskaya, Ekaterina; Dahlén, Rahil; van der Flier, Wiesje; Scheltens, Philip; Duits, Floor; Hansson, Oskar; Teunissen, Charlotte; Blennow, Kaj; Zetterberg, Henrik; Gobom, Johan

2017-10-17

We present a new, quantification-driven proteomic approach to identifying biomarkers. In contrast to the identification-driven approach, limited in scope to peptides that are identified by database searching in the first step, all MS data are considered to select biomarker candidates. The endopeptidome of cerebrospinal fluid from 40 Alzheimer's disease (AD) patients, 40 subjects with mild cognitive impairment, and 40 controls with subjective cognitive decline was analyzed using multiplex isobaric labeling. Spectral clustering was used to match MS/MS spectra. The top biomarker candidate cluster (215% higher in AD compared to controls, area under ROC curve = 0.96) was identified as a fragment of pleiotrophin located near the protein's C-terminus. Analysis of another cohort (n = 60 over four clinical groups) verified that the biomarker was increased in AD patients while no change in controls, Parkinson's disease or progressive supranuclear palsy was observed. The identification of the novel biomarker pleiotrophin 151-166 demonstrates that our quantification-driven proteomic approach is a promising method for biomarker discovery, which may be universally applicable in clinical proteomics.

Validation and implementation of liquid chromatographic-mass spectrometric (LC-MS) methods for the quantification of tenofovir prodrugs.

PubMed

Hummert, Pamela; Parsons, Teresa L; Ensign, Laura M; Hoang, Thuy; Marzinke, Mark A

2018-04-15

The nucleotide reverse transcriptase inhibitor tenofovir (TFV) is widely administered in a disoproxil prodrug form (tenofovir disoproxil fumarate, TDF) for HIV management and prevention. Recently, novel prodrugs tenofovir alafenamide fumarate (TAF) and hexadecyloxypropyl tenofovir (CMX157) have been pursued for HIV treatment while minimizing adverse effects associated with systemic TFV exposure. Dynamic and sensitive bioanalytical tools are required to characterize the pharmacokinetics of these prodrugs in systemic circulation. Two parallel methods have been developed, one to combinatorially quantify TAF and TFV, and a second method for CMX157 quantification, in plasma. K 2 EDTA plasma was spiked with TAF and TFV, or CMX157. Following the addition of isotopically labeled internal standards and sample extraction via solid phase extraction (TAF and TFV) or protein precipitation (CMX157), samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. For TAF and TFV, separation occurred using a Zorbax Eclipse Plus C18 Narrow Bore RR, 2.1 × 50 mm, 3.5 μm column and analytes were detected on an API5000 mass analyzer; CMX157 was separated using a Kinetex C8, 2.1 × 50 mm, 2.6 μm column and quantified using an API4500 mass spectrometer. Methods were validated according to FDA Bioanalytical Method Validation guidelines. Analytical methods: were optimized for the multiplexed monitoring of TAF and TFV, and CMX157 in plasma. The lower limits of quantification (LLOQs) for TAF, TFV, and CMX157 were 0.03, 1.0, and 0.25 ng/mL, respectively. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs ≤ 14.4% and %DEVs ≤ ± 7.95%, respectively. Stability and matrix effects studies were also performed. All results were acceptable and in accordance with the recommended guidelines for bioanalytical methods. Assays were also applied to quantify in vivo concentrations of prodrugs and TFV in a preclinical study post-rectal administration. Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the multiplexed quantification TAF and TFV, as well as an independent assay for CMX157 quantification, in plasma. The described methods meet sufficient throughput criteria to support large research trials. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantification of plasma exosome is a potential prognostic marker for esophageal squamous cell carcinoma.

PubMed

Matsumoto, Yasunori; Kano, Masayuki; Akutsu, Yasunori; Hanari, Naoyuki; Hoshino, Isamu; Murakami, Kentaro; Usui, Akihiro; Suito, Hiroshi; Takahashi, Masahiko; Otsuka, Ryota; Xin, Hu; Komatsu, Aki; Iida, Keiko; Matsubara, Hisahiro

2016-11-01

Exosomes play important roles in cancer progression. Although its contents (e.g., proteins and microRNAs) have been focused on in cancer research, particularly as potential diagnostic markers, the exosome behavior and methods for exosome quantification remain unclear. In the present study, we analyzed the tumor-derived exosome behavior and assessed the quantification of exosomes in patient plasma as a biomarker for esophageal squamous cell carcinoma (ESCC). A CD63-GFP expressing human ESCC cell line (TE2-CD63-GFP) was made by transfection, and mouse subcutaneous tumor models were established. Fluorescence imaging was performed on tumors and plasma exosomes harvested from mice. GFP-positive small vesicles were confirmed in the plasma obtained from TE2-CD63-GFP tumor-bearing mice. Patient plasma was collected in Chiba University Hospital (n=86). Exosomes were extracted from 100 µl of the plasma and quantified by acetylcholinesterase (AChE) activity. The relationship between exosome quantification and the patient clinical characteristics was assessed. The quantification of exosomes isolated from the patient plasma revealed that esophageal cancer patients (n=66) expressed higher exosome levels than non-malignant patients (n=20) (P=0.0002). Although there was no correlation between the tumor progression and the exosome levels, exosome number was the independent prognostic marker and low levels of exosome predicted a poor prognosis (P=0.03). In conclusion, exosome levels may be useful as an independent prognostic factor for ESCC patients.

An Optimized Informatics Pipeline for Mass Spectrometry-Based Peptidomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Chaochao; Monroe, Matthew E.; Xu, Zhe

2015-12-26

Comprehensive MS analysis of peptidome, the intracellular and intercellular products of protein degradation, has the potential to provide novel insights on endogenous proteolytic processing and their utility in disease diagnosis and prognosis. Along with the advances in MS instrumentation, a plethora of proteomics data analysis tools have been applied for direct use in peptidomics; however an evaluation of the currently available informatics pipelines for peptidomics data analysis has yet to be reported. In this study, we set off by evaluating the results of several popular MS/MS database search engines including MS-GF+, SEQUEST and MS-Align+ for peptidomics data analysis, followed bymore » identification and label-free quantification using the well-established accurate mass and time (AMT) tag and newly developed informed quantification (IQ) approaches, both based on direct LC-MS analysis. Our result demonstrated that MS-GF+ outperformed both SEQUEST and MS-Align+ in identifying peptidome peptides. Using a database established from the MS-GF+ peptide identifications, both the AMT tag and IQ approaches provided significantly deeper peptidome coverage and less missing value for each individual data set than the MS/MS methods, while achieving robust label-free quantification. Besides having an excellent correlation with the AMT tag quantification results, IQ also provided slightly higher peptidome coverage than AMT. Taken together, we propose an optimal informatics pipeline combining MS-GF+ for initial database searching with IQ (or AMT) for identification and label-free quantification for high-throughput, comprehensive and quantitative peptidomics analysis.« less

Simultaneous quantification of 21 water soluble vitamin circulating forms in human plasma by liquid chromatography-mass spectrometry.

PubMed

Meisser Redeuil, Karine; Longet, Karin; Bénet, Sylvie; Munari, Caroline; Campos-Giménez, Esther

2015-11-27

This manuscript reports a validated analytical approach for the quantification of 21 water soluble vitamins and their main circulating forms in human plasma. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. Instrumental lower limits of detection and quantification reached <0.1-10nM and 0.2-25nM, respectively. Commercially available pooled human plasma was used to build matrix-matched calibration curves ranging 2-500, 5-1250, 20-5000 or 150-37500nM depending on the analyte. The overall performance of the method was considered adequate, with 2.8-20.9% and 5.2-20.0% intra and inter-day precision, respectively and averaged accuracy reaching 91-108%. Recovery experiments were also performed and reached in average 82%. This analytical approach was then applied for the quantification of circulating water soluble vitamins in human plasma single donor samples. The present report provides a sensitive and reliable approach for the quantification of water soluble vitamins and main circulating forms in human plasma. In the future, the application of this analytical approach will give more confidence to provide a comprehensive assessment of water soluble vitamins nutritional status and bioavailability studies in humans. Copyright © 2015 Elsevier B.V. All rights reserved.

Automated renal histopathology: digital extraction and quantification of renal pathology

NASA Astrophysics Data System (ADS)

Sarder, Pinaki; Ginley, Brandon; Tomaszewski, John E.

2016-03-01

The branch of pathology concerned with excess blood serum proteins being excreted in the urine pays particular attention to the glomerulus, a small intertwined bunch of capillaries located at the beginning of the nephron. Normal glomeruli allow moderate amount of blood proteins to be filtered; proteinuric glomeruli allow large amount of blood proteins to be filtered. Diagnosis of proteinuric diseases requires time intensive manual examination of the structural compartments of the glomerulus from renal biopsies. Pathological examination includes cellularity of individual compartments, Bowman's and luminal space segmentation, cellular morphology, glomerular volume, capillary morphology, and more. Long examination times may lead to increased diagnosis time and/or lead to reduced precision of the diagnostic process. Automatic quantification holds strong potential to reduce renal diagnostic time. We have developed a computational pipeline capable of automatically segmenting relevant features from renal biopsies. Our method first segments glomerular compartments from renal biopsies by isolating regions with high nuclear density. Gabor texture segmentation is used to accurately define glomerular boundaries. Bowman's and luminal spaces are segmented using morphological operators. Nuclei structures are segmented using color deconvolution, morphological processing, and bottleneck detection. Average computation time of feature extraction for a typical biopsy, comprising of ~12 glomeruli, is ˜69 s using an Intel(R) Core(TM) i7-4790 CPU, and is ~65X faster than manual processing. Using images from rat renal tissue samples, automatic glomerular structural feature estimation was reproducibly demonstrated for 15 biopsy images, which contained 148 individual glomeruli images. The proposed method holds immense potential to enhance information available while making clinical diagnoses.

Influence of physical and chemical properties of HTSXT-FTIR samples on the quality of prediction models developed to determine absolute concentrations of total proteins, carbohydrates and triglycerides: a preliminary study on the determination of their absolute concentrations in fresh microalgal biomass.

PubMed

Serrano León, Esteban; Coat, Rémy; Moutel, Benjamin; Pruvost, Jérémy; Legrand, Jack; Gonçalves, Olivier

2014-11-01

Absolute concentrations of total macromolecules (triglycerides, proteins and carbohydrates) in microorganisms can be rapidly measured by FTIR spectroscopy, but caution is needed to avoid non-specific experimental bias. Here, we assess the limits within which this approach can be used on model solutions of macromolecules of interest. We used the Bruker HTSXT-FTIR system. Our results show that the solid deposits obtained after the sampling procedure present physical and chemical properties that influence the quality of the absolute concentration prediction models (univariate and multivariate). The accuracy of the models was degraded by a factor of 2 or 3 outside the recommended concentration interval of 0.5-35 µg spot(-1). Change occurred notably in the sample hydrogen bond network, which could, however, be controlled using an internal probe (pseudohalide anion). We also demonstrate that for aqueous solutions, accurate prediction of total carbohydrate quantities (in glucose equivalent) could not be made unless a constant amount of protein was added to the model solution (BSA). The results of the prediction model for more complex solutions, here with two components: glucose and BSA, were very encouraging, suggesting that this FTIR approach could be used as a rapid quantification method for mixtures of molecules of interest, provided the limits of use of the HTSXT-FTIR method are precisely known and respected. This last finding opens the way to direct quantification of total molecules of interest in more complex matrices.

Characterization of the Distance Relationship Between Localized Serotonin Receptors and Glia Cells on Fluorescence Microscopy Images of Brain Tissue.

PubMed

Jacak, Jaroslaw; Schaller, Susanne; Borgmann, Daniela; Winkler, Stephan M

2015-08-01

We here present two new methods for the characterization of fluorescent localization microscopy images obtained from immunostained brain tissue sections. Direct stochastic optical reconstruction microscopy images of 5-HT1A serotonin receptors and glial fibrillary acidic proteins in healthy cryopreserved brain tissues are analyzed. In detail, we here present two image processing methods for characterizing differences in receptor distribution on glial cells and their distribution on neural cells: One variant relies on skeleton extraction and adaptive thresholding, the other on k-means based discrete layer segmentation. Experimental results show that both methods can be applied for distinguishing classes of images with respect to serotonin receptor distribution. Quantification of nanoscopic changes in relative protein expression on particular cell types can be used to analyze degeneration in tissues caused by diseases or medical treatment.

Quantitative Analysis of Energy Metabolic Pathways in MCF-7 Breast Cancer Cells by Selected Reaction Monitoring Assay*

PubMed Central

Drabovich, Andrei P.; Pavlou, Maria P.; Dimitromanolakis, Apostolos; Diamandis, Eleftherios P.

2012-01-01

To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells. PMID:22535206

Symbiosis induces widespread changes in the proteome of the model cnidarian Aiptasia.

PubMed

Oakley, Clinton A; Ameismeier, Michael F; Peng, Lifeng; Weis, Virginia M; Grossman, Arthur R; Davy, Simon K

2016-07-01

Coral reef ecosystems are metabolically founded on the mutualism between corals and photosynthetic dinoflagellates of the genus Symbiodinium. The glass anemone Aiptasia sp. has become a tractable model for this symbiosis, and recent advances in genetic information have enabled the use of mass spectrometry-based proteomics in this model. We utilized label-free liquid chromatography electrospray-ionization tandem mass spectrometry to analyze the effects of symbiosis on the proteomes of symbiotic and aposymbiotic Aiptasia. We identified and obtained relative quantification of more than 3,300 proteins in 1,578 protein clusters, with 81 protein clusters showing significantly different expression between symbiotic states. Symbiotic anemones showed significantly higher expression of proteins involved in lipid storage and transport, nitrogen transport and cycling, intracellular trafficking, endocytosis and inorganic carbon transport. These changes reflect shifts in host metabolism and nutrient reserves due to increased nutritional exchange with the symbionts, as well as mechanisms for supplying inorganic nutrients to the algae. Aposymbiotic anemones exhibited increased expression of multiple systems responsible for mediating reactive oxygen stress, suggesting that the host derives direct or indirect protection from oxidative stress while in symbiosis. Aposymbiotic anemones also increased their expression of an array of proteases and chitinases, indicating a metabolic shift from autotrophy to heterotrophy. These results provide a comprehensive Aiptasia proteome with more direct relative quantification of protein abundance than transcriptomic methods. The extension of "omics" techniques to this model system will allow more powerful studies of coral physiology, ecosystem function, and the effects of biotic and abiotic stress on the coral-dinoflagellate mutualism. © 2015 John Wiley & Sons Ltd.

Determination of total antioxidant capacity of milk by CUPRAC and ABTS methods with separate characterisation of milk protein fractions.

PubMed

Çekiç, Sema Demirci; Demir, Aslı; Başkan, Kevser Sözgen; Tütem, Esma; Apak, Reşat

2015-05-01

Most milk-applied antioxidant assays in literature are based on the isolation and quantification of individual antioxidative compounds, whereas total antioxidant capacity (TAC) gives a more holistic picture due to cooperative action of antioxidants. Recently, the cupric reducing antioxidant capacity (CUPRAC) method has been modified to measure the antioxidant capacities of thiol-containing proteins, where the classical ammonium acetate buffer - that may otherwise precipitate proteins- was replaced with concentrated urea buffer (able to expose embedded thiol groups of proteins to oxidative attack) adjusted to pH 7.0. Thus, antioxidant capacity of milk was investigated with two competing TAC assays, namely CUPRAC and ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid))/persulphate, because only these assays were capable of evaluating protein contribution to the observed TAC value. As milk fat caused turbidity, experiments were carried out with skim milk or defatted milk samples. To determine TAC, modified CUPRAC method was applied to whole milk, separated and redissolved protein fractions, and the remaining liquid phase after necessary operations. Both TAC methods were investigated for their dilution sensitivity and antioxidant power assessment of separate milk fractions such as casein and whey. Proteins like β-lactoglobulin and casein (but not simple thiols) exhibited enhanced CUPRAC reactivity with surfactant (SDS) addition. Addition of milk protein fractions to whole skim milk produced significant 'negative-biased' deviations (up to -26% relative standard error) from TAC absorbance additivity in the application of the ABTS method, as opposed to that of the CUPRAC method less affected by chemical deviations from Beer's law thereby producing much smaller deviations from additivity (i.e. the property of additivity is valid when the measured TAC of a mixture is equal to the sum of individual antioxidant capacities of its constituents).

Quantification of Confocal Images Using LabVIEW for Tissue Engineering Applications

PubMed Central

Sfakis, Lauren; Kamaldinov, Tim; Larsen, Melinda; Castracane, James

2016-01-01

Quantifying confocal images to enable location of specific proteins of interest in three-dimensional (3D) is important for many tissue engineering (TE) applications. Quantification of protein localization is essential for evaluation of specific scaffold constructs for cell growth and differentiation for application in TE and tissue regeneration strategies. Although obtaining information regarding protein expression levels is important, the location of proteins within cells grown on scaffolds is often the key to evaluating scaffold efficacy. Functional epithelial cell monolayers must be organized with apicobasal polarity with proteins specifically localized to the apical or basolateral regions of cells in many organs. In this work, a customized program was developed using the LabVIEW platform to quantify protein positions in Z-stacks of confocal images of epithelial cell monolayers. The program's functionality is demonstrated through salivary gland TE, since functional salivary epithelial cells must correctly orient many proteins on the apical and basolateral membranes. Bio-LabVIEW Image Matrix Evaluation (Bio-LIME) takes 3D information collected from confocal Z-stack images and processes the fluorescence at each pixel to determine cell heights, nuclei heights, nuclei widths, protein localization, and cell count. As a demonstration of its utility, Bio-LIME was used to quantify the 3D location of the Zonula occludens-1 protein contained within tight junctions and its change in 3D position in response to chemical modification of the scaffold with laminin. Additionally, Bio-LIME was used to demonstrate that there is no advantage of sub-100 nm poly lactic-co-glycolic acid nanofibers over 250 nm fibers for epithelial apicobasal polarization. Bio-LIME will be broadly applicable for quantification of proteins in 3D that are grown in many different contexts. PMID:27758134

Quantification of Confocal Images Using LabVIEW for Tissue Engineering Applications.

PubMed

Sfakis, Lauren; Kamaldinov, Tim; Larsen, Melinda; Castracane, James; Khmaladze, Alexander

2016-11-01

Quantifying confocal images to enable location of specific proteins of interest in three-dimensional (3D) is important for many tissue engineering (TE) applications. Quantification of protein localization is essential for evaluation of specific scaffold constructs for cell growth and differentiation for application in TE and tissue regeneration strategies. Although obtaining information regarding protein expression levels is important, the location of proteins within cells grown on scaffolds is often the key to evaluating scaffold efficacy. Functional epithelial cell monolayers must be organized with apicobasal polarity with proteins specifically localized to the apical or basolateral regions of cells in many organs. In this work, a customized program was developed using the LabVIEW platform to quantify protein positions in Z-stacks of confocal images of epithelial cell monolayers. The program's functionality is demonstrated through salivary gland TE, since functional salivary epithelial cells must correctly orient many proteins on the apical and basolateral membranes. Bio-LabVIEW Image Matrix Evaluation (Bio-LIME) takes 3D information collected from confocal Z-stack images and processes the fluorescence at each pixel to determine cell heights, nuclei heights, nuclei widths, protein localization, and cell count. As a demonstration of its utility, Bio-LIME was used to quantify the 3D location of the Zonula occludens-1 protein contained within tight junctions and its change in 3D position in response to chemical modification of the scaffold with laminin. Additionally, Bio-LIME was used to demonstrate that there is no advantage of sub-100 nm poly lactic-co-glycolic acid nanofibers over 250 nm fibers for epithelial apicobasal polarization. Bio-LIME will be broadly applicable for quantification of proteins in 3D that are grown in many different contexts.

Colorimetric Quantification and in Situ Detection of Collagen

ERIC Educational Resources Information Center

Esteban, Francisco J.; del Moral, Maria L.; Sanchez-Lopez, Ana M.; Blanco, Santos; Jimenez, Ana; Hernandez, Raquel; Pedrosa, Juan A.; Peinado, Maria A.

2005-01-01

A simple multidisciplinary and inexpensive laboratory exercise is proposed, in which the undergraduate student may correlate biochemical and anatomical findings. The entire practical session can be completed in one 2.5-3 hour laboratory period, and consists of the quantification of collagen and total protein content from tissue sections--without…

In vivo study on the neurotransmitters and their metabolites change in depressive disorder rat plasma by ultra high performance liquid chromatography coupled to tandem mass spectrometry.

PubMed

Zhao, Longshan; Zheng, Shuning; Su, Guangyue; Lu, Xiumei; Yang, Jingyu; Xiong, Zhili; Wu, Chunfu

2015-04-15

A sensitive and versatile, ultra-high performance, liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method coupled to pre-column derivatization for the simultaneous determination of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA), norepinephrine (NE), homovanillic acid (HVA), γ-aminobutyric acid (GABA) and glutamic acid (Glu) was developed and validated in rat plasma. The analytes were dansylated under strong alkaline conditions after protein precipitation extraction, which were analyzed on a BEH C18 column using a gradient elution. The lower limit of quantification (LLOQ) values for 5-HT, 5-HIAA, DA, NE, HVA, GABA and Glu were 1.00, 1.00, 0.991, 0.992, 1.02, 1000, and 5030 pmol/mL, respectively. Good linearity was obtained (r > 0.99) and the intra- and inter-day precisions of the method (relative standard deviation, RSD%) were lower than 12%. The method was novel, sensitive and specific which can provide an alternative method for the quantification of neurotransmitters and their metabolites in plasma samples. Copyright © 2015 Elsevier B.V. All rights reserved.

ICPD-A New Peak Detection Algorithm for LC/MS

PubMed Central

2010-01-01

Background The identification and quantification of proteins using label-free Liquid Chromatography/Mass Spectrometry (LC/MS) play crucial roles in biological and biomedical research. Increasing evidence has shown that biomarkers are often low abundance proteins. However, LC/MS systems are subject to considerable noise and sample variability, whose statistical characteristics are still elusive, making computational identification of low abundance proteins extremely challenging. As a result, the inability of identifying low abundance proteins in a proteomic study is the main bottleneck in protein biomarker discovery. Results In this paper, we propose a new peak detection method called Information Combining Peak Detection (ICPD ) for high resolution LC/MS. In LC/MS, peptides elute during a certain time period and as a result, peptide isotope patterns are registered in multiple MS scans. The key feature of the new algorithm is that the observed isotope patterns registered in multiple scans are combined together for estimating the likelihood of the peptide existence. An isotope pattern matching score based on the likelihood probability is provided and utilized for peak detection. Conclusions The performance of the new algorithm is evaluated based on protein standards with 48 known proteins. The evaluation shows better peak detection accuracy for low abundance proteins than other LC/MS peak detection methods. PMID:21143790

Respiratory Mucosal Proteome Quantification in Human Influenza Infections.

PubMed

Marion, Tony; Elbahesh, Husni; Thomas, Paul G; DeVincenzo, John P; Webby, Richard; Schughart, Klaus

2016-01-01

Respiratory influenza virus infections represent a serious threat to human health. Underlying medical conditions and genetic make-up predispose some influenza patients to more severe forms of disease. To date, only a few studies have been performed in patients to correlate a selected group of cytokines and chemokines with influenza infection. Therefore, we evaluated the potential of a novel multiplex micro-proteomics technology, SOMAscan, to quantify proteins in the respiratory mucosa of influenza A and B infected individuals. The analysis included but was not limited to quantification of cytokines and chemokines detected in previous studies. SOMAscan quantified more than 1,000 secreted proteins in small nasal wash volumes from infected and healthy individuals. Our results illustrate the utility of micro-proteomic technology for analysis of proteins in small volumes of respiratory mucosal samples. Furthermore, when we compared nasal wash samples from influenza-infected patients with viral load ≥ 2(8) and increased IL-6 and CXCL10 to healthy controls, we identified 162 differentially-expressed proteins between the two groups. This number greatly exceeds the number of DEPs identified in previous studies in human influenza patients. Most of the identified proteins were associated with the host immune response to infection, and changes in protein levels of 151 of the DEPs were significantly correlated with viral load. Most important, SOMAscan identified differentially expressed proteins heretofore not associated with respiratory influenza infection in humans. Our study is the first report for the use of SOMAscan to screen nasal secretions. It establishes a precedent for micro-proteomic quantification of proteins that reflect ongoing response to respiratory infection.

Respiratory Mucosal Proteome Quantification in Human Influenza Infections

PubMed Central

Marion, Tony; Elbahesh, Husni; Thomas, Paul G.; DeVincenzo, John P.; Webby, Richard; Schughart, Klaus

2016-01-01

Respiratory influenza virus infections represent a serious threat to human health. Underlying medical conditions and genetic make-up predispose some influenza patients to more severe forms of disease. To date, only a few studies have been performed in patients to correlate a selected group of cytokines and chemokines with influenza infection. Therefore, we evaluated the potential of a novel multiplex micro-proteomics technology, SOMAscan, to quantify proteins in the respiratory mucosa of influenza A and B infected individuals. The analysis included but was not limited to quantification of cytokines and chemokines detected in previous studies. SOMAscan quantified more than 1,000 secreted proteins in small nasal wash volumes from infected and healthy individuals. Our results illustrate the utility of micro-proteomic technology for analysis of proteins in small volumes of respiratory mucosal samples. Furthermore, when we compared nasal wash samples from influenza-infected patients with viral load ≥ 28 and increased IL-6 and CXCL10 to healthy controls, we identified 162 differentially-expressed proteins between the two groups. This number greatly exceeds the number of DEPs identified in previous studies in human influenza patients. Most of the identified proteins were associated with the host immune response to infection, and changes in protein levels of 151 of the DEPs were significantly correlated with viral load. Most important, SOMAscan identified differentially expressed proteins heretofore not associated with respiratory influenza infection in humans. Our study is the first report for the use of SOMAscan to screen nasal secretions. It establishes a precedent for micro-proteomic quantification of proteins that reflect ongoing response to respiratory infection. PMID:27088501

Colonization State Influences the Hemocyte Proteome in a Beneficial Squid–Vibrio Symbiosis*

PubMed Central

Schleicher, Tyler R.; VerBerkmoes, Nathan C.; Shah, Manesh; Nyholm, Spencer V.

2014-01-01

The squid Euprymna scolopes and the luminescent bacterium Vibrio fischeri form a highly specific beneficial light organ symbiosis. Not only does the host have to select V. fischeri from the environment, but it must also prevent subsequent colonization by non-symbiotic microorganisms. Host macrophage-like hemocytes are believed to play a role in mediating the symbiosis with V. fischeri. Previous studies have shown that the colonization state of the light organ influences the host's hemocyte response to the symbiont. To further understand the molecular mechanisms behind this process, we used two quantitative mass-spectrometry-based proteomic techniques, isobaric tags for relative and absolute quantification (iTRAQ) and label-free spectral counting, to compare and quantify the adult hemocyte proteomes from colonized (sym) and uncolonized (antibiotic-treated/cured) squid. Overall, iTRAQ allowed for the quantification of 1,024 proteins with two or more peptides. Thirty-seven unique proteins were determined to be significantly different between sym and cured hemocytes (p value < 0.05), with 20 more abundant proteins and 17 less abundant in sym hemocytes. The label-free approach resulted in 1,241 proteins that were identified in all replicates. Of 185 unique proteins present at significantly different amounts in sym hemocytes (as determined by spectral counting), 92 were more abundant and 93 were less abundant. Comparisons between iTRAQ and spectral counting revealed that 30 of the 37 proteins quantified via iTRAQ exhibited trends similar to those identified by the label-free method. Both proteomic techniques mutually identified 16 proteins that were significantly different between the two groups of hemocytes (p value < 0.05). The presence of V. fischeri in the host light organ influenced the abundance of proteins associated with the cytoskeleton, adhesion, lysosomes, proteolysis, and the innate immune response. These data provide evidence that colonization by V. fischeri alters the hemocyte proteome and reveals proteins that may be important for maintaining host–symbiont specificity. PMID:25038065

Automatic computational labeling of glomerular textural boundaries

NASA Astrophysics Data System (ADS)

Ginley, Brandon; Tomaszewski, John E.; Sarder, Pinaki

2017-03-01

The glomerulus, a specialized bundle of capillaries, is the blood filtering unit of the kidney. Each human kidney contains about 1 million glomeruli. Structural damages in the glomerular micro-compartments give rise to several renal conditions; most severe of which is proteinuria, where excessive blood proteins flow freely to the urine. The sole way to confirm glomerular structural damage in renal pathology is by examining histopathological or immunofluorescence stained needle biopsies under a light microscope. However, this method is extremely tedious and time consuming, and requires manual scoring on the number and volume of structures. Computational quantification of equivalent features promises to greatly ease this manual burden. The largest obstacle to computational quantification of renal tissue is the ability to recognize complex glomerular textural boundaries automatically. Here we present a computational pipeline to accurately identify glomerular boundaries with high precision and accuracy. The computational pipeline employs an integrated approach composed of Gabor filtering, Gaussian blurring, statistical F-testing, and distance transform, and performs significantly better than standard Gabor based textural segmentation method. Our integrated approach provides mean accuracy/precision of 0.89/0.97 on n = 200Hematoxylin and Eosin (HE) glomerulus images, and mean 0.88/0.94 accuracy/precision on n = 200 Periodic Acid Schiff (PAS) glomerulus images. Respective accuracy/precision of the Gabor filter bank based method is 0.83/0.84 for HE and 0.78/0.8 for PAS. Our method will simplify computational partitioning of glomerular micro-compartments hidden within dense textural boundaries. Automatic quantification of glomeruli will streamline structural analysis in clinic, and can help realize real time diagnoses and interventions.

Serum apolipoprotein A-1 quantification by LC–MS with a SILAC internal standard reveals reduced levels in smokers

PubMed Central

Wang, Qingqing; Zhang, Suhong; Guo, Lili; Busch, Christine M; Jian, Wenying; Weng, Naidong; Snyder, Nathaniel W; Rangiah, Kannan; Mesaros, Clementina; Blair, Ian A

2015-01-01

Background: Absolute quantification of protein biomarkers such as serum apolipoprotein A1 by both immunoassays and LC–MS can provide misleading results. Results: Recombinant ApoA-1 internal standard was prepared using stable isotope labeling by amino acids in cell culture with [13C615N2]-lysine and [13C915N1]-tyrosine in human cells. A stable isotope dilution LC–MS method for serum ApoA-1 was validated and levels analyzed for 50 nonsmokers and 50 smokers. Conclusion: The concentration of ApoA-1 in nonsmokers was 169.4 mg/dl with an 18.4% reduction to 138.2 mg/dl in smokers. The validated assay will have clinical utility for assessing effects of smoking cessation and therapeutic or dietary interventions in high-risk populations. PMID:26394123

Spatial Normalization of Reverse Phase Protein Array Data

PubMed Central

Kaushik, Poorvi; Molinelli, Evan J.; Miller, Martin L.; Wang, Weiqing; Korkut, Anil; Liu, Wenbin; Ju, Zhenlin; Lu, Yiling; Mills, Gordon; Sander, Chris

2014-01-01

Reverse phase protein arrays (RPPA) are an efficient, high-throughput, cost-effective method for the quantification of specific proteins in complex biological samples. The quality of RPPA data may be affected by various sources of error. One of these, spatial variation, is caused by uneven exposure of different parts of an RPPA slide to the reagents used in protein detection. We present a method for the determination and correction of systematic spatial variation in RPPA slides using positive control spots printed on each slide. The method uses a simple bi-linear interpolation technique to obtain a surface representing the spatial variation occurring across the dimensions of a slide. This surface is used to calculate correction factors that can normalize the relative protein concentrations of the samples on each slide. The adoption of the method results in increased agreement between technical and biological replicates of various tumor and cell-line derived samples. Further, in data from a study of the melanoma cell-line SKMEL-133, several slides that had previously been rejected because they had a coefficient of variation (CV) greater than 15%, are rescued by reduction of CV below this threshold in each case. The method is implemented in the R statistical programing language. It is compatible with MicroVigene and SuperCurve, packages commonly used in RPPA data analysis. The method is made available, along with suggestions for implementation, at http://bitbucket.org/rppa_preprocess/rppa_preprocess/src. PMID:25501559

A Quantitative Microscopy Technique for Determining the Number of Specific Proteins in Cellular Compartments

PubMed Central

Mutch, Sarah A.; Gadd, Jennifer C.; Fujimoto, Bryant S.; Kensel-Hammes, Patricia; Schiro, Perry G.; Bajjalieh, Sandra M.; Chiu, Daniel T.

2013-01-01

This protocol describes a method to determine both the average number and variance of proteins in the few to tens of copies in isolated cellular compartments, such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number but lack information on the variance or are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling the cellular compartment with fluorescent primary-secondary antibody complexes, TIRF (total internal reflection fluorescence) microscopy imaging of the cellular compartment, digital image analysis, and deconvolution of the fluorescence intensity data. A minimum of 2.5 days is required to complete the labeling, imaging, and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes. PMID:22094731

Analysis of Carbohydrate and Fatty Acid Marker Abundance in Ricin Toxin Preparations for Forensic Information

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colburn, Heather A.; Wunschel, David S.; Kreuzer-Martin, Helen W.

2010-07-15

One challenge in the forensic analysis of ricin samples is determining the method and extent of sample preparation. Ricin purification from the source castor seeds is essentially a protein purification through removal of the non-protein fractions of the seed. Two major, non-protein constituents in the seed are the castor oil and carbohydrates. Ricinoleic acid is a relatively unique fatty acid in nature and is the most abundant component of castor oil, which comprises roughly half the seed weight. The carbohydrate component comprises roughly half of the remaining “mash” left after oil and hull removal. We used derivatization of carbohydrate andmore » fatty acid markers followed by identification and quantification using gas chromatography/mass spectrometry (GC/MS) to assess compositional changes in ricin samples purified by different methods. The loss of ricinoleic acid indicated steps for oil removal had occurred. Changes to the carbohydrate content of the sample were also observed following protein precipitation. The differential loss of arabinose relative to mannose indicated removal of the major carbohydrate fraction of the seed and enrichment of the protein content. Taken together, these changes in fatty acid and carbohydrate abundance are indicative of the preparation method used for each sample.« less

A new approach for rapid detection and typing of serum monoclonal components.

PubMed

Cacoub, P; Camproux, A C; Thiolières, J M; Assogba, U; Hausfater, P; Mallet, A; Foglietti, M J; Piette, J C; Bernard, M

2000-12-01

When used independently, none of the routine methods to explore serum monoclonal components (MC), including: serum protein electrophoresis (SPE), immunoelectrophoresis (IEP), kappa to lambda ratio (KLR) and immunofixation (IFE), provides a comprehensive quantitative and qualitative identification of the MC. In the past few years the concept of 'protein profile', based on immunonephelometric quantifications of serum proteins, has become widely used. It consists of a qualitative and quantitative graphic representation of numerous serum proteins including immunoglobulins. Aim of study was to develop a multidimensional model based exclusively on protein profiles labeled the protein profile prediction method (PPPM) to improve routine MC detection and typing. Serum samples from 127 hospitalized patients and 99 healthy blood donors were submitted to all of the following: SPE, IFE, KLR and a protein profile (which included IgM, IgA, IgG, kappa and lambda chain detections and quantification). The presence of a MC using IFE was chosen as the gold standard. Healthy donors and patients were randomly divided into two groups defined as testing and validation groups. A logistic model was designed based on the protein profiles of the testing group leading to the determination of a threshold value (called Z(r)) for MC detection. It was then tested to detect MC in the validation group. Using IFE, 73 MC were found in the 127 hospitalized patients. Using the threshold value for MC detection of Z(r)=1.86, the PPPM showed greater sensitivity (94.6%) in detecting a MC compared to either SPE (64.8%) or KLR (89.2%). This result was obtained without diminished specificity (80.8%). The association of SPE or KLR to PPPM did not significantly increase the sensitivity of the PPPM. In the validation group, for samples which had a high predictive probability of a MC using PPPM, the correct MC typing was identified in up to 77% of sera using PPPM only. These results may be interesting in helping to determine when supplementary IFE analysis is required to qualitatively analyze a MC. PPPM allows MC detection with great sensitivity. The immune protein profile dramatically increases the sensitivity of either SPE and/or KLR in detecting MC and may also allow heavy and light chain typing.

Quantification of Acetaminophen and Its Metabolites in Plasma Using UPLC-MS: Doors Open to Therapeutic Drug Monitoring in Special Patient Populations.

PubMed

Flint, Robert B; Mian, Paola; van der Nagel, Bart; Slijkhuis, Nuria; Koch, Birgit C P

2017-04-01

Acetaminophen (APAP, paracetamol) is the most commonly used drug for pain and fever in both the United States and Europe and is considered safe when used at registered dosages. Nevertheless, differences between specific populations lead to remarkable changes in exposure to potentially toxic metabolites. Furthermore, extended knowledge is required on metabolite formation after intoxication, to optimize antidote treatment. Therefore, the authors aimed to develop and validate a quick and easy analytical method for simultaneous quantification of APAP, APAP-glucuronide, APAP-sulfate, APAP-cysteine, APAP-glutathione, APAP-mercapturate, and protein-derived APAP-cysteine in human plasma by ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry. The internal standard was APAP-D4 for all analytes. Chromatographic separation was achieved with a reversed-phase Acquity ultraperformance liquid chromatography HSS T3 column with a runtime of only 4.5 minutes per injected sample. Gradient elution was performed with a mobile phase consisting of ammonium acetate, formic acid in Milli-Q ultrapure water or in methanol at flow rate of 0.4 mL/minute. A plasma volume of only 10 μL was required to achieve both adequate accuracy and precision. Calibration curves of all 6 analytes were linear. All analytes were stable for at least 48 hours in the autosampler; the high quality control of APAP-glutathione was stable for 24 hours. The method was validated according to the U.S. Food and Drug Administration guidelines. This method allows quantification of APAP and 6 metabolites, which serves purposes for research, as well as therapeutic drug monitoring. The advantage of this method is the combination of minimal injection volume, a short runtime, an easy sample preparation method, and the ability to quantify APAP and all 6 metabolites.

On-line sample cleanup and enrichment chromatographic technique for the determination of ambroxol in human serum.

PubMed

Emara, Samy; Kamal, Maha; Abdel Kawi, Mohamed

2012-02-01

A sensitive and efficient on-line clean up and pre-concentration method has been developed using column-switching technique and protein-coated µ-Bondapak CN silica pre-column for quantification of ambroxol (AM) in human serum. The method is performed by direct injection of serum sample onto a protein-coated µ-Bondapak CN silica pre-column, where AM is pre-concentrated and retained, while proteins and very polar constituents are washed to waste using a phosphate buffer saline (pH 7.4). The retained analyte on the pre-column is directed onto a C(18) analytical column for separation, with a mobile phase consisting of a mixture of methanol and distilled deionized water (containing 1% triethylamine adjusted to pH 3.5 with ortho-phosphoric acid) in the ratio of 50:50 (v/v). Detection is performed at 254 nm. The calibration curve is linear over the concentration range of 12-120 ng/mL (r(2) = 0.9995). The recovery, selectivity, linearity, precision, and accuracy of the method are convenient for pharmacokinetic studies or routine assays.

Universal absolute quantification of biomolecules using element mass spectrometry and generic standards.

PubMed

Calderón-Celis, Francisco; Sanz-Medel, Alfredo; Encinar, Jorge Ruiz

2018-01-23

We present a novel and highly sensitive ICP-MS approach for absolute quantification of all important target biomolecule containing P, S, Se, As, Br, and/or I (e.g., proteins and phosphoproteins, metabolites, pesticides, drugs), under the same simple instrumental conditions and without requiring any specific and/or isotopically enriched standard.

Independent Research Projects Using Protein Extraction: Affordable Ways to Inquire, Discover & Publish for Undergraduate Students

ERIC Educational Resources Information Center

Pu, Rongsun

2010-01-01

This article describes how to use protein extraction, quantification, and analysis in the undergraduate teaching laboratory to engage students in inquiry-based, discovery-driven learning. Detailed instructions for obtaining proteins from animal tissues, using BCA assay to quantify the proteins, and data analysis are provided. The experimental…

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

PubMed Central

Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

2014-01-01

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

PubMed

Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

2016-01-29

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

Microplate Bioassay for Nisin in Foods, Based on Nisin-Induced Green Fluorescent Protein Fluorescence

PubMed Central

Reunanen, J.; Saris, P. E. J.

2003-01-01

A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 μg of nisin in cheese, and 1 μg of nisin per ml in salad dressings. PMID:12839802

A comparative proteomics method for multiple samples based on a 18O-reference strategy and a quantitation and identification-decoupled strategy.

PubMed

Wang, Hongbin; Zhang, Yongqian; Gui, Shuqi; Zhang, Yong; Lu, Fuping; Deng, Yulin

2017-08-15

Comparisons across large numbers of samples are frequently necessary in quantitative proteomics. Many quantitative methods used in proteomics are based on stable isotope labeling, but most of these are only useful for comparing two samples. For up to eight samples, the iTRAQ labeling technique can be used. For greater numbers of samples, the label-free method has been used, but this method was criticized for low reproducibility and accuracy. An ingenious strategy has been introduced, comparing each sample against a 18 O-labeled reference sample that was created by pooling equal amounts of all samples. However, it is necessary to use proportion-known protein mixtures to investigate and evaluate this new strategy. Another problem for comparative proteomics of multiple samples is the poor coincidence and reproducibility in protein identification results across samples. In present study, a method combining 18 O-reference strategy and a quantitation and identification-decoupled strategy was investigated with proportion-known protein mixtures. The results obviously demonstrated that the 18 O-reference strategy had greater accuracy and reliability than other previously used comparison methods based on transferring comparison or label-free strategies. By the decoupling strategy, the quantification data acquired by LC-MS and the identification data acquired by LC-MS/MS are matched and correlated to identify differential expressed proteins, according to retention time and accurate mass. This strategy made protein identification possible for all samples using a single pooled sample, and therefore gave a good reproducibility in protein identification across multiple samples, and allowed for optimizing peptide identification separately so as to identify more proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous quantification of Myelin Basic Protein and Tau proteins in cerebrospinal fluid and serum of Multiple Sclerosis patients using nanoimmunosensor.

PubMed

Derkus, Burak; Acar Bozkurt, Pinar; Tulu, Metin; Emregul, Kaan C; Yucesan, Canan; Emregul, Emel

2017-03-15

This study was aimed at the development of an immunosensor for the simultaneous quantification of Myelin Basic Protein (MBP) and Tau proteins in cerebrospinal fluid (CSF) and serum, obtained from Multiple Sclerosis (MS) patients. The newly developed GO/pPG/anti-MBP/anti-Tau nanoimmunosensor has been established by immobilization of MBP and Tau antibodies. The newly developed nanoimmunosensor was tested, optimized and characterized using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The developed nanoimmunosensor was seen to have detection limits of 0.30nM for MBP and 0.15nM for Tau proteins which were sufficient for the levels to be analysed in neuro-clinic. The clinical study performed using CSF and serum of MS patients showed that the designed nanoimmunosensor was capable of detecting the proteins properly, that were essentially proven by ELISA. Copyright © 2016 Elsevier B.V. All rights reserved.

Absolute quantification of protein NP24 in tomato fruit by liquid chromatography/tandem mass spectrometry using stable isotope-labelled tryptic peptide standard.

PubMed

Ippoushi, Katsunari; Sasanuma, Motoe; Oike, Hideaki; Kobori, Masuko; Maeda-Yamamoto, Mari

2015-04-15

Protein NP24 is a thaumatin-like protein contained in tomato (Lycopersicon esculentum Mill.). This protein is reported to be a putative tomato allergen and is listed as a food allergen in Structural Database of Allergenic Proteins (SDAP). In this research, we developed the quantitative analysis of NP24 by employing the protein absolute quantification (AQUA) technology composed of stable isotope-labelled internal standard (SIIS) peptide (GQTWVINAPR[(13)C6,(15)N4]) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). A linear relationship (r(2)>0.99) was found throughout the concentration range (2.0-500 fmol/μL). The coefficients of variation (CVs) measured on each of the five days when NP24 contained in the tomato skin was analysed did not exceed 13%. Our developed assay of NP24 will contribute to the allergological examination of tomato and its derived products. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation of the reliability of maize reference assays for GMO quantification.

PubMed

Papazova, Nina; Zhang, David; Gruden, Kristina; Vojvoda, Jana; Yang, Litao; Buh Gasparic, Meti; Blejec, Andrej; Fouilloux, Stephane; De Loose, Marc; Taverniers, Isabel

2010-03-01

A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize.

Application of fluorescence resonance energy transfer in protein studies

PubMed Central

Ma, Linlin; Yang, Fan; Zheng, Jie

2014-01-01

Since the physical process of fluorescence resonance energy transfer (FRET) was elucidated more than six decades ago, this peculiar fluorescence phenomenon has turned into a powerful tool for biomedical research due to its compatibility in scale with biological molecules as well as rapid developments in novel fluorophores and optical detection techniques. A wide variety of FRET approaches have been devised, each with its own advantages and drawbacks. Especially in the last decade or so, we are witnessing a flourish of FRET applications in biological investigations, many of which exemplify clever experimental design and rigorous analysis. Here we review the current stage of FRET methods development with the main focus on its applications in protein studies in biological systems, by summarizing the basic components of FRET techniques, most established quantification methods, as well as potential pitfalls, illustrated by example applications. PMID:25368432

Assessment of partial coalescence in whippable oil-in-water food emulsions.

PubMed

Petrut, Raul Flaviu; Danthine, Sabine; Blecker, Christophe

2016-03-01

Partial coalescence influences to a great extent the properties of final food products such as ice cream and whipped toppings. In return, the partial coalescence occurrence and development are conditioned, in such systems, by the emulsion's intrinsic properties (e.g. solid fat content, fat crystal shape and size), formulation (e.g. protein content, surfactants presence) and extrinsic factors (e.g. cooling rate, shearing). A set of methods is available for partial coalescence investigation and quantification. These methods are critically reviewed in this paper, balancing the weaknesses of the methods in terms of structure alteration (for turbidity, dye dilution, etc.) and assumptions made for mathematical models (for particle size determination) with their advantages (good repeatability, high sensitivity, etc.). With the methods proposed in literature, the partial coalescence investigations can be conducted quantitatively and/or qualitatively. Good correlation were observed between some of the quantitative methods such as dye dilution, calorimetry, fat particle size; while a poor correlation was found in the case of solvent extraction method with other quantitative methods. The most suitable way for partial coalescence quantification was implied to be the fat particle size method, which would give results with a high degree of confidence if used in combination with a microscopic technique for the confirmation of partial coalescence as the main destabilization mechanism. Copyright © 2015 Elsevier B.V. All rights reserved.

An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies using Isobaric Tags

PubMed Central

Cologna, Stephanie M.; Crutchfield, Christopher A.; Searle, Brian C.; Blank, Paul S.; Toth, Cynthia L.; Ely, Alexa M.; Picache, Jaqueline A.; Backlund, Peter S.; Wassif, Christopher A.; Porter, Forbes D.; Yergey, Alfred L.

2017-01-01

Protein quantification, identification and abundance determination are important aspects of proteome characterization and are crucial in understanding biological mechanisms and human diseases. Different strategies are available to quantify proteins using mass spectrometric detection, and most are performed at the peptide level and include both targeted and un-targeted methodologies. Discovery-based or un-targeted approaches oftentimes use covalent tagging strategies (i.e., iTRAQ®, TMT™) where reporter ion signals collected in the tandem MS experiment are used for quantification. Herein we investigate the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The experimental design and data analysis approach described is simple and straightforward, which allows researchers to optimize data collection and proper analysis within a laboratory. iTRAQ reporter ion signals were normalized within each spectrum to remove peptide biases. An advantage of this approach is that missing reporter ion values can be accepted for purposes of protein identification and quantification with the need for ANOVA analysis. We investigate the distribution of reporter ion peak areas in an equimolar system and a mock biological system and provide recommendations for establishing fold-change cutoff values at the peptide level for iTRAQ datasets. These data provide a unique dataset available to the community for informatics training and analysis. PMID:26288259

Automated Quantification and Integrative Analysis of 2D and 3D Mitochondrial Shape and Network Properties

PubMed Central

Nikolaisen, Julie; Nilsson, Linn I. H.; Pettersen, Ina K. N.; Willems, Peter H. G. M.; Lorens, James B.; Koopman, Werner J. H.; Tronstad, Karl J.

2014-01-01

Mitochondrial morphology and function are coupled in healthy cells, during pathological conditions and (adaptation to) endogenous and exogenous stress. In this sense mitochondrial shape can range from small globular compartments to complex filamentous networks, even within the same cell. Understanding how mitochondrial morphological changes (i.e. “mitochondrial dynamics”) are linked to cellular (patho) physiology is currently the subject of intense study and requires detailed quantitative information. During the last decade, various computational approaches have been developed for automated 2-dimensional (2D) analysis of mitochondrial morphology and number in microscopy images. Although these strategies are well suited for analysis of adhering cells with a flat morphology they are not applicable for thicker cells, which require a three-dimensional (3D) image acquisition and analysis procedure. Here we developed and validated an automated image analysis algorithm allowing simultaneous 3D quantification of mitochondrial morphology and network properties in human endothelial cells (HUVECs). Cells expressing a mitochondria-targeted green fluorescence protein (mitoGFP) were visualized by 3D confocal microscopy and mitochondrial morphology was quantified using both the established 2D method and the new 3D strategy. We demonstrate that both analyses can be used to characterize and discriminate between various mitochondrial morphologies and network properties. However, the results from 2D and 3D analysis were not equivalent when filamentous mitochondria in normal HUVECs were compared with circular/spherical mitochondria in metabolically stressed HUVECs treated with rotenone (ROT). 2D quantification suggested that metabolic stress induced mitochondrial fragmentation and loss of biomass. In contrast, 3D analysis revealed that the mitochondrial network structure was dissolved without affecting the amount and size of the organelles. Thus, our results demonstrate that 3D imaging and quantification are crucial for proper understanding of mitochondrial shape and topology in non-flat cells. In summary, we here present an integrative method for unbiased 3D quantification of mitochondrial shape and network properties in mammalian cells. PMID:24988307

Reproducible Tissue Homogenization and Protein Extraction for Quantitative Proteomics Using MicroPestle-Assisted Pressure-Cycling Technology.

PubMed

Shao, Shiying; Guo, Tiannan; Gross, Vera; Lazarev, Alexander; Koh, Ching Chiek; Gillessen, Silke; Joerger, Markus; Jochum, Wolfram; Aebersold, Ruedi

2016-06-03

The reproducible and efficient extraction of proteins from biopsy samples for quantitative analysis is a critical step in biomarker and translational research. Recently, we described a method consisting of pressure-cycling technology (PCT) and sequential windowed acquisition of all theoretical fragment ions-mass spectrometry (SWATH-MS) for the rapid quantification of thousands of proteins from biopsy-size tissue samples. As an improvement of the method, we have incorporated the PCT-MicroPestle into the PCT-SWATH workflow. The PCT-MicroPestle is a novel, miniaturized, disposable mechanical tissue homogenizer that fits directly into the microTube sample container. We optimized the pressure-cycling conditions for tissue lysis with the PCT-MicroPestle and benchmarked the performance of the system against the conventional PCT-MicroCap method using mouse liver, heart, brain, and human kidney tissues as test samples. The data indicate that the digestion of the PCT-MicroPestle-extracted proteins yielded 20-40% more MS-ready peptide mass from all tissues tested with a comparable reproducibility when compared to the conventional PCT method. Subsequent SWATH-MS analysis identified a higher number of biologically informative proteins from a given sample. In conclusion, we have developed a new device that can be seamlessly integrated into the PCT-SWATH workflow, leading to increased sample throughput and improved reproducibility at both the protein extraction and proteomic analysis levels when applied to the quantitative proteomic analysis of biopsy-level samples.

Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhou; Adams, Rachel M; Chourey, Karuna

2012-01-01

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaricmore » chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.« less

Quantification of Ligand Binding to G-Protein Coupled Receptors on Cell Membranes by Ellipsometry

PubMed Central

Kriechbaumer, Verena; Nabok, Alexei; Widdowson, Robert; Smith, David P.; Abell, Ben M.

2012-01-01

G-protein-coupled receptors (GPCRs) are prime drug targets and targeted by approximately 60% of current therapeutic drugs such as β-blockers, antipsychotics and analgesics. However, no biophysical methods are available to quantify their interactions with ligand binding in a native environment. Here, we use ellipsometry to quantify specific interactions of receptors within native cell membranes. As a model system, the GPCR-ligand CXCL12α and its receptor CXCR4 are used. Human-derived Ishikawa cells were deposited onto gold coated slides via Langmuir-Schaefer film deposition and interactions between the receptor CXCR4 on these cells and its ligand CXCL12α were detected via total internal reflection ellipsometry (TIRE). This interaction could be inhibited by application of the CXCR4-binding drug AMD3100. Advantages of this approach are that it allows measurement of interactions in a lipid environment without the need for labelling, protein purification or reconstitution of membrane proteins. This technique is potentially applicable to a wide variety of cell types and their membrane receptors, providing a novel method to determine ligand or drug interactions targeting GPCRs and other membrane proteins. PMID:23049983

Large-scale isotype-specific quantification of Serum amyloid A 1/2 by multiple reaction monitoring in crude sera.

PubMed

Sung, Hye-Jin; Jeon, Seon-Ae; Ahn, Jung-Mo; Seul, Kyung-Jo; Kim, Jin Young; Lee, Ju Yeon; Yoo, Jong Shin; Lee, Soo-Youn; Kim, Hojoong; Cho, Je-Yoel

2012-04-03

Quantification is an essential step in biomarker development. Multiple reaction monitoring (MRM) is a new modified mass spectrometry-based quantification technology that does not require antibody development. Serum amyloid A (SAA) is a positive acute-phase protein identified as a lung cancer biomarker in our previous study. Acute SAA exists in two isoforms with highly similar (92%) amino acid sequences. Until now, studies of SAA have been unable to distinguish between SAA1 and SAA2. To overcome the unavailability of a SAA2-specific antibody, we developed MRM methodology for the verification of SAA1 and SAA2 in clinical crude serum samples from 99 healthy controls and 100 lung adenocarcinoma patients. Differential measurement of SAA1 and SAA2 was made possible for the first time with the developed isotype-specific MRM method. Most healthy control samples had small or no MS/MS peaks of the targeted peptides otherwise, higher peak areas with 10- to 34-fold increase over controls were detected in lung cancer samples. In addition, our SAA1 MRM data demonstrated good agreement with the SAA1 enzyme-linked immunosorbent assay (ELISA) data. Finally, successful quantification of SAA2 in crude serum by MRM, for the first time, shows that SAA2 can be a good biomarker for the detection of lung cancers. Copyright © 2012 Elsevier B.V. All rights reserved.

Subnuclear foci quantification using high-throughput 3D image cytometry

NASA Astrophysics Data System (ADS)

Wadduwage, Dushan N.; Parrish, Marcus; Choi, Heejin; Engelward, Bevin P.; Matsudaira, Paul; So, Peter T. C.

2015-07-01

Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell (5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.

A novel strategy using MASCOT Distiller for analysis of cleavable isotope-coded affinity tag data to quantify protein changes in plasma.

PubMed

Leung, Kit-Yi; Lescuyer, Pierre; Campbell, James; Byers, Helen L; Allard, Laure; Sanchez, Jean-Charles; Ward, Malcolm A

2005-08-01

A novel strategy consisting of cleavable Isotope-Coded Affinity Tag (cICAT) combined with MASCOT Distiller was evaluated as a tool for the quantification of proteins in "abnormal" patient plasma, prepared by pooling samples from patients with acute stroke. Quantification of all light and heavy cICAT-labelled peptide ion pairs was obtained using MASCOT Distiller combined with a proprietary software. Peptides displaying differences were selected for identification by MS. These preliminary results show the promise of our approach to identify potential biomarkers.

Fluorescent detection of C-reactive protein using polyamide beads

NASA Astrophysics Data System (ADS)

Jagadeesh, Shreesha; Chen, Lu; Aitchison, Stewart

2016-03-01

Bacterial infection causes Sepsis which is one of the leading cause of mortality in hospitals. This infection can be quantified from blood plasma using C - reactive protein (CRP). A quick diagnosis at the patient's location through Point-of- Care (POC) testing could give doctors the confidence to prescribe antibiotics. In this paper, the development and testing of a bead-based procedure for CRP quantification is described. The size of the beads enable them to be trapped in wells without the need for magnetic methods of immobilization. Large (1.5 mm diameter) Polyamide nylon beads were used as the substrate for capturing CRP from pure analyte samples. The beads captured CRP either directly through adsorption or indirectly by having specific capture antibodies on their surface. Both methods used fluorescent imaging techniques to quantify the protein. The amount of CRP needed to give a sufficient fluorescent signal through direct capture method was found suitable for identifying bacterial causes of infection. Similarly, viral infections could be quantified by the more sensitive indirect capture method. This bead-based assay can be potentially integrated as a disposable cartridge in a POC device due to its passive nature and the small quantities needed.

Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

NASA Astrophysics Data System (ADS)

Wang, Evelyn H.; Combe, Peter C.; Schug, Kevin A.

2016-05-01

Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 μg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

Matrix Assisted Laser Desorption Ionization Mass Spectrometric Analysis of Bacillus anthracis: From Fingerprint Analysis of the Bacterium to Quantification of its Toxins in Clinical Samples

NASA Astrophysics Data System (ADS)

Woolfitt, Adrian R.; Boyer, Anne E.; Quinn, Conrad P.; Hoffmaster, Alex R.; Kozel, Thomas R.; de, Barun K.; Gallegos, Maribel; Moura, Hercules; Pirkle, James L.; Barr, John R.

A range of mass spectrometry-based techniques have been used to identify, characterize and differentiate Bacillus anthracis, both in culture for forensic applications and for diagnosis during infection. This range of techniques could usefully be considered to exist as a continuum, based on the degrees of specificity involved. We show two examples here, a whole-organism fingerprinting method and a high-specificity assay for one unique protein, anthrax lethal factor.

Immunoturbidimetric quantification of serum immunoglobulin G concentration in foals.

PubMed

Bauer, J E; Brooks, T P

1990-08-01

Immunoturbidimetric determination of serum IgG concentration in foals was compared with the reference methods of single radial immunodiffusion and serum protein electrophoresis. High positive correlations were discovered when the technique was compared with either of these reference methods. The zinc sulfate turbidity test for serum IgG estimation was also evaluated. Although a positive correlation was discovered when the latter method was compared with reference methods, it was not as strong as the correlation between reference methods and the immunoturbidimetric method. The immunoturbidimetric method used in this study is specific and precise for equine serum IgG determination. It is rapid and, thus, is advantageous when timely evaluation of critically ill foals is necessary. The technique should be adaptable to various spectrophotometers and microcomputers for widespread application in veterinary medicine.

A validated Fourier transform infrared spectroscopy method for quantification of total lactones in Inula racemosa and Andrographis paniculata.

PubMed

Shivali, Garg; Praful, Lahorkar; Vijay, Gadgil

2012-01-01

Fourier transform infrared (FT-IR) spectroscopy is a technique widely used for detection and quantification of various chemical moieties. This paper describes the use of the FT-IR spectroscopy technique for the quantification of total lactones present in Inula racemosa and Andrographis paniculata. To validate the FT-IR spectroscopy method for quantification of total lactones in I. racemosa and A. paniculata. Dried and powdered I. racemosa roots and A. paniculata plant were extracted with ethanol and dried to remove ethanol completely. The ethanol extract was analysed in a KBr pellet by FT-IR spectroscopy. The FT-IR spectroscopy method was validated and compared with a known spectrophotometric method for quantification of lactones in A. paniculata. By FT-IR spectroscopy, the amount of total lactones was found to be 2.12 ± 0.47% (n = 3) in I. racemosa and 8.65 ± 0.51% (n = 3) in A. paniculata. The method showed comparable results with a known spectrophotometric method used for quantification of such lactones: 8.42 ± 0.36% (n = 3) in A. paniculata. Limits of detection and quantification for isoallantolactone were 1 µg and 10 µg respectively; for andrographolide they were 1.5 µg and 15 µg respectively. Recoveries were over 98%, with good intra- and interday repeatability: RSD ≤ 2%. The FT-IR spectroscopy method proved linear, accurate, precise and specific, with low limits of detection and quantification, for estimation of total lactones, and is less tedious than the UV spectrophotometric method for the compounds tested. This validated FT-IR spectroscopy method is readily applicable for the quality control of I. racemosa and A. paniculata. Copyright © 2011 John Wiley & Sons, Ltd.

Crowdsourcing scoring of immunohistochemistry images: Evaluating Performance of the Crowd and an Automated Computational Method

NASA Astrophysics Data System (ADS)

Irshad, Humayun; Oh, Eun-Yeong; Schmolze, Daniel; Quintana, Liza M.; Collins, Laura; Tamimi, Rulla M.; Beck, Andrew H.

2017-02-01

The assessment of protein expression in immunohistochemistry (IHC) images provides important diagnostic, prognostic and predictive information for guiding cancer diagnosis and therapy. Manual scoring of IHC images represents a logistical challenge, as the process is labor intensive and time consuming. Since the last decade, computational methods have been developed to enable the application of quantitative methods for the analysis and interpretation of protein expression in IHC images. These methods have not yet replaced manual scoring for the assessment of IHC in the majority of diagnostic laboratories and in many large-scale research studies. An alternative approach is crowdsourcing the quantification of IHC images to an undefined crowd. The aim of this study is to quantify IHC images for labeling of ER status with two different crowdsourcing approaches, image-labeling and nuclei-labeling, and compare their performance with automated methods. Crowdsourcing- derived scores obtained greater concordance with the pathologist interpretations for both image-labeling and nuclei-labeling tasks (83% and 87%), as compared to the pathologist concordance achieved by the automated method (81%) on 5,338 TMA images from 1,853 breast cancer patients. This analysis shows that crowdsourcing the scoring of protein expression in IHC images is a promising new approach for large scale cancer molecular pathology studies.

High throughput, parallel imaging and biomarker quantification of human spermatozoa by ImageStream flow cytometry.

PubMed

Buckman, Clayton; George, Thaddeus C; Friend, Sherree; Sutovsky, Miriam; Miranda-Vizuete, Antonio; Ozanon, Christophe; Morrissey, Phil; Sutovsky, Peter

2009-12-01

Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.

The speciation of the proteome

PubMed Central

Jungblut, Peter R; Holzhütter, Hermann G; Apweiler, Rolf; Schlüter, Hartmut

2008-01-01

Introduction In proteomics a paradox situation developed in the last years. At one side it is basic knowledge that proteins are post-translationally modified and occur in different isoforms. At the other side the protein expression concept disclaims post-translational modifications by connecting protein names directly with function. Discussion Optimal proteome coverage is today reached by bottom-up liquid chromatography/mass spectrometry. But quantification at the peptide level in shotgun or bottom-up approaches by liquid chromatography and mass spectrometry is completely ignoring that a special peptide may exist in an unmodified form and in several-fold modified forms. The acceptance of the protein species concept is a basic prerequisite for meaningful quantitative analyses in functional proteomics. In discovery approaches only top-down analyses, separating the protein species before digestion, identification and quantification by two-dimensional gel electrophoresis or protein liquid chromatography, allow the correlation between changes of a biological situation and function. Conclusion To obtain biological relevant information kinetics and systems biology have to be performed at the protein species level, which is the major challenge in proteomics today. PMID:18638390

Comparison of Quantitative Mass Spectrometry Platforms for Monitoring Kinase ATP Probe Uptake in Lung Cancer.

PubMed

Hoffman, Melissa A; Fang, Bin; Haura, Eric B; Rix, Uwe; Koomen, John M

2018-01-05

Recent developments in instrumentation and bioinformatics have led to new quantitative mass spectrometry platforms including LC-MS/MS with data-independent acquisition (DIA) and targeted analysis using parallel reaction monitoring mass spectrometry (LC-PRM), which provide alternatives to well-established methods, such as LC-MS/MS with data-dependent acquisition (DDA) and targeted analysis using multiple reaction monitoring mass spectrometry (LC-MRM). These tools have been used to identify signaling perturbations in lung cancers and other malignancies, supporting the development of effective kinase inhibitors and, more recently, providing insights into therapeutic resistance mechanisms and drug repurposing opportunities. However, detection of kinases in biological matrices can be challenging; therefore, activity-based protein profiling enrichment of ATP-utilizing proteins was selected as a test case for exploring the limits of detection of low-abundance analytes in complex biological samples. To examine the impact of different MS acquisition platforms, quantification of kinase ATP uptake following kinase inhibitor treatment was analyzed by four different methods: LC-MS/MS with DDA and DIA, LC-MRM, and LC-PRM. For discovery data sets, DIA increased the number of identified kinases by 21% and reduced missingness when compared with DDA. In this context, MRM and PRM were most effective at identifying global kinome responses to inhibitor treatment, highlighting the value of a priori target identification and manual evaluation of quantitative proteomics data sets. We compare results for a selected set of desthiobiotinylated peptides from PRM, MRM, and DIA and identify considerations for selecting a quantification method and postprocessing steps that should be used for each data acquisition strategy.

Autologous Blood Transfusion in Sports: Emerging Biomarkers.

PubMed

Salamin, Olivier; De Angelis, Sara; Tissot, Jean-Daniel; Saugy, Martial; Leuenberger, Nicolas

2016-07-01

Despite being prohibited by the World Anti-Doping Agency, blood doping through erythropoietin injection or blood transfusion is frequently used by athletes to increase oxygen delivery to muscles and enhance performance. In contrast with allogeneic blood transfusion and erythropoietic stimulants, there is presently no direct method of detection for autologous blood transfusion (ABT) doping. Blood reinfusion is currently monitored with individual follow-up of hematological variables via the athlete biological passport, which requires further improvement. Microdosage is undetectable, and suspicious profiles in athletes are often attributed to exposure to altitude, heat stress, or illness. Additional indirect biomarkers may increase the sensitivity and specificity of the longitudinal approach. The emergence of "-omics" strategies provides new opportunities to discover biomarkers for the indirect detection of ABT. With the development of direct quantitative methods, transcriptomics based on microRNA or messenger RNA expression is a promising approach. Because blood donation and blood reinfusion alter iron metabolism, quantification of proteins involved in metal metabolism, such as hepcidin, may be applied in an "ironomics" strategy to improve the detection of ABT. As red blood cell (RBC) storage triggers changes in membrane proteins, proteomic methods have the potential to identify the presence of stored RBCs in blood. Alternatively, urine matrix can be used for the quantification of the plasticizer di(2-ethyhexyl)phthalate and its metabolites that originate from blood storage bags, suggesting recent blood transfusion, and have an important degree of sensitivity and specificity. This review proposes that various indirect biomarkers should be applied in combination with mathematical approaches for longitudinal monitoring aimed at improving ABT detection. Copyright © 2016 Elsevier Inc. All rights reserved.

Rapid identification and quantification of tumor cells using an electrocatalytic method based on gold nanoparticles.

PubMed

de la Escosura-Muñiz, Alfredo; Sánchez-Espinel, Christian; Díaz-Freitas, Belén; González-Fernández, Africa; Maltez-da Costa, Marisa; Merkoçi, Arben

2009-12-15

There is a high demand for simple, rapid, efficient, and user-friendly alternative methods for the detection of cells in general and, in particular, for the detection of cancer cells. A biosensor able to detect cells would be an all-in-one dream device for such applications. The successful integration of nanoparticles into cell detection assays could allow for the development of this novel class of cell sensors. Indeed, their application could well have a great future in diagnostics, as well as other fields. As an example of a novel biosensor, we report here an electrocatalytic device for the specific identification of tumor cells that quantifies gold nanoparticles (AuNPs) coupled with an electrotransducing platform/sensor. Proliferation and adherence of tumor cells are achieved on the electrotransducer/detector, which consists of a mass-produced screen-printed carbon electrode (SPCE). In situ identification/quantification of tumor cells is achieved with a detection limit of 4000 cells per 700 microL of suspension. This novel and selective cell-sensing device is based on the reaction of cell surface proteins with specific antibodies conjugated with AuNPs. Final detection requires only a couple of minutes, taking advantage of the catalytic properties of AuNPs on hydrogen evolution. The proposed detection method does not require the chemical agents used in most existing assays for the detection of AuNPs. It allows for the miniaturization of the system and is much cheaper than other expensive and sophisticated methods used for tumor cell detection. We envisage that this device could operate in a simple way as an immunosensor or DNA sensor. Moreover, it could be used, even by inexperienced staff, for the detection of protein molecules or DNA strands.

Entropy based quantification of Ki-67 positive cell images and its evaluation by a reader study

NASA Astrophysics Data System (ADS)

Niazi, M. Khalid Khan; Pennell, Michael; Elkins, Camille; Hemminger, Jessica; Jin, Ming; Kirby, Sean; Kurt, Habibe; Miller, Barrie; Plocharczyk, Elizabeth; Roth, Rachel; Ziegler, Rebecca; Shana'ah, Arwa; Racke, Fred; Lozanski, Gerard; Gurcan, Metin N.

2013-03-01

Presence of Ki-67, a nuclear protein, is typically used to measure cell proliferation. The quantification of the Ki-67 proliferation index is performed visually by the pathologist; however, this is subject to inter- and intra-reader variability. Automated techniques utilizing digital image analysis by computers have emerged. The large variations in specimen preparation, staining, and imaging as well as true biological heterogeneity of tumor tissue often results in variable intensities in Ki-67 stained images. These variations affect the performance of currently developed methods. To optimize the segmentation of Ki-67 stained cells, one should define a data dependent transformation that will account for these color variations instead of defining a fixed linear transformation to separate different hues. To address these issues in images of tissue stained with Ki-67, we propose a methodology that exploits the intrinsic properties of CIE L∗a∗b∗ color space to translate this complex problem into an automatic entropy based thresholding problem. The developed method was evaluated through two reader studies with pathology residents and expert hematopathologists. Agreement between the proposed method and the expert pathologists was good (CCC = 0.80).

Quantification of seven β-lactam antibiotics and two β-lactamase inhibitors in human plasma using a validated UPLC-MS/MS method.

PubMed

Carlier, Mieke; Stove, Veronique; Roberts, Jason A; Van de Velde, Eric; De Waele, Jan J; Verstraete, Alain G

2012-11-01

There is an increasing interest in monitoring plasma concentrations of β-lactam antibiotics. The objective of this work was to develop and validate a rapid ultra-performance liquid chromatographic method with tandem mass spectrometric detection (UPLC-MS/MS) for simultaneous quantification of amoxicillin, ampicillin, cefuroxime, cefazolin, ceftazidime, meropenem, piperacillin, clavulanic acid and tazobactam. Sample clean-up included protein precipitation with acetonitrile and back-extraction of acetonitrile with dichloromethane. Six deuterated β-lactam antibiotics were used as internal standards. Chromatographic separation was performed on a Waters ACQUITY UPLC system using a BEH C(18) column (1.7 μm, 100 mm×2.1 mm) applying a binary gradient elution of water and acetonitrile both containing 0.1% formic acid. The total run time was 5.5 min. The developed method was validated in terms of precision, accuracy, linearity, matrix effect and recovery. The assay has now been successfully used to determine concentrations of amoxicillin/clavulanic acid, cefuroxime and meropenem in plasma samples from intensive care patients. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Absolute quantification of prion protein (90-231) using stable isotope-labeled chymotryptic peptide standards in a LC-MRM AQUA workflow

PubMed Central

Sturm, Robert; Kreitinger, Gloria; Booth, Clarissa; Smith, Lloyd; Pedersen, Joel; Li, Lingjun

2012-01-01

Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiological agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at sub-femtomole levels while animal bioassays, cell culture, and in vitro conversion assays offer ultrasensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein’s signature peptide, often with sub-femtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors’ knowledge, this is the first report of the use of a non-tryptic peptide in a LC-MRM AQUA workflow. PMID:22714949

EpiProfile Quantifies Histone Peptides With Modifications by Extracting Retention Time and Intensity in High-resolution Mass Spectra*

PubMed Central

Yuan, Zuo-Fei; Lin, Shu; Molden, Rosalynn C.; Cao, Xing-Jun; Bhanu, Natarajan V.; Wang, Xiaoshi; Sidoli, Simone; Liu, Shichong; Garcia, Benjamin A.

2015-01-01

Histone post-translational modifications contribute to chromatin function through their chemical properties which influence chromatin structure and their ability to recruit chromatin interacting proteins. Nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC-MS/MS) has emerged as the most suitable technology for global histone modification analysis because of the high sensitivity and the high mass accuracy of this approach that provides confident identification. However, analysis of histones with this method is even more challenging because of the large number and variety of isobaric histone peptides and the high dynamic range of histone peptide abundances. Here, we introduce EpiProfile, a software tool that discriminates isobaric histone peptides using the distinguishing fragment ions in their tandem mass spectra and extracts the chromatographic area under the curve using previous knowledge about peptide retention time. The accuracy of EpiProfile was evaluated by analysis of mixtures containing different ratios of synthetic histone peptides. In addition to label-free quantification of histone peptides, EpiProfile is flexible and can quantify different types of isotopically labeled histone peptides. EpiProfile is unique in generating layouts (i.e. relative retention time) of histone peptides when compared with manual quantification of the data and other programs (such as Skyline), filling the need of an automatic and freely available tool to quantify labeled and non-labeled modified histone peptides. In summary, EpiProfile is a valuable nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry-based quantification tool for histone peptides, which can also be adapted to analyze nonhistone protein samples. PMID:25805797

Urine Sample Preparation in 96-Well Filter Plates for Quantitative Clinical Proteomics

PubMed Central

2015-01-01

Urine is an important, noninvasively collected body fluid source for the diagnosis and prognosis of human diseases. Liquid chromatography mass spectrometry (LC-MS) based shotgun proteomics has evolved as a sensitive and informative technique to discover candidate disease biomarkers from urine specimens. Filter-aided sample preparation (FASP) generates peptide samples from protein mixtures of cell lysate or body fluid origin. Here, we describe a FASP method adapted to 96-well filter plates, named 96FASP. Soluble urine concentrates containing ∼10 μg of total protein were processed by 96FASP and LC-MS resulting in 700–900 protein identifications at a 1% false discovery rate (FDR). The experimental repeatability, as assessed by label-free quantification and Pearson correlation analysis for shared proteins among replicates, was high (R ≥ 0.97). Application to urinary pellet lysates which is of particular interest in the context of urinary tract infection analysis was also demonstrated. On average, 1700 proteins (±398) were identified in five experiments. In a pilot study using 96FASP for analysis of eight soluble urine samples, we demonstrated that protein profiles of technical replicates invariably clustered; the protein profiles for distinct urine donors were very different from each other. Robust, highly parallel methods to generate peptide mixtures from urine and other body fluids are critical to increase cost-effectiveness in clinical proteomics projects. This 96FASP method has potential to become a gold standard for high-throughput quantitative clinical proteomics. PMID:24797144

Microfluidic extraction and microarray detection of biomarkers from cancer tissue slides

NASA Astrophysics Data System (ADS)

Nguyen, H. T.; Dupont, L. N.; Jean, A. M.; Géhin, T.; Chevolot, Y.; Laurenceau, E.; Gijs, M. A. M.

2018-03-01

We report here a new microfluidic method allowing for the quantification of human epidermal growth factor receptor 2 (HER2) expression levels from formalin-fixed breast cancer tissues. After partial extraction of proteins from the tissue slide, the extract is routed to an antibody (Ab) microarray for HER2 titration by fluorescence. Then the HER2-expressing cell area is evaluated by immunofluorescence (IF) staining of the tissue slide and used to normalize the fluorescent HER2 signal measured from the Ab microarray. The number of HER2 gene copies measured by fluorescence in situ hybridization (FISH) on an adjacent tissue slide is concordant with the normalized HER2 expression signal. This work is the first study implementing biomarker extraction and detection from cancer tissue slides using microfluidics in combination with a microarray system, paving the way for further developments towards multiplex and precise quantification of cancer biomarkers.

A force-based, parallel assay for the quantification of protein-DNA interactions.

PubMed

Limmer, Katja; Pippig, Diana A; Aschenbrenner, Daniela; Gaub, Hermann E

2014-01-01

Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

PubMed Central

Shi, Tujin; Su, Dian; Liu, Tao; Tang, Keqi; Camp, David G.; Qian, Wei-Jun; Smith, Richard D.

2012-01-01

Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for e.g., detecting low-abundance biomarkers likely present at the low ng/mL to pg/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in cells or tissues. Herein we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides including posttranslational modifications (PTMs), as well as advances in MS instrumentation which have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low to sub- ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed. PMID:22577010

Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Su, Dian; Liu, Tao

2012-04-01

Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for e.g., detecting low-abundance biomarkers likely present at the pg/mL to low ng/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in the cells or tissues. Herein we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides or their posttranslational modifications (PTMs), as well as advances in MS instrumentation, whichmore » have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low to sub- ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed.« less

The dual role of paramagnetic particles for integrated lysis and measurement in a rapid immunoassay for intracellular proteins.

PubMed

Sharif, Elham; Kiely, Janice; Wraith, Patrick; Luxton, Richard

2013-05-01

A novel, integrated lysis and immunoassay methodology and system for intracellular protein measurement are described. The method uses paramagnetic particles both as a lysis agent and assay label resulting in a rapid test requiring minimal operator intervention, the test being homogeneous and completed in less than 10 min. A design study highlights the critical features of the magnetic detection system used to quantify the paramagnetic particles and a novel frequency-locked loop-based magnetometer is presented. A study of paramagnetic particle enhanced lysis demonstrates that the technique is more than twice as efficient at releasing intracellular protein as ultrasonic lysis alone. Results are presented for measurements of intracellular prostate specific antigen in an LNCAP cell line. This model was selected to demonstrate the rapidity and efficiency of intracellular protein quantification. It was shown that, on average, LNCAP cells contained 0.43 fg of prostate specific antigen. This system promises an attractive solution for applications that require a rapid determination of intracellular proteins.

Simple, miniaturized blood plasma extraction method.

PubMed

Kim, Jin-Hee; Woenker, Timothy; Adamec, Jiri; Regnier, Fred E

2013-12-03

A rapid plasma extraction technology that collects a 2.5 μL aliquot of plasma within three minutes from a finger-stick derived drop of blood was evaluated. The utility of the plasma extraction cards used was that a paper collection disc bearing plasma was produced that could be air-dried in fifteen minutes and placed in a mailing envelop for transport to an analytical laboratory. This circumvents the need for venipuncture and blood collection in specialized vials by a phlebotomist along with centrifugation and refrigerated storage. Plasma extraction was achieved by applying a blood drop to a membrane stack through which plasma was drawn by capillary action. During the course of plasma migration to a collection disc at the bottom of the membrane stack blood cells were removed by a combination of adsorption and filtration. After the collection disc filled with an aliquot of plasma the upper membranes were stripped from the collection card and the collection disc was air-dried. Intercard differences in the volume of plasma collected varied approximately 1% while volume variations of less than 2% were seen with hematocrit levels ranging from 20% to 71%. Dried samples bearing metabolites and proteins were then extracted from the disc and analyzed. 25-Hydroxy vitamin D was quantified by LC-MS/MS analysis following derivatization with a secosteroid signal enhancing tag that imparted a permanent positive charge to the vitamin and reduced the limit of quantification (LOQ) to 1 pg of collected vitamin on the disc; comparable to values observed with liquid-liquid extraction (LLE) of a venipuncture sample. A similar study using conventional proteomics methods and spectral counting for quantification was conducted with yeast enolase added to serum as an internal standard. The LOQ with extracted serum samples for enolase was 1 μM, linear from 1 to 40 μM, the highest concentration examined. In all respects protein quantification with extracted serum samples was comparable to that observed with serum samples obtained by venipuncture.

Quantification of soy protein using the isotope method (δ(13)C and δ(15)N) for commercial brands of beef hamburger.

PubMed

Ducatti, Rhani; de Almeida Nogueira Pinto, José Paes; Sartori, Maria Márcia Pereira; Ducatti, Carlos

2016-12-01

Hamburgers (beef patties) may be adulterated through the overuse of protein extenders. Among vegetables, soy protein is the best substitute for animal protein. These ingredients help to reduce the cost of producing a final product, and they maximize profits for fraudulent industries. Moreover, the ingestion of soy or other non-meat proteins by allergic individuals may present a health risk. In addition, monitoring by supervisory bodies is hampered by a lack of appropriate analytical methodologies. Within this context, the aim of this study was to determine and quantify the levels of added soy protein by determination of (15)N and (13)C stable isotopes. A total of 100 beef hamburger samples from 10 commercial brands were analyzed. Only three samples of the G brand were within the standards set the Brazilian legislation. The remaining 97 samples from 10 commercial brands contained >4% soy protein; therefore, they are adulterated and not in compliance with the current legislation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-Based Untargeted Quantitative Proteomic Approach To Identify Change of the Plasma Proteins by Salbutamol Abuse in Beef Cattle.

PubMed

Zhang, Kai; Tang, Chaohua; Liang, Xiaowei; Zhao, Qingyu; Zhang, Junmin

2018-01-10

Salbutamol, a selective β 2 -agonist, endangers the safety of animal products as a result of illegal use in food animals. In this study, an iTRAQ-based untargeted quantitative proteomic approach was applied to screen potential protein biomarkers in plasma of cattle before and after treatment with salbutamol for 21 days. A total of 62 plasma proteins were significantly affected by salbutamol treatment, which can be used as potential biomarkers to screen for the illegal use of salbutamol in beef cattle. Enzyme-linked immunosorbent assay measurements of five selected proteins demonstrated the reliability of iTRAQ-based proteomics in screening of candidate biomarkers among the plasma proteins. The plasma samples collected before and after salbutamol treatment were well-separated by principal component analysis (PCA) using the differentially expressed proteins. These results suggested that an iTRAQ-based untargeted quantitative proteomic strategy combined with PCA pattern recognition methods can discriminate differences in plasma protein profiles collected before and after salbutamol treatment.

Characterization and quantification of grape variety by means of shikimic acid concentration and protein fingerprint in still white wines.

PubMed

Chabreyrie, David; Chauvet, Serge; Guyon, François; Salagoïty, Marie-Hélène; Antinelli, Jean-François; Medina, Bernard

2008-08-27

Protein profiles, obtained by high-performance capillary electrophoresis (HPCE) on white wines previously dialyzed, combined with shikimic acid concentration and multivariate analysis, were used for the determination of grape variety composition of a still white wine. Six varieties were studied through monovarietal wines elaborated in the laboratory: Chardonnay (24 samples), Chenin (24), Petit Manseng (7), Sauvignon (37), Semillon (24), and Ugni Blanc (9). Homemade mixtures were elaborated from authentic monovarietal wines according to a Plackett-Burman sampling plan. After protein peak area normalization, a matrix was elaborated containing protein results of wines (mixtures and monovarietal). Partial least-squares processing was applied to this matrix allowing the elaboration of a model that provided a varietal quantification precision of around 20% for most of the grape varieties studied. The model was applied to commercial samples from various geographical origins, providing encouraging results for control purposes.

Understanding the Effects of Genotype, Growing Year, and Breeding on Tunisian Durum Wheat Allergenicity. 1. The Baker's Asthma Case.

PubMed

Boukid, Fatma; Prandi, Barbara; Sforza, Stefano; Sayar, Rhouma; Seo, Yong Weon; Mejri, Mondher; Yacoubi, Ines

2017-07-19

Baker's asthma is a serious airway disease triggered by wheat protein CM3 α-amylase/trypsin inhibitor. The purpose of the present study was to investigate the impact of genotype and crop year on allergen CM3 α-amylase/trypsin inhibitor associated with baker's asthma. A historical series of Tunisian durum wheat (100 accessions), derived from three crop years, was used to compare the amount of CM3 from landraces to advanced cultivars. CM3 protein quantification was assessed after an enzymatic cleavage of the soluble protein extracts on a UPLC/ESI-MS system, using a marker peptide for its quantification. Combined data analysis of variance revealed an important effect of genotype, crop year, and their interaction. The CM3 allergenic proteins were found to significantly vary among studied genotypes, as confirmed by genetic variability, coefficient of variance, heritability, and genetic advance.

Analysis of DNA interactions using single-molecule force spectroscopy.

PubMed

Ritzefeld, Markus; Walhorn, Volker; Anselmetti, Dario; Sewald, Norbert

2013-06-01

Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein-DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide- and protein-DNA interactions are given.

Direct analysis of ethylene glycol in human serum on the basis of analyte adduct formation and liquid chromatography-tandem mass spectrometry.

PubMed

Dziadosz, Marek

2018-01-01

The aim of this work was to develop a fast, cost-effective and time-saving liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method for the analysis of ethylene glycol (EG) in human serum. For these purposes, the formation/fragmentation of an EG adduct ion with sodium and sodium acetate was applied in the positive electrospray mode for signal detection. Adduct identification was performed with appropriate infusion experiments based on analyte solutions prepared in different concentrations. Corresponding analyte adduct ions and adduct ion fragments could be identified both for EG and the deuterated internal standard (EG-D4). Protein precipitation was used as sample preparation. The analysis of the supernatant was performed with a Luna 5μm C18 (2) 100A, 150mm×2mm analytical column and a mobile phase consisting of 95% A (H 2 O/methanol=95/5, v/v) and 5% B (H 2 O/methanol=3/97, v/v), both with 10mmolL -1 ammonium acetate and 0.1% acetic acid. Method linearity was examined in the range of 100-4000μg/mL and the calculated limit of detection/quantification was 35/98μg/mL. However, on the basis of the signal to noise ratio, quantification was recommended at a limit of 300μg/mL. Additionally, the examined precision, accuracy, stability, selectivity and matrix effect demonstrated that the method is a practicable alternative for EG quantification in human serum. In comparison to other methods based on liquid chromatography, the strategy presented made for the first time the EG analysis without analyte derivatisation possible. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and validation of a high-performance liquid chromatography-tandem mass spectrometry assay quantifying vemurafenib in human plasma.

PubMed

Nijenhuis, C M; Rosing, H; Schellens, J H M; Beijnen, J H

2014-01-01

Vemurafenib is an inhibitor of mutated serine/threonine-protein kinase B-Raf (BRAF) and is registered as Zelboraf(®) for the treatment of adult patients with BRAF V600 mutation-positive unresectable or metastatic melanoma. To support Therapeutic Drug Monitoring (TDM) and clinical trials, we developed and validated a method for the quantification of vemurafenib in human plasma. Additionally two LC-MS systems with different detectors were tested: the TSQ Quantum Ultra and the API3000. Human plasma samples were collected in the clinic and stored at nominally -20°C. Vemurafenib was isolated from plasma by liquid-liquid extraction, separated on a C18 column with gradient elution, and analysed with triple quadrupole mass spectrometry in positive-ion mode. A stable isotope was used as internal standard for the quantification. Ranging from 1 to 100μg/ml the assay was linear with correlation coefficients (r(2)) of 0.9985 or better. Inter-assay and intra-assay accuracies were within ±7.6% of the nominal concentration; inter-assay and intra-assay precision were within ≤9.3% of the nominal concentration. In addition all results were within the acceptance criteria of the US FDA and the latest EMA guidelines for method validation for both MS detectors. In conclusion, the presented analytical method for vemurafenib in human plasma was successfully validated and the performance of the two LC-MS systems for this assay was comparable. In addition the method was successfully applied to evaluate the pharmacokinetic quantification of vemurafenib in cancer patients treated with vemurafenib. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of Two Analytical Methods Based on Reverse Phase Chromatographic and SDS-PAGE Gel for Assessment of Deglycosylation Yield in N-Glycan Mapping.

PubMed

Eckard, Anahita D; Dupont, David R; Young, Johnie K

2018-01-01

N -lined glycosylation is one of the critical quality attributes (CQA) for biotherapeutics impacting the safety and activity of drug product. Changes in pattern and level of glycosylation can significantly alter the intrinsic properties of the product and, therefore, have to be monitored throughout its lifecycle. Therefore fast, precise, and unbiased N -glycan mapping assay is desired. To ensure these qualities, using analytical methods that evaluate completeness of deglycosylation is necessary. For quantification of deglycosylation yield, methods such as reduced liquid chromatography-mass spectrometry (LC-MS) and reduced capillary gel electrophoresis (CGE) have been commonly used. Here we present development of two additional methods to evaluate deglycosylation yield: one based on LC using reverse phase (RP) column and one based on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE gel) with offline software (GelAnalyzer). With the advent of rapid deglycosylation workflows in the market for N -glycan profiling replacing overnight incubation, we have aimed to quantify the level of deglycosylation in a selected rapid deglycosylation workflow. Our results have shown well resolved peaks of glycosylated and deglycosylated protein species with RP-LC method allowing simple quantification of deglycosylation yield of protein with high confidence. Additionally a good correlation, ≥0.94, was found between deglycosylation yields estimated by RP-LC method and that of reduced SDS-PAGE gel method with offline software. Evaluation of rapid deglycosylation protocol from GlycanAssure™ HyPerformance assay kit performed on fetuin and RNase B has shown complete deglycosylation within the recommended protocol time when evaluated with these techniques. Using this kit, N -glycans from NIST mAb were prepared in 1.4 hr and analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh performance LC (UHPLC) equipped with a fluorescence detector (FLD). 37 peaks were resolved with good resolution. Excellent sample preparation repeatability was found with relative standard deviation (RSD) of <5% for peaks with >0.5% relative area.

Liquid chromatography-tandem mass spectrometry for the quantification of flurbiprofen in human plasma and its application in a study of bioequivalence.

PubMed

Mei, Chenghan; Li, Bin; Yin, Qiangfeng; Jin, Jing; Xiong, Ting; He, Wenjuan; Gao, Xiujuan; Xu, Rong; Zhou, Piqi; Zheng, Heng; Chen, Hui

2015-07-01

A simple, quick and accurate LC-MS/MS method for the quantification of flurbiprofen in human plasma with indomethacin as internal standard (IS) was developed and validated. Samples were treated with methanol to precipitate proteins, then separated on a Ultimate C18 column (5μm, 2.1×50mm) with a gradient elusion process. Mobile phase A was comprised of water and formic acid, mobile phase B was comprised of acetonitrile and formic acid. Multi reaction monitoring (MRM) signals were saved on a negative ionization electrospray mass spectrometer. The calibration curve showed good linearity in the range of 40.00-10000.00μg/L (r(2)=0.998). Intra-day RE was 0.2-2.2%. Inter-day RE was 0.5-3.4%. The samples showed good stability under the study conditions. No significant matrix effect was observed. The established method was then applied to a bioequivalence study of a flurbiprofen axetil formulation. Copyright © 2015 Elsevier B.V. All rights reserved.

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin.

PubMed

Damle, Sagar; Hanser, Bridget; Davidson, Eric H; Fraser, Scott E

2006-11-15

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.

Sugar nucleotide quantification by liquid chromatography tandem mass spectrometry reveals a distinct profile in Plasmodium falciparum sexual stage parasites

PubMed Central

López-Gutiérrez, Borja; Dinglasan, Rhoel R.

2017-01-01

The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties in obtaining sufficient amounts of biological material have hampered the study of specific metabolic pathways in the malaria parasite. Thus, for example, the pools of sugar nucleotides required to fuel glycosylation reactions have never been studied in-depth in well-synchronized asexual parasites or in other stages of its life cycle. These metabolites are of critical importance, especially considering the renewed interest in the presence of N-, O-, and other glycans in key parasite proteins. In this work, we adapted a liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on the use of porous graphitic carbon (PGC) columns and MS-friendly solvents to quantify sugar nucleotides in the malaria parasite. We report the thorough quantification of the pools of these metabolites throughout the intraerythrocytic cycle of P. falciparum. The sensitivity of the method enabled, for the first time, the targeted analysis of these glycosylation precursors in gametocytes, the parasite sexual stages that are transmissible to the mosquito vector. PMID:28104756

Microbubble Enzyme-Linked Immunosorbent Assay for the Detection of Targeted Microbubbles in in Vitro Static Binding Assays.

PubMed

Wischhusen, Jennifer; Padilla, Frederic

2017-07-01

Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities. This study proposes a new method to reproducibly quantify MB binding based on enzyme-linked immunosorbent assay (ELISA), in which bound MBs are revealed with an enzyme-linked antibody. MB-ELISA was adapted to in vitro static binding assays, incubating the MBs in inverted position or by agitation, and compared with microscopy. The specificity and sensitivity of MB-ELISA enable the reliable quantification of MB binding in a rapid, high-throughput and whole-well analysis, facilitating the characterization of new targeted contrast agents. Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Differential Mass Spectrometry Profiles of Tau Protein in the Cerebrospinal Fluid of Patients with Alzheimer's Disease, Progressive Supranuclear Palsy, and Dementia with Lewy Bodies.

PubMed

Barthélemy, Nicolas R; Gabelle, Audrey; Hirtz, Christophe; Fenaille, François; Sergeant, Nicolas; Schraen-Maschke, Susanna; Vialaret, Jérôme; Buée, Luc; Junot, Christophe; Becher, François; Lehmann, Sylvain

2016-01-01

Microtubule-associated Tau proteins are major actors in neurological disorders, the so-called tauopathies. In some of them, and specifically in Alzheimer's disease (AD), hyperphosphorylated forms of Tau aggregate into neurofibrillary tangles. Following and understanding the complexity of Tau's molecular profile with its multiple isoforms and post-translational modifications represent an important issue, and a major analytical challenge. Immunodetection methods are, in fact, limited by the number, specificity, sensitivity, and capturing property of the available antibodies. Mass spectrometry (MS) has recently allowed protein quantification in complex biological fluids using isotope-labeled recombinant standard for absolute quantification (PSAQ). To study Tau proteins, which are found at very low concentrations within the cerebrospinal fluid (CSF), we relied on an innovative two-step pre-fractionation strategy, which was not dependent on immuno-enrichment. We then developed a sensitive multiplex peptide detection capability using targeted high-resolution MS to quantify Tau-specific peptides covering its entire sequence. This approach was used on a clinical cohort of patients with AD, progressive supranuclear palsy (PSP), and dementia with Lewy body (DLB) and with control non-neurodegenerative disorders. We uncovered a common CSF Tau molecular profile characterized by a predominance of central core expression and 1N/3R isoform detection. While PSP and DLB tau profiles showed minimal changes, AD was characterized by a unique pattern with specific modifications of peptide distribution. Taken together these results provide important information on Tau biology for future therapeutic interventions, and improved molecular diagnosis of tauopathies.

A research design for the quantification of the neuropeptides substance p and calcitonin gene-related Peptide in rat skin using Western blot analysis.

PubMed

Lapin, Guilherme Abbud Franco; Hochman, Bernardo; Nishioka, Michele Akemi; Maximino, Jessica Ruivo; Chadi, Gerson; Ferreira, Lydia Masako

2015-06-01

To describe and standardize a protocol that overcomes the technical limitations of Western blot (WB) analysis in the quantification of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) following nociceptive stimuli in rat skin. Male Wistar rats (Rattus norvegicus albinus) weighing 250 to 350 g were used in this study. Elements of WB analysis were adapted by using specific manipulation of samples, repeated cycles of freezing and thawing, more thorough maceration, and a more potent homogenizer; increasing lytic reagents; promoting greater inhibition of protease activity; and using polyvinylidene fluoride membranes as transfer means for skin-specific protein. Other changes were also made to adapt the WB analysis to a rat model. University research center. Western blot analysis adapted to a rat model. This research design has proven effective in collecting and preparing skin samples to quantify SP and CGRP using WB analysis in rat skin. This study described a research design that uses WB analysis as a reproducible, technically accessible, and cost-effective method for the quantification of SP and CGRP in rat skin that overcomes technical biases.

Evaluation of empirical rule of linearly correlated peptide selection (ERLPS) for proteotypic peptide-based quantitative proteomics.

PubMed

Liu, Kehui; Zhang, Jiyang; Fu, Bin; Xie, Hongwei; Wang, Yingchun; Qian, Xiaohong

2014-07-01

Precise protein quantification is essential in comparative proteomics. Currently, quantification bias is inevitable when using proteotypic peptide-based quantitative proteomics strategy for the differences in peptides measurability. To improve quantification accuracy, we proposed an "empirical rule for linearly correlated peptide selection (ERLPS)" in quantitative proteomics in our previous work. However, a systematic evaluation on general application of ERLPS in quantitative proteomics under diverse experimental conditions needs to be conducted. In this study, the practice workflow of ERLPS was explicitly illustrated; different experimental variables, such as, different MS systems, sample complexities, sample preparations, elution gradients, matrix effects, loading amounts, and other factors were comprehensively investigated to evaluate the applicability, reproducibility, and transferability of ERPLS. The results demonstrated that ERLPS was highly reproducible and transferable within appropriate loading amounts and linearly correlated response peptides should be selected for each specific experiment. ERLPS was used to proteome samples from yeast to mouse and human, and in quantitative methods from label-free to O18/O16-labeled and SILAC analysis, and enabled accurate measurements for all proteotypic peptide-based quantitative proteomics over a large dynamic range. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recent application of quantification II in Japanese medical research.

PubMed Central

Suzuki, T; Kudo, A

1979-01-01

Hayashi's Quantification II is a method of multivariate discrimination analysis to manipulate attribute data as predictor variables. It is very useful in the medical research field for estimation, diagnosis, prognosis, evaluation of epidemiological factors, and other problems based on multiplicity of attribute data. In Japan, this method is so well known that most of the computer program packages include the Hayashi Quantification, but it seems to be yet unfamiliar with the method for researchers outside Japan. In view of this situation, we introduced 19 selected articles of recent applications of the Quantification II in Japanese medical research. In reviewing these papers, special mention is made to clarify how the researchers were satisfied with findings provided by the method. At the same time, some recommendations are made about terminology and program packages. Also a brief discussion of the background of the quantification methods is given with special reference to the Behaviormetric Society of Japan. PMID:540587

Quantification of Paclitaxel and Polyaspartate Paclitaxel Conjugate in Beagle Plasma: Application to a Pharmacokinetic Study.

PubMed

Gao, Yangyang; Chen, Junying; Zhang, Xiqian; Xie, Huiru; Wang, Yanran; Guo, Shuquan

2017-03-01

An LC-MS/MS method for the determination of polyaspartate paclitaxel conjugate (PASP-PTX) and paclitaxel (PTX) in dog plasma with cephalomannine (Internal Standard for PASP-PTX, IS-I) and clopidogrel bisulfate (Internal Standard for PTX, IS-II) as the internal standards was developed and validated. Plasma samples of PASP-PTX were extracted by ethyl acetate following the hydrolysis reaction, while protein precipitation was used for the extraction of PTX using acetonitrile. Analytes were separated by a CAPCELL PAK C18 MG II column using a gradient elution with the mobile phase (A) 5 mM ammonium containing 0.1% formic acid, and (B) acetonitrile. Quantification was performed by monitoring the m/z transitions of 286.2/105.0 for PASP-PTX, 264.2/83.0 for IS-I, 854.4/286.0 for PTX, and 322.1/184.1 for IS-II in the ESI positive mode. This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The lower limit of quantification was 0.15 µg/mL for PASP-PTX and 0.01 µg/mL for PTX, and the calibration curves were linear over 0.15-300 µg/mL for PASP-PTX and over 0.01-10 µg/mL for PTX. The samples were stable under all the tested conditions. The method was successfully applied to study the pharmacokinetic profiles of PASP-PTX and PTX in beagles following intravenous administration of PASP-PTX. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Quantitative real-time PCR as a sensitive protein-protein interaction quantification method and a partial solution for non-accessible autoactivator and false-negative molecule analysis in the yeast two-hybrid system.

PubMed

Maier, Richard H; Maier, Christina J; Hintner, Helmut; Bauer, Johann W; Onder, Kamil

2012-12-01

Many functional proteomic experiments make use of high-throughput technologies such as mass spectrometry combined with two-dimensional polyacrylamide gel electrophoresis and the yeast two-hybrid (Y2H) system. Currently there are even automated versions of the Y2H system available that can be used for proteome-wide research. The Y2H system has the capacity to deliver a profusion of Y2H positive colonies from a single library screen. However, subsequent analysis of these numerous primary candidates with complementary methods can be overwhelming. Therefore, a method to select the most promising candidates with strong interaction properties might be useful to reduce the number of candidates requiring further analysis. The method described here offers a new way of quantifying and rating the performance of positive Y2H candidates. The novelty lies in the detection and measurement of mRNA expression instead of proteins or conventional Y2H genetic reporters. This method correlates well with the direct genetic reporter readouts usually used in the Y2H system, and has greater sensitivity for detecting and quantifying protein-protein interactions (PPIs) than the conventional Y2H system, as demonstrated by detection of the Y2H false-negative PPI of RXR/PPARG. Approximately 20% of all proteins are not suitable for the Y2H system, the so-called autoactivators. A further advantage of this method is the possibility to evaluate molecules that usually cannot be analyzed in the Y2H system, exemplified by a VDR-LXXLL motif peptide interaction. Copyright © 2012 Elsevier Inc. All rights reserved.

ROCS: a Reproducibility Index and Confidence Score for Interaction Proteomics Studies

PubMed Central

2012-01-01

Background Affinity-Purification Mass-Spectrometry (AP-MS) provides a powerful means of identifying protein complexes and interactions. Several important challenges exist in interpreting the results of AP-MS experiments. First, the reproducibility of AP-MS experimental replicates can be low, due both to technical variability and the dynamic nature of protein interactions in the cell. Second, the identification of true protein-protein interactions in AP-MS experiments is subject to inaccuracy due to high false negative and false positive rates. Several experimental approaches can be used to mitigate these drawbacks, including the use of replicated and control experiments and relative quantification to sensitively distinguish true interacting proteins from false ones. Methods To address the issues of reproducibility and accuracy of protein-protein interactions, we introduce a two-step method, called ROCS, which makes use of Indicator Prey Proteins to select reproducible AP-MS experiments, and of Confidence Scores to select specific protein-protein interactions. The Indicator Prey Proteins account for measures of protein identifiability as well as protein reproducibility, effectively allowing removal of outlier experiments that contribute noise and affect downstream inferences. The filtered set of experiments is then used in the Protein-Protein Interaction (PPI) scoring step. Prey protein scoring is done by computing a Confidence Score, which accounts for the probability of occurrence of prey proteins in the bait experiments relative to the control experiment, where the significance cutoff parameter is estimated by simultaneously controlling false positives and false negatives against metrics of false discovery rate and biological coherence respectively. In summary, the ROCS method relies on automatic objective criterions for parameter estimation and error-controlled procedures. Results We illustrate the performance of our method by applying it to five previously published AP-MS experiments, each containing well characterized protein interactions, allowing for systematic benchmarking of ROCS. We show that our method may be used on its own to make accurate identification of specific, biologically relevant protein-protein interactions, or in combination with other AP-MS scoring methods to significantly improve inferences. Conclusions Our method addresses important issues encountered in AP-MS datasets, making ROCS a very promising tool for this purpose, either on its own or in conjunction with other methods. We anticipate that our methodology may be used more generally in proteomics studies and databases, where experimental reproducibility issues arise. The method is implemented in the R language, and is available as an R package called “ROCS”, freely available from the CRAN repository http://cran.r-project.org/. PMID:22682516

A step-by-step protocol for assaying protein carbonylation in biological samples.

PubMed

Colombo, Graziano; Clerici, Marco; Garavaglia, Maria Elisa; Giustarini, Daniela; Rossi, Ranieri; Milzani, Aldo; Dalle-Donne, Isabella

2016-04-15

Protein carbonylation represents the most frequent and usually irreversible oxidative modification affecting proteins. This modification is chemically stable and this feature is particularly important for storage and detection of carbonylated proteins. Many biochemical and analytical methods have been developed during the last thirty years to assay protein carbonylation. The most successful method consists on protein carbonyl (PCO) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and consequent spectrophotometric assay. This assay allows a global quantification of PCO content due to the ability of DNPH to react with carbonyl giving rise to an adduct able to absorb at 366 nm. Similar approaches were also developed employing chromatographic separation, in particular HPLC, and parallel detection of absorbing adducts. Subsequently, immunological techniques, such as Western immunoblot or ELISA, have been developed leading to an increase of sensitivity in protein carbonylation detection. Currently, they are widely employed to evaluate change in total protein carbonylation and eventually to highlight the specific proteins undergoing selective oxidation. In the last decade, many mass spectrometry (MS) approaches have been developed for the identification of the carbonylated proteins and the relative amino acid residues modified to carbonyl derivatives. Although these MS methods are much more focused and detailed due to their ability to identify the amino acid residues undergoing carbonylation, they still require too expensive equipments and, therefore, are limited in distribution. In this protocol paper, we summarise and comment on the most diffuse protocols that a standard laboratory can employ to assess protein carbonylation; in particular, we describe step-by-step the different protocols, adding suggestions coming from our on-bench experience. Copyright © 2015 Elsevier B.V. All rights reserved.

Noninvasive diagnosis of intraamniotic infection: proteomic biomarkers in vaginal fluid.

PubMed

Hitti, Jane; Lapidus, Jodi A; Lu, Xinfang; Reddy, Ashok P; Jacob, Thomas; Dasari, Surendra; Eschenbach, David A; Gravett, Michael G; Nagalla, Srinivasa R

2010-07-01

We analyzed the vaginal fluid proteome to identify biomarkers of intraamniotic infection among women in preterm labor. Proteome analysis was performed on vaginal fluid specimens from women with preterm labor, using multidimensional liquid chromatography, tandem mass spectrometry, and label-free quantification. Enzyme immunoassays were used to quantify candidate proteins. Classification accuracy for intraamniotic infection (positive amniotic fluid bacterial culture and/or interleukin-6 >2 ng/mL) was evaluated using receiver-operator characteristic curves obtained by logistic regression. Of 170 subjects, 30 (18%) had intraamniotic infection. Vaginal fluid proteome analysis revealed 338 unique proteins. Label-free quantification identified 15 proteins differentially expressed in intraamniotic infection, including acute-phase reactants, immune modulators, high-abundance amniotic fluid proteins and extracellular matrix-signaling factors; these findings were confirmed by enzyme immunoassay. A multi-analyte algorithm showed accurate classification of intraamniotic infection. Vaginal fluid proteome analyses identified proteins capable of discriminating between patients with and without intraamniotic infection. Copyright (c) 2010 Mosby, Inc. All rights reserved.

Bulk derivatization and direct injection of human cerebrospinal fluid for trace-level quantification of endogenous estrogens using trap-and-elute liquid chromatography with tandem mass spectrometry.

PubMed

Fan, Hui; Papouskova, Barbora; Lemr, Karel; Wigginton, Jane G; Schug, Kevin A

2014-08-01

Although there are existing methods for determining estrogen in human bodily fluids including blood plasma and serum, very little information is available regarding estrogen levels in human cerebrospinal fluid (CSF), which is critical to assess in studies of neuroprotective functions and diffusion of neuroprotective estrogens across the blood-brain barrier. To address this problem, a liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of four endogenous estrogens (estrone, 17α-estradiol, 17β-estradiol, and estriol) in human CSF was developed. An aliquot (300 μL) of human CSF was bulk derivatized using dansyl chloride in the sample and 10 μL was directly injected onto a restricted-access media trap column for protein removal. No off-line sample extraction or cleanup was needed. The limits of detection of estrone, 17α-estradiol, 17β-estradiol, and estriol were 17, 28, 13, and 30 pg/mL, respectively, which is in the parts-per-trillion regime. The method was then applied to human CSF collected from ischemic trauma patients. Endogenous estrogens were detected and quantified, demonstrating the effectiveness of this method. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantification of Training and Competition Loads in Endurance Sports: Methods and Applications.

PubMed

Mujika, Iñigo

2017-04-01

Training quantification is basic to evaluate an endurance athlete's responses to training loads, ensure adequate stress/recovery balance, and determine the relationship between training and performance. Quantifying both external and internal workload is important, because external workload does not measure the biological stress imposed by the exercise sessions. Generally used quantification methods include retrospective questionnaires, diaries, direct observation, and physiological monitoring, often based on the measurement of oxygen uptake, heart rate, and blood lactate concentration. Other methods in use in endurance sports include speed measurement and the measurement of power output, made possible by recent technological advances such as power meters in cycling and triathlon. Among subjective methods of quantification, rating of perceived exertion stands out because of its wide use. Concurrent assessments of the various quantification methods allow researchers and practitioners to evaluate stress/recovery balance, adjust individual training programs, and determine the relationships between external load, internal load, and athletes' performance. This brief review summarizes the most relevant external- and internal-workload-quantification methods in endurance sports and provides practical examples of their implementation to adjust the training programs of elite athletes in accordance with their individualized stress/recovery balance.

GMDD: a database of GMO detection methods.

PubMed

Dong, Wei; Yang, Litao; Shen, Kailin; Kim, Banghyun; Kleter, Gijs A; Marvin, Hans J P; Guo, Rong; Liang, Wanqi; Zhang, Dabing

2008-06-04

Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed. GMO Detection method Database (GMDD) has collected almost all the previous developed and reported GMOs detection methods, which have been grouped by different strategies (screen-, gene-, construct-, and event-specific), and also provide a user-friendly search service of the detection methods by GMO event name, exogenous gene, or protein information, etc. In this database, users can obtain the sequences of exogenous integration, which will facilitate PCR primers and probes design. Also the information on endogenous genes, certified reference materials, reference molecules, and the validation status of developed methods is included in this database. Furthermore, registered users can also submit new detection methods and sequences to this database, and the newly submitted information will be released soon after being checked. GMDD contains comprehensive information of GMO detection methods. The database will make the GMOs analysis much easier.

Detection of Protein Modifications and Counterfeit Protein Pharmaceuticals Using iTRAQ and MALDI TOF/TOF Mass Spectrometry: Studies with Insulins

PubMed Central

Ye, Hongping; Hill, John; Kauffman, John; Gryniewicz, Connie; Han, Xianlin

2013-01-01

iTRAQ (isotope tags for relative and absolute quantification) reagent coupled with MALDI TOF/TOF mass spectrometric analysis has been evaluated as both a qualitative and quantitative method for the detection of modifications to active pharmaceutical ingredients derived from recombinant DNA technologies, and as a method to detect counterfeit drug products. Five types of insulin (human, bovine, porcine, Lispro, Lantus®) were used as model products in the study because of their minor variations in amino acid sequence. Several experiments were conducted in which each insulin variant was separately digested with Glu-C, and the digestate was labeled with one of four different iTRAQ reagents. All digestates were then combined for desalting and MALDI TOF/TOF mass spectrometric analysis. When the digestion procedure was optimized, the insulin sequence coverage was 100%. Five different types of insulin were readily differentiated, including Human insulin (P28K29) and Lispro (K28P29), which only differ by the interchange of two contiguous residues. Moreover, quantitative analyses show that the results obtained from the iTRAQ method agree well with those determined by other conventional methods. Collectively, the iTRAQ method can be used as a qualitative and quantitative technique for the detection of protein modification and counterfeiting. PMID:18489896

BioInfra.Prot: A comprehensive proteomics workflow including data standardization, protein inference, expression analysis and data publication.

PubMed

Turewicz, Michael; Kohl, Michael; Ahrens, Maike; Mayer, Gerhard; Uszkoreit, Julian; Naboulsi, Wael; Bracht, Thilo; Megger, Dominik A; Sitek, Barbara; Marcus, Katrin; Eisenacher, Martin

2017-11-10

The analysis of high-throughput mass spectrometry-based proteomics data must address the specific challenges of this technology. To this end, the comprehensive proteomics workflow offered by the de.NBI service center BioInfra.Prot provides indispensable components for the computational and statistical analysis of this kind of data. These components include tools and methods for spectrum identification and protein inference, protein quantification, expression analysis as well as data standardization and data publication. All particular methods of the workflow which address these tasks are state-of-the-art or cutting edge. As has been shown in previous publications, each of these methods is adequate to solve its specific task and gives competitive results. However, the methods included in the workflow are continuously reviewed, updated and improved to adapt to new scientific developments. All of these particular components and methods are available as stand-alone BioInfra.Prot services or as a complete workflow. Since BioInfra.Prot provides manifold fast communication channels to get access to all components of the workflow (e.g., via the BioInfra.Prot ticket system: bioinfraprot@rub.de) users can easily benefit from this service and get support by experts. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Assay Development for the Determination of Phosphorylation Stoichiometry using MRM methods with and without Phosphatase Treatment: Application to Breast Cancer Signaling Pathways

PubMed Central

Domanski, Dominik; Murphy, Leigh C.; Borchers, Christoph H.

2010-01-01

We have developed a phosphatase-based phosphopeptide quantitation (PPQ) method for determining phosphorylation stoichiometry in complex biological samples. This PPQ method is based on enzymatic dephosphorylation, combined with specific and accurate peptide identification and quantification by multiple reaction monitoring (MRM) detection with stable-isotope-labeled standard peptides. In contrast with the classical MRM methods for the quantitation of phosphorylation stoichiometry, the PPQ-MRM method needs only one non-phosphorylated SIS (stable isotope-coded standard) and two analyses (one for the untreated and one for the phosphatase-treated sample), from which the expression and modification levels can accurately be determined. From these analyses, the % phosphorylation can be determined. In this manuscript, we compare the PPQ-MRM method with an MRM method without phosphatase, and demonstrate the application of these methods to the detection and quantitation of phosphorylation of the classic phosphorylated breast cancer biomarkers (ERα and HER2), and for phosphorylated RAF and ERK1, which also contain phosphorylation sites with important biological implications. Using synthetic peptides spiked into a complex protein digest, we were able to use our PPQ-MRM method to accurately determine the total phosphorylation stoichiometry on specific peptides, as well as the absolute amount of the peptide and phosphopeptide present. Analyses of samples containing ERα protein revealed that the PPQ-MRM is capable of determining phosphorylation stoichiometry in proteins from cell lines, and is in good agreement with determinations obtained using the direct MRM approach in terms of phosphorylation and total protein amount. PMID:20524616

Molecular characterization of multivalent bioconjugates by size-exclusion chromatography with multiangle laser light scattering.

PubMed

Pollock, Jacob F; Ashton, Randolph S; Rode, Nikhil A; Schaffer, David V; Healy, Kevin E

2012-09-19

The degree of substitution and valency of bioconjugate reaction products are often poorly judged or require multiple time- and product-consuming chemical characterization methods. These aspects become critical when analyzing and optimizing the potency of costly polyvalent bioactive conjugates. In this study, size-exclusion chromatography with multiangle laser light scattering was paired with refractive index detection and ultraviolet spectroscopy (SEC-MALS-RI-UV) to characterize the reaction efficiency, degree of substitution, and valency of the products of conjugation of either peptides or proteins to a biopolymer scaffold, i.e., hyaluronic acid (HyA). Molecular characterization was more complete compared to estimates from a protein quantification assay, and exploitation of this method led to more accurate deduction of the molecular structures of polymer bioconjugates. Information obtained using this technique can improve macromolecular engineering design principles and help to better understand multivalent macromolecular interactions in biological systems.

A review on recent developments in mass spectrometry instrumentation and quantitative tools advancing bacterial proteomics.

PubMed

Van Oudenhove, Laurence; Devreese, Bart

2013-06-01

Proteomics has evolved substantially since its early days, some 20 years ago. In this mini-review, we aim to provide an overview of general methodologies and more recent developments in mass spectrometric approaches used for relative and absolute quantitation of proteins. Enhancement of sensitivity of the mass spectrometers as well as improved sample preparation and protein fractionation methods are resulting in a more comprehensive analysis of proteomes. We also document some upcoming trends for quantitative proteomics such as the use of label-free quantification methods. Hopefully, microbiologists will continue to explore proteomics as a tool in their research to understand the adaptation of microorganisms to their ever changing environment. We encourage them to incorporate some of the described new developments in mass spectrometry to facilitate their analyses and improve the general knowledge of the fascinating world of microorganisms.

Molecular characterization of multivalent bioconjugates by size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALS)

PubMed Central

Pollock, Jacob F.; Ashton, Randolph S.; Rode, Nikhil A.; Schaffer, David V.; Healy, Kevin E.

2013-01-01

The degree of substitution and valency of bioconjugate reaction products are often poorly judged or require multiple time- and product- consuming chemical characterization methods. These aspects become critical when analyzing and optimizing the potency of costly polyvalent bioactive conjugates. In this study, size-exclusion chromatography with multi-angle laser light scattering was paired with refractive index detection and ultraviolet spectroscopy (SEC-MALS-RI-UV) to characterize the reaction efficiency, degree of substitution, and valency of the products of conjugation of either peptides or proteins to a biopolymer scaffold, i.e., hyaluronic acid (HyA). Molecular characterization was more complete compared to estimates from a protein quantification assay, and exploitation of this method led to more accurate deduction of the molecular structures of polymer bioconjugates. Information obtained using this technique can improve macromolecular engineering design principles and better understand multivalent macromolecular interactions in biological systems. PMID:22794081

Quantifying protein interface footprinting by hydroxyl radical oxidation and molecular dynamics simulation: application to galectin-1.

PubMed

Charvátová, Olga; Foley, B Lachele; Bern, Marshall W; Sharp, Joshua S; Orlando, Ron; Woods, Robert J

2008-11-01

Biomolecular surface mapping methods offer an important alternative method for characterizing protein-protein and protein-ligand interactions in cases in which it is not possible to determine high-resolution three-dimensional (3D) structures of complexes. Hydroxyl radical footprinting offers a significant advance in footprint resolution compared with traditional chemical derivatization. Here we present results of footprinting performed with hydroxyl radicals generated on the nanosecond time scale by laser-induced photodissociation of hydrogen peroxide. We applied this emerging method to a carbohydrate-binding protein, galectin-1. Since galectin-1 occurs as a homodimer, footprinting was employed to characterize the interface of the monomeric subunits. Efficient analysis of the mass spectrometry data for the oxidized protein was achieved with the recently developed ByOnic (Palo Alto, CA) software that was altered to handle the large number of modifications arising from side-chain oxidation. Quantification of the level of oxidation has been achieved by employing spectral intensities for all of the observed oxidation states on a per-residue basis. The level of accuracy achievable from spectral intensities was determined by examination of mixtures of synthetic peptides related to those present after oxidation and tryptic digestion of galectin-1. A direct relationship between side-chain solvent accessibility and level of oxidation emerged, which enabled the prediction of the level of oxidation given the 3D structure of the protein. The precision of this relationship was enhanced through the use of average solvent accessibilities computed from 10 ns molecular dynamics simulations of the protein.

A multiplexed quantitative proteomics approach for investigating protein expression in the developing central nervous system.

PubMed

Orme, Rowan P; Gates, Monte A; Fricker-Gates, Rosemary A

2010-08-15

Cell transplantation using stem cell-derived neurons is commonly viewed as a candidate therapy for neurodegenerative diseases. However, methods for differentiating stem cells into homogenous populations of neurons suitable for transplant remain elusive. This suggests that there are as yet unknown signalling factors working in vivo to specify neuronal cell fate during development. These factors could be manipulated to better differentiate stem cells into neural populations useful for therapeutic transplantation. Here a quantitative proteomics approach is described for investigating cell signalling in the developing central nervous system (CNS), using the embryonic ventral mesencephalon as a model. Briefly, total protein was extracted from embryonic ventral midbrain tissue before, during and after the birth of dopaminergic neurons, and digested using trypsin. Two-dimensional liquid chromatography, coupled with tandem mass spectrometry, was then used to identify proteins from the tryptic peptides. Isobaric tagging for relative and absolute quantification (iTRAQ) reagents were used to label the tryptic peptides and facilitate relative quantitative analysis. The success of the experiment was confirmed by the identification of proteins known to be expressed in the developing ventral midbrain, as well as by Western blotting, and immunolabelling of embryonic tissue sections. This method of protein discovery improves upon previous attempts to identify novel signalling factors through microarray analysis. Importantly, the methods described here could be applied to virtually any aspect of development. (c) 2010 Elsevier B.V. All rights reserved.

First detection and quantification of N(δ)-monomethylarginine, a structural isomer of N(G)-monomethylarginine, in humans using MS(3).

PubMed

Martens-Lobenhoffer, Jens; Bode-Böger, Stefanie M; Clement, Bernd

2016-01-15

The L-arginine metabolites methylated at the guanidino moiety, such as N(G)-monomethyl-L-arginine (LNMMA), asymmetric N(G),N(G)-dimethyl-L-arginine (ADMA), and symmetric N(G),N(G')-dimethyl-L-arginine (SDMA), are long known to be present in human plasma. Far less is known about the structural isomer of LNMMA, N(δ)-monomethyl-L-arginine (δ-MMA). In prior work, it has been detected in yeast proteins, but it has not been investigated in mammalian plasma or cells. In this work, we present a method for the simultaneous and unambiguous quantification of LNMMA and δ-MMA in human plasma that is capable of detecting δ-MMA separately from LNMMA. The method comprises a simple protein precipitation sample preparation, hydrophilic interaction liquid chromatography (HILIC) gradient elution on an unmodified silica column, and triple stage mass spectrometric detection. Stable isotope-labeled D6-SDMA was used as internal standard. The calibration ranges were 25-1000 nmol/L for LNMMA and 5-350 nmol/L for δ-MMA. The intra- and inter-batch precision determinations resulted in relative standard deviations of less than 12% for both compounds with accuracies of less than 6% deviation from the expected values. In a pilot study enrolling 10 healthy volunteers, mean concentrations of 48.0 ± 7.4 nmol/L for LNMMA and 27.4 ± 7.7 nmol/L for δ-MMA were found. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparison of total protein concentration in skeletal muscle as measured by the Bradford and Lowry assays.

PubMed

Seevaratnam, Rajini; Patel, Barkha P; Hamadeh, Mazen J

2009-06-01

The Lowry and Bradford assays are the most commonly used methods of total protein quantification, yet vary in several aspects. To date, no comparisons have been made in skeletal muscle. We compared total protein concentrations of mouse red and white gastrocnemius, reagent stability, protein stability and range of linearity using both assays. The Lowry averaged protein concentrations 15% higher than the Bradford with a moderate correlation (r = 0.36, P = 0.01). However, Bland-Altman analysis revealed considerable bias (15.8 +/- 29.7%). Both Lowry reagents and its protein-reagent interactions were less stable over time than the Bradford. The linear range of concentration was smaller for the Lowry (0.05-0.50 mg/ml) than the Bradford (0-2.0 mg/ml). We conclude that the Bradford and Lowry measures of total protein concentration in skeletal muscle are not interchangeable. The Bradford and Lowry assays have various strengths and weaknesses in terms of substance interference and protein size. However, the Bradford provides greater reagent stability, protein-reagent stability and range of linearity, and requires less time to analyse compared to the Lowry assay.

The Analytical Chemistry of Drug Monitoring in Athletes

NASA Astrophysics Data System (ADS)

Bowers, Larry D.

2009-07-01

The detection and deterrence of the abuse of performance-enhancing drugs in sport are important to maintaining a level playing field among athletes and to decreasing the risk to athletes’ health. The World Anti-Doping Program consists of six documents, three of which play a role in analytical development: The World Anti-Doping Code, The List of Prohibited Substances and Methods, and The International Standard for Laboratories. Among the classes of prohibited substances, three have given rise to the most recent analytical developments in the field: anabolic agents; peptide and protein hormones; and methods to increase oxygen delivery to the tissues, including recombinant erythropoietin. Methods for anabolic agents, including designer steroids, have been enhanced through the use of liquid chromatography/tandem mass spectrometry and gas chromatography/combustion/isotope-ratio mass spectrometry. Protein and peptide identification and quantification have benefited from advances in liquid chromatography/tandem mass spectrometry. Incorporation of techniques such as flow cytometry and isoelectric focusing have supported the detection of blood doping.

Detection of proteins using a colorimetric bio-barcode assay.

PubMed

Nam, Jwa-Min; Jang, Kyung-Jin; Groves, Jay T

2007-01-01

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).

Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography.

PubMed

Ibáñez-Vea, María; Huang, Honggang; Martínez de Morentin, Xabier; Pérez, Estela; Gato, Maria; Zuazo, Miren; Arasanz, Hugo; Fernández-Irigoyen, Joaquin; Santamaría, Enrique; Fernandez-Hinojal, Gonzalo; Larsen, Martin R; Escors, David; Kochan, Grazyna

2018-03-02

Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO 2 ) chromatography enrichment prior to nLC-MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.

Absolute Quantification of Selected Proteins in the Human Osteoarthritic Secretome

PubMed Central

Peffers, Mandy J.; Beynon, Robert J.; Clegg, Peter D.

2013-01-01

Osteoarthritis (OA) is characterized by a loss of extracellular matrix which is driven by catabolic cytokines. Proteomic analysis of the OA cartilage secretome enables the global study of secreted proteins. These are an important class of molecules with roles in numerous pathological mechanisms. Although cartilage studies have identified profiles of secreted proteins, quantitative proteomics techniques have been implemented that would enable further biological questions to be addressed. To overcome this limitation, we used the secretome from human OA cartilage explants stimulated with IL-1β and compared proteins released into the media using a label-free LC-MS/MS-based strategy. We employed QconCAT technology to quantify specific proteins using selected reaction monitoring. A total of 252 proteins were identified, nine were differentially expressed by IL-1 β stimulation. Selected protein candidates were quantified in absolute amounts using QconCAT. These findings confirmed a significant reduction in TIMP-1 in the secretome following IL-1β stimulation. Label-free and QconCAT analysis produced equivocal results indicating no effect of cytokine stimulation on aggrecan, cartilage oligomeric matrix protein, fibromodulin, matrix metalloproteinases 1 and 3 or plasminogen release. This study enabled comparative protein profiling and absolute quantification of proteins involved in molecular pathways pertinent to understanding the pathogenesis of OA. PMID:24132152

Application of targeted proteomics to metabolically engineered Escherichia coli.

PubMed

Singh, Pragya; Batth, Tanveer S; Juminaga, Darmawi; Dahl, Robert H; Keasling, Jay D; Adams, Paul D; Petzold, Christopher J

2012-04-01

As synthetic biology matures to compete with chemical transformation of commodity and high-value compounds, a wide variety of well-characterized biological parts are needed to facilitate system design. Protein quantification based on selected-reaction monitoring (SRM) mass spectrometry compliments metabolite and transcript analysis for system characterization and optimizing flux through engineered pathways. By using SRM quantification, we assayed red fluorescent protein (RFP) expressed from plasmids containing several inducible and constitutive promoters and subsequently assessed protein production from the same promoters driving expression of eight mevalonate pathway proteins in Escherichia coli. For each of the promoter systems, the protein level for the first gene in the operon followed that of RFP, however, the levels of proteins produced from genes farther from the promoter were much less consistent. Second, we used targeted proteomics to characterize tyrosine biosynthesis pathway proteins after removal of native regulation. The changes were not expected to cause significant impact on protein levels, yet significant variation in protein abundance was observed and tyrosine production for these strains spanned a range from less than 1 mg/L to greater than 250 mg/L. Overall, our results underscore the importance of targeted proteomics for determining accurate protein levels in engineered systems and fine-tuning metabolic pathways. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein loss in human hair from combination straightening and coloring treatments.

PubMed

França-Stefoni, Simone Aparecida; Dario, Michelli Ferrera; Sá-Dias, Tânia Cristina; Bedin, Valcinir; de Almeida, Adriano José; Baby, André Rolim; Velasco, Maria Valéria R

2015-09-01

Hair chemical treatments, such as dyeing and straightening products, are known to cause damage that can be assessed by protein loss. The aim of this study was to evaluate the hair protein loss caused by combined chemical treatments (dye and relaxer) using the validated bicinchoninic acid (BCA) method. Three kinds of straighteners, based on ammonium thioglycolate, guanidine hydroxide and sodium hydroxide, were evaluated and the least harmful combination indicated. Caucasian virgin dark brown hair tresses were treated with developed natural brown color oxidative hair dyeing and/or straightening commercial products based on ammonium thioglycolate, sodium hydroxide, or guanidine hydroxide. Protein loss quantification was assessed by the validated BCA method which has several advantages for quantifying protein loss in chemically treated hair. When both treatments (straightening and dyeing) were combined, a higher negative effect was observed, particularly for dyed hair treated with sodium hydroxide. In this case, a 356% increase in protein loss relative to virgin hair was observed and 208% in relation to only dyed hair. The combination of dying and relaxers based on ammonium thioglycolate or guanidine hydroxide caused a small increase in protein loss, suggesting that these straightening products could be the best alternatives for individuals wishing to combine both treatments. These results indicated that when application of both types of products is desired, ammonium thioglycolate or guanidine hydroxide should be chosen for the straightening process. © 2015 Wiley Periodicals, Inc.

Intracellular protein determination using droplet-based immunoassays.

PubMed

Martino, Chiara; Zagnoni, Michele; Sandison, Mairi E; Chanasakulniyom, Mayuree; Pitt, Andrew R; Cooper, Jonathan M

2011-07-01

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells.

Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues*

PubMed Central

Bruderer, Roland; Bernhardt, Oliver M.; Gandhi, Tejas; Miladinović, Saša M.; Cheng, Lin-Yang; Messner, Simon; Ehrenberger, Tobias; Zanotelli, Vito; Butscheid, Yulia; Escher, Claudia; Vitek, Olga; Rinner, Oliver; Reiter, Lukas

2015-01-01

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)1-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling. PMID:25724911

A label-free internal standard method for the differential analysis of bioactive lupin proteins using nano HPLC-Chip coupled with Ion Trap mass spectrometry.

PubMed

Brambilla, Francesca; Resta, Donatella; Isak, Ilena; Zanotti, Marco; Arnoldi, Anna

2009-01-01

Quantitative proteomics based on MS is useful for pointing out the differences in some food proteomes relevant to human nutrition. Stable isotope label-free (SIF) techniques are suitable for comparing an unlimited number of samples by the use of relatively simple experimental workflows. We have developed an internal standard label-free method based on the intensities of peptide precursor ions from MS/MS spectra, collected in data dependent runs, for the simultaneous qualitative characterization and relative quantification of storage proteins of Lupinus albus seeds in protein extracts of four lupin cultivars (cv Adam, Arés, Lucky, Multitalia). The use of an innovative microfluidic system, the HPLC-Chip, coupled with a classical IT mass spectrometer, has allowed a complete qualitative characterization of all proteins. In particular, the homology search mode has permitted to identify single amino acid substitutions in the sequences of vicilins (beta-conglutin precursor and vicilin-like protein). The MS/MS sequencing of substituted peptides confirms the high heterogeneity of vicilins according to the peculiar characteristics of the vicilin-encoding gene family. Two suitable bioinformatics parameters were optimized for the differential analyses of the main bioactive proteins: the "normalized protein average of common reproducible peptides" (N-ACRP) for gamma-conglutin, which is a homogeneous protein, and the "normalized protein mean peptide spectral intensity" (N-MEAN) for the highly heterogenous class of the vicilins.

Absolute quantification of Medicago truncatula sucrose synthase isoforms and N-metabolism enzymes in symbiotic root nodules and the detection of novel nodule phosphoproteins by mass spectrometry

PubMed Central

Wienkoop, Stefanie; Larrainzar, Estíbaliz; Glinski, Mirko; González, Esther M.; Arrese-Igor, Cesar; Weckwerth, Wolfram

2008-01-01

Mass spectrometry (MS) has become increasingly important for tissue specific protein quantification at the isoform level, as well as for the analysis of protein post-translational regulation mechanisms and turnover rates. Thanks to the development of high accuracy mass spectrometers, peptide sequencing without prior knowledge of the amino acid sequence—de novo sequencing—can be performed. In this work, absolute quantification of a set of key enzymes involved in carbon and nitrogen metabolism in Medicago truncatula ‘Jemalong A17’ root nodules is presented. Among them, sucrose synthase (SuSy; EC 2.4.1.13), one of the central enzymes in sucrose cleavage in root nodules, has been further characterized and the relative phosphorylation state of the three most abundant isoforms has been quantified. De novo sequencing provided sequence information of a so far unidentified peptide, most probably belonging to SuSy2, the second most abundant isoform in M. truncatula root nodules. TiO2-phosphopeptide enrichment led to the identification of not only a phosphorylation site at Ser11 in SuSy1, but also of several novel phosphorylation sites present in other root nodule proteins such as alkaline invertase (AI; EC 3.2.1.26) and an RNA-binding protein. PMID:18772307

In-depth evaluation of software tools for data-independent acquisition based label-free quantification.

PubMed

Kuharev, Jörg; Navarro, Pedro; Distler, Ute; Jahn, Olaf; Tenzer, Stefan

2015-09-01

Label-free quantification (LFQ) based on data-independent acquisition workflows currently experiences increasing popularity. Several software tools have been recently published or are commercially available. The present study focuses on the evaluation of three different software packages (Progenesis, synapter, and ISOQuant) supporting ion mobility enhanced data-independent acquisition data. In order to benchmark the LFQ performance of the different tools, we generated two hybrid proteome samples of defined quantitative composition containing tryptically digested proteomes of three different species (mouse, yeast, Escherichia coli). This model dataset simulates complex biological samples containing large numbers of both unregulated (background) proteins as well as up- and downregulated proteins with exactly known ratios between samples. We determined the number and dynamic range of quantifiable proteins and analyzed the influence of applied algorithms (retention time alignment, clustering, normalization, etc.) on quantification results. Analysis of technical reproducibility revealed median coefficients of variation of reported protein abundances below 5% for MS(E) data for Progenesis and ISOQuant. Regarding accuracy of LFQ, evaluation with synapter and ISOQuant yielded superior results compared to Progenesis. In addition, we discuss reporting formats and user friendliness of the software packages. The data generated in this study have been deposited to the ProteomeXchange Consortium with identifier PXD001240 (http://proteomecentral.proteomexchange.org/dataset/PXD001240). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Statistical inference from multiple iTRAQ experiments without using common reference standards.

PubMed

Herbrich, Shelley M; Cole, Robert N; West, Keith P; Schulze, Kerry; Yager, James D; Groopman, John D; Christian, Parul; Wu, Lee; O'Meally, Robert N; May, Damon H; McIntosh, Martin W; Ruczinski, Ingo

2013-02-01

Isobaric tags for relative and absolute quantitation (iTRAQ) is a prominent mass spectrometry technology for protein identification and quantification that is capable of analyzing multiple samples in a single experiment. Frequently, iTRAQ experiments are carried out using an aliquot from a pool of all samples, or "masterpool", in one of the channels as a reference sample standard to estimate protein relative abundances in the biological samples and to combine abundance estimates from multiple experiments. In this manuscript, we show that using a masterpool is counterproductive. We obtain more precise estimates of protein relative abundance by using the available biological data instead of the masterpool and do not need to occupy a channel that could otherwise be used for another biological sample. In addition, we introduce a simple statistical method to associate proteomic data from multiple iTRAQ experiments with a numeric response and show that this approach is more powerful than the conventionally employed masterpool-based approach. We illustrate our methods using data from four replicate iTRAQ experiments on aliquots of the same pool of plasma samples and from a 406-sample project designed to identify plasma proteins that covary with nutrient concentrations in chronically undernourished children from South Asia.

Quantifying construction and demolition waste: An analytical review

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Zezhou; Yu, Ann T.W., E-mail: bsannyu@polyu.edu.hk; Shen, Liyin

2014-09-15

Highlights: • Prevailing C and D waste quantification methodologies are identified and compared. • One specific methodology cannot fulfill all waste quantification scenarios. • A relevance tree for appropriate quantification methodology selection is proposed. • More attentions should be paid to civil and infrastructural works. • Classified information is suggested for making an effective waste management plan. - Abstract: Quantifying construction and demolition (C and D) waste generation is regarded as a prerequisite for the implementation of successful waste management. In literature, various methods have been employed to quantify the C and D waste generation at both regional and projectmore » levels. However, an integrated review that systemically describes and analyses all the existing methods has yet to be conducted. To bridge this research gap, an analytical review is conducted. Fifty-seven papers are retrieved based on a set of rigorous procedures. The characteristics of the selected papers are classified according to the following criteria - waste generation activity, estimation level and quantification methodology. Six categories of existing C and D waste quantification methodologies are identified, including site visit method, waste generation rate method, lifetime analysis method, classification system accumulation method, variables modelling method and other particular methods. A critical comparison of the identified methods is given according to their characteristics and implementation constraints. Moreover, a decision tree is proposed for aiding the selection of the most appropriate quantification method in different scenarios. Based on the analytical review, limitations of previous studies and recommendations of potential future research directions are further suggested.« less

Quantification of egg proteome changes during fertilization in sterlet Acipenser ruthenus.

PubMed

Niksirat, Hamid; Andersson, Liselotte; Golpour, Amin; Chupani, Latifeh; James, Peter

2017-08-19

Eggs of sterlet are discharged outside into ambient aquatic environment where egg activation and fertilization occur. Effects of different activation media including freshwater and clay suspension on protein abundances of egg were quantified in sterlet Acipenser ruthenus. In-gel digestion and high resolution mass spectrometry were used for label-free protein quantification in the eggs of five females. No significant (p > 0.05) difference was found between protein abundances in eggs activated with different media. However, results showed significant (p < 0.05, fold change ≥2) reduction in the abundances of nine proteins including six glycoproteins, enolase and heat shock protein in activated groups compared to freshly ovulated eggs as control. The fact that abundance of proteasome subunit alpha significantly reduced only in eggs which were activated by clay suspension suggests that activation medium can somehow intervene with protein regulation during fertilization. In conclusion, external fertilization in sturgeon egg is accompanied by huge release of proteins into the external environment that may participate in the construction of a transient microenvironment around egg for attraction and protection of spermatozoa to ensure ensuing fertilization. Data are available via ProteomeXchange with identifier PXD006232. Copyright © 2017 Elsevier Inc. All rights reserved.

Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris.

PubMed

Garcia-Ortega, Xavier; Reyes, Cecilia; Montesinos, José Luis; Valero, Francisco

2015-01-01

The most commonly used cell disruption procedures may present lack of reproducibility, which introduces significant errors in the quantification of intracellular components. In this work, an approach consisting in the definition of an overall key performance indicator (KPI) was implemented for a lab scale high-pressure homogenizer (HPH) in order to determine the disruption settings that allow the reliable quantification of a wide sort of intracellular components. This innovative KPI was based on the combination of three independent reporting indicators: decrease of absorbance, release of total protein, and release of alkaline phosphatase activity. The yeast Pichia pastoris growing on methanol was selected as model microorganism due to it presents an important widening of the cell wall needing more severe methods and operating conditions than Escherichia coli and Saccharomyces cerevisiae. From the outcome of the reporting indicators, the cell disruption efficiency achieved using HPH was about fourfold higher than other lab standard cell disruption methodologies, such bead milling cell permeabilization. This approach was also applied to a pilot plant scale HPH validating the methodology in a scale-up of the disruption process. This innovative non-complex approach developed to evaluate the efficacy of a disruption procedure or equipment can be easily applied to optimize the most common disruption processes, in order to reach not only reliable quantification but also recovery of intracellular components from cell factories of interest.