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Free flow electrophoresis separation and AMS quantitation of 14C-naphthalene-protein adducts  

NASA Astrophysics Data System (ADS)

Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose-(concentration)dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 ?Ci) of 14C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 h post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with 14C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.

Buchholz, Bruce A.; Haack, Kurt W.; Sporty, Jennifer L.; Buckpitt, Alan R.; Morin, Dexter



ProteinAnalysis Electrophoresis  

E-print Network

ProteinDetectionKit(Colorimetric)--GLYCO-PRO GoldSolution,Colloidal--50755 OilRedO--O9755 PonceauSSolution--P7170 ProteoSilverTM--PROT-SIL1 ProteoSilver (G 1041). #12;ProteinAnalysis Electrophoresis 104 ELECTROPHORESIS Silver Stain Markers Designed for molecular weight determinations on silver stained gels, Silver Stain SDS

Lebendiker, Mario


Protein Electrophoresis/Immunofixation Electrophoresis  


... abnormal protein production, such as multiple myeloma or multiple sclerosis Sample Required? A blood sample drawn from a ... To search for the characteristic banding seen in multiple sclerosis ; the presence of multiple distinct bands in the ...


Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry  

PubMed Central

Background Certain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated. Results Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction. Conclusions This is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the analysis revealed additional layers of complexity to the wheat flour proteome that should be considered when evaluating quantitative 2-DE data. PMID:24517725



Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry  

Technology Transfer Automated Retrieval System (TEKTRAN)

Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication and further separated by size exclusion chromatography into monomeric and polymeric fractions. Proteins in each fraction were analyzed by quantitative two-dimensional gel...


Quantitative determination of native proteins in individual human erythrocytes by capillary zone electrophoresis with laser-induced fluorescence detection  

SciTech Connect

Intracellular fluid within single human erythrocytes is analyzed by capillary electrophoresis with laser-excited native protein fluorescence. Good signal-to-noise is achieved, allowing even minor components to be quantified. Non-Gaussian distributions were found for total protein, fraction carbonic anhydrase, fraction hemoglobin A[sub 0], and an unidentified component. Variations among a group of 29 cells for each quantity are as much as 1 order of magnitude, even though erythrocytes are known to be fairly homogeneous in size distribution. Variations in fraction hemoglobin A[sub 0] reflect differences in in vitro oxidation rates to methemoglobin. A positive correlation was observed between carbonic anhydrase and hemoglobin A[sub 0] for individual cells. This is consistent with the presence of erythrocytes of different ages within the population, with the older cells being less capable of maintaining enzyme activity and preventing oxidative damage. 35 refs., 10 figs., 1 tab.

Lee, T.T.; Yeung, E.S. (Ames Lab., IA (United States) Iowa State Univ., Ames (United States))



Non-denaturating isoelectric focusing gel electrophoresis for uranium-protein complexes quantitative analysis with LA-ICP MS.  


A non-denaturating isoelectric focusing (ND-IEF) gel electrophoresis protocol has been developed to study and identify uranium (U)-protein complexes with laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS) and electrospray ionization mass spectrometry (ESI-MS). The ND-IEF-LA-ICP MS methodology set-up was initiated using in vitro U-protein complex standards (i.e., U-bovine serum albumin and U-transferrin) allowing the assessment of U recovery to 64.4?±?0.4 %. This methodology enabled the quantification of U-protein complexes at 9.03?±?0.23, 15.27?±?0.36, and 177.31?±?25.51 nmol U L(-1) in digestive gland cytosols of the crayfish, Procambarus clarkii, exposed respectively to 0, 0.12, and 2.5 ?mol of waterborne depleted U L(-1) during 10 days. ND-IEF-LA-ICP MS limit of detection was 19.3 pmol U L(-1). Elemental ICP MS signals obtained both in ND-IEF electropherograms and in size exclusion chromatograms of in vivo U-protein complexes revealed interactions between U- and Fe- and Cu-proteins. Moreover, three proteins (hemocyanin, pseudohemocyanin-2, and arginine kinase) out of 42 were identified as potential uranium targets in waterborne-exposed crayfish cytosols by microbore reversed phase chromatography coupled to molecular mass spectrometry (µRPC-ESI-MS/MS) after ND-IEF separation. PMID:23665639

Xu, Ming; Frelon, Sandrine; Simon, Olivier; Lobinski, Ryszard; Mounicou, Sandra



Quantitative analysis of electrophoresis data: novel curve fitting methodology and its application to the determination of a protein-DNA binding constant.  

PubMed Central

A computer program, GelExplorer, which uses a new methodology for obtaining quantitative information about electrophoresis has been developed. It provides a straightforward, easy-to-use graphical interface, and includes a number of features which offer significant advantages over existing methods for quantitative gel analysis. The method uses curve fitting with a nonlinear least-squares optimization to deconvolute overlapping bands. Unlike most curve fitting approaches, the data is treated in two dimensions, fitting all the data across the entire width of the lane. This allows for accurate determination of the intensities of individual, overlapping bands, and in particular allows imperfectly shaped bands to be accurately modeled. Experiments described in this paper demonstrate empirically that the Lorentzian lineshape reproduces the contours of an individual gel band and provides a better model than the Gaussian function for curve fitting of electrophoresis bands. Results from several fitting applications are presented and a discussion of the sources and magnitudes of uncertainties in the results is included. Finally, the method is applied to the quantitative analysis of a hydroxyl radical footprint titration experiment to obtain the free energy of binding of the lambda repressor protein to the OR1 operator DNA sequence. PMID:9016637

Shadle, S E; Allen, D F; Guo, H; Pogozelski, W K; Bashkin, J S; Tullius, T D



Protein electrophoresis - serum  


... alpha-2, beta, and gamma globulins. In general, alpha and gamma globulin protein levels increase when there is inflammation in ... globulin: 0.1 to 0.3 g/dL Alpha-2 globulin: 0.6 to 1.0 g/dL Beta globulin: 0.7 to 1.2 g/dL Gamma globulin: 0.7 to 1.6 g/dL ...


Protein Charge Ladders, Capillary Electrophoresis, and  

E-print Network

Protein Charge Ladders, Capillary Electrophoresis, and the Role of Electrostatics in Biomolecular Introduction Life rests on a web of molecular recognition;1,2 the folding of proteins, the hybridization the recognition site of the protein. Hydrophobic interactions, although still a challenge to describe

Prentiss, Mara


SDS capillary gel electrophoresis of proteins in microfabricated channels  

PubMed Central

Analysis of variations in the concentrations or structures of biomolecules (e.g., mRNAs, proteins, peptides, natural products) that occur either naturally or in response to environmental or genetic perturbations can provide important insight into complex biological processes. Many biological samples are mixtures that require a separation step before quantitation of variations in the individual components. Two-dimensional denaturing gel electrophoresis has been used very effectively to separate complex mixtures of proteins, but it is time consuming and requires considerable amounts of sample. Microchannel-based separations have proven very effective in rapidly separating small amounts of nucleic acids; more recently, isoelectric focusing of proteins also has been adapted to the microchannel format. Here, we describe microchannel-based SDS capillary gel electrophoresis of proteins and demonstrate the speed and high resolution it provides. This development is an important step toward the miniaturization and integration of multidimensional and array separation methods for complex protein mixtures. PMID:10318890

Yao, Shao; Anex, Deon S.; Caldwell, W. Brett; Arnold, Don W.; Smith, Katherine B.; Schultz, Peter G.



Two-dimensional electrophoresis of membrane proteins  

Microsoft Academic Search

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due\\u000a to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional\\u000a two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples\\u000a that applies isoelectric focusing (IEF) in the first dimension and

Ralf J. Braun; Norbert Kinkl; Monika Beer; Marius Ueffing



Two-Dimensional Electrophoresis of Serum Proteins  

Microsoft Academic Search

A METHOD of zone electrophoresis in starch gels has recently been described by one of us1. The high degree of resolution obtained with this method when applied to serum appears to be due to the use of a supporting medium the pore size of which approaches the molecular dimensions of some of the proteins involved, so that resolution by molecular

O. Smithies; M. D. POULIK



Accessing Protein Methyltransferase and Demethylase Enzymology Using Microfluidic Capillary Electrophoresis  

PubMed Central

Summary The discovery of small molecules targeting the > 80 enzymes that add (methyltransferases) or remove (demethylases) methyl marks from lysine and arginine residues, most notably present in histone tails, may yield unprecedented chemotherapeutic agents and facilitate regenerative medicine. To better enable chemical exploration of these proteins, we have developed a novel and highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes and the kinetics of their inhibition. This technology separates small biomolecules, i.e., peptides, based on their charge-to-mass ratio. Methylation, however, does not alter the charge of peptide substrates. To overcome this limitation, we have employed a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules. PMID:20659682

Wigle, Tim J.; Provencher, Laurel M.; Norris, Jacqueline L.; Jin, Jian; Brown, Peter J.; Frye, Stephen V.; Janzen, William P.



Simplification and improvement of protein detection in two-dimensional electrophoresis gels with SERVA HPE™ lightning red.  


A new fluorescent amino-reactive dye has been tested for both labelling proteins prior to electrophoretic separations and between the two steps of two-dimensional electrophoresis. A series of experiments showed, that the labelling of lysines with this dye is compatible with all standard additives used for sample preparation, including reducing substances and carrier ampholytes. Using this dye for pre-labelling considerably simplifies the electrophoresis and detection workflow and provides highly sensitive and quantitative visualisation of proteins. PMID:23786184

Griebel, Anja; Obermaier, Christian; Westermeier, Reiner; Moche, Martin; Büttner, Knut




NSDL National Science Digital Library

Electrophoresis involves the movement of electrically charged substances under the influence of an electric field. This website demonstrates electrophoresis by providing a java applet which virtually applies an electric field across an agarose or polyacrylamide electrophoresis gel in which biological macromolecules are placed. The applet shows how the molecules move and at the end of the experiment plots the logarithm of the molecular weight versus the logarithm of the distance moved. This tutorial is one of a large collection of tutorials on electricity and magnetism available from Molecular Expressions.

Davidson, Michael



Mass Estimation of Native Proteins by Blue Native Electrophoresis  

PubMed Central

Blue native electrophoresis is one of the most popular techniques for mass estimation of native membrane proteins, but the use of non-optimal mass markers and acrylamide gels can compromise accuracy and reliability of the results. We present short protocols taking 10–30 min to prepare optimal sets of membrane protein markers from chicken, rat, mouse, and bovine heart. Especially heart materials from local supermarkets or butcher's shops, e.g. chicken or bovine heart, are ideal sources of high mass membrane protein standards. Considerable discrepancies between the migration behavior of membrane and soluble markers suggest using membrane protein markers for mass estimation of membrane proteins. Soluble standard proteins can be used, with some limitations, when soluble proteins are the focus. Principles and general rules for the determination of mass and oligomeric state of native membrane and soluble proteins are elaborated, and potential pitfalls are discussed. PMID:20173216

Wittig, Ilka; Beckhaus, Tobias; Wumaier, Zibiernisha; Karas, Michael; Schägger, Hermann



Protein Separation by Capillary Gel Electrophoresis: A Review  

PubMed Central

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples. PMID:22122927

Zhu, Zaifang; Lu, Joann J.; Liu, Shaorong



Relative quantitative comparisons of the extracellular protein profiles of Staphylococcus aureus UAMS-1 and its sarA, agr, and sarA agr regulatory mutants using one-dimensional polyacrylamide gel electrophoresis and nanocapillary liquid chromatography coupled with tandem mass spectrometry.  


One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography coupled with mass spectrometry was used to analyze proteins isolated from Staphylococcus aureus UAMS-1 after 3, 6, 12, and 24 h of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more abundant early on in growth, while proteins known to be secreted into the surrounding milieu were more abundant late in growth. In addition, proteins from spent media and cell lysates of strain UAMS-1 and its isogenic sarA, agr, and sarA agr regulatory mutant strains during exponential growth were identified, and their relative abundances were compared. Extracellular proteins known to be regulated by the global regulators sarA and agr displayed protein levels in accordance with what is known regarding the effects of these regulators. For example, cysteine protease (SspB), endopeptidase (SspA), staphopain (ScpA), and aureolysin (Aur) were higher in abundance in the sarA and sarA agr mutants than in strain UAMS-1. The immunoglobulin G (IgG)-binding protein (Sbi), immunodominant staphylococcal antigen A (IsaA), IgG-binding protein A (Spa), and the heme-iron-binding protein (IsdA) were most abundant in the agr mutant background. Proteins whose abundance was decreased in the sarA mutant included fibrinogen-binding protein (Fib [Efb]), IsaA, lipase 1 and 2, and two proteins identified as putative leukocidin F and S subunits of the two-component leukotoxin family. Collectively, this approach identified 1,263 proteins (matches of two peptides or more) and provided a convenient and reliable way of identifying proteins and comparing their relative abundances. PMID:18539737

Jones, Richard C; Deck, Joanna; Edmondson, Ricky D; Hart, Mark E



Relative Quantitative Comparisons of the Extracellular Protein Profiles of Staphylococcus aureus UAMS-1 and Its sarA, agr, and sarA agr Regulatory Mutants Using One-Dimensional Polyacrylamide Gel Electrophoresis and Nanocapillary Liquid Chromatography Coupled with Tandem Mass Spectrometry ? †  

PubMed Central

One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography coupled with mass spectrometry was used to analyze proteins isolated from Staphylococcus aureus UAMS-1 after 3, 6, 12, and 24 h of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more abundant early on in growth, while proteins known to be secreted into the surrounding milieu were more abundant late in growth. In addition, proteins from spent media and cell lysates of strain UAMS-1 and its isogenic sarA, agr, and sarA agr regulatory mutant strains during exponential growth were identified, and their relative abundances were compared. Extracellular proteins known to be regulated by the global regulators sarA and agr displayed protein levels in accordance with what is known regarding the effects of these regulators. For example, cysteine protease (SspB), endopeptidase (SspA), staphopain (ScpA), and aureolysin (Aur) were higher in abundance in the sarA and sarA agr mutants than in strain UAMS-1. The immunoglobulin G (IgG)-binding protein (Sbi), immunodominant staphylococcal antigen A (IsaA), IgG-binding protein A (Spa), and the heme-iron-binding protein (IsdA) were most abundant in the agr mutant background. Proteins whose abundance was decreased in the sarA mutant included fibrinogen-binding protein (Fib [Efb]), IsaA, lipase 1 and 2, and two proteins identified as putative leukocidin F and S subunits of the two-component leukotoxin family. Collectively, this approach identified 1,263 proteins (matches of two peptides or more) and provided a convenient and reliable way of identifying proteins and comparing their relative abundances. PMID:18539737

Jones, Richard C.; Deck, Joanna; Edmondson, Ricky D.; Hart, Mark E.



High Resolution Two-Dimensional Electrophoresis of Proteins*  

PubMed Central

Summary A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources. Proteins are separated according to isoelectric point by isoelectric focusing in the first dimension, and according to molecular weight by sodium dodecyl sulfate electrophoresis in the second dimension. Since these two parameters are unrelated, it is possible to obtain an almost uniform distribution of protein spots across a two-dimensional gel. This technique has resolved 1100 different components from Escherichia coli and should be capable of resolving a maximum of 5000 proteins. A protein containing as little as one disintegration per min of either 14C or 35S can be detected by autoradiography. A protein which constitutes 10?4 to 10?5% of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation is sufficient to permit each spot on one separation to be matched with a spot on a different separation. This technique provides a method for estimation (at the described sensitivities) of the number of proteins made by any biological system. This system can resolve proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge. Proteins whose charge is changed by missense mutations can be identified. A detailed description of the methods as well as the characteristics of this system are presented. PMID:236308

O'Farrell, Patrick H.



Procedures for two-dimensional electrophoresis of proteins  

SciTech Connect

High-resolution two-dimensional gel electrophoresis (2DE) of proteins, using isoelectric focusing in the first dimension and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) in the second, was first described in 1975. In the 20 years since those publications, numerous modifications of the original method have evolved. The ISO-DALT system of 2DE is a high-throughput approach that has stood the test of time. The problem of casting many isoelectric focusing gels and SDS-PAGE slab gels (up to 20) in a reproducible manner has been solved by the use of the techniques and equipment described in this manual. The ISO-DALT system of two-dimensional gel electrophoresis originated in the late 1970s and has been modified many times to improve its high-resolution, high-throughput capabilities. This report provides the detailed procedures used with the current ISO-DALT system to prepare, run, stain, and photograph two-dimensional gels for protein analysis.

Tollaksen, S.L.; Giometti, C.S.



High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture(SILAC), two-dimensional gel electrophoresis(2DE) and nano-liquid chromatograpohy coupled to an LTQ-OrbitrapMass spectrometer.  


The proteomics field has shifted over recent years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel-free workflows because of the tremendous developments in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry. However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined stable isotope labeling with amino acids in cell culture of controls and apoptotic HeLa cells with 2-DE and the subsequent analysis of tryptic peptides via nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer to obtain quantitative data using the methods with the highest resolving power on all levels of the proteomics workflow. More than 1,200 proteins with more than 2,700 protein species were identified and quantified from 816 Coomassie Brilliant Blue G-250 stained 2-DE spots. About half of the proteins were identified and quantified only in single 2-DE spots. The majority of spots revealed one to five proteins; however, in one 2-DE spot, up to 23 proteins were identified. Only half of the 2-DE spots represented a dominant protein with more than 90% of the whole protein amount. Consequently, quantification based on staining intensities in 2-DE gels would in approximately half of the spots be imprecise, and minor components could not be quantified. These problems are circumvented by quantification using stable isotope labeling with amino acids in cell culture. Despite challenges, as shown in detail for lamin A/C and vimentin, the quantitative changes of protein species can be detected. The combination of 2-DE with high-resolution nano-liquid chromatography-mass spectrometry allowed us to identify proteomic changes in apoptotic cells that would be unobservable using any of the other previously employed proteomic workflows. PMID:23033477

Thiede, Bernd; Koehler, Christian J; Strozynski, Margarita; Treumann, Achim; Stein, Robert; Zimny-Arndt, Ursula; Schmid, Monika; Jungblut, Peter R



Capillary zone electrophoresis-mass spectrometry of peptides and proteins  

SciTech Connect

Capillary zone electrophoresis (CZE) is attracting extensive attention as a fast, high resolution analytical and micro-preparative separations technique for systems of biological interest. In zone electrophoresis, a column is filled with a single electrolyte having a specific conductivity. The mixture of substances to be separated is applied as a narrow band to the head of a buffer filled column in a band whose width is much less than the length of the column and at a concentration too low to affect the buffer conductivity. An electric field is then applied across the length of the column and the individual substances migrate and separate according to their net electrophoretic velocities. Zone electrophoresis carried out in small diameter (<100 fused silica capillaries is a relatively new approach to the high resolution separation of aqueous samples. Very small volume samples (picoliter range) with separation efficiencies on the order of 10/sup 6/ theoretical plates for amino acids have been achieved. The method can be further enhanced by the dynamic combination of detection sensitivity and selectivity offered by mass spectrometry (MS). The on-line marriage of mass spectrometry to CZE is accomplished by an atmospheric pressure electrospray ionization source interface. Our research efforts have demonstrated that proteins with MW's greater than 100 kDa can be analyzed using a conventional quadrupole mass spectrometer with an upper m/z limit of only 1700. 6 refs.

Loo, J.A.; Udseth, H.R.; Smith, R.D.



Comparison of the Serum Protein Fractions of the Newly Hatched Chick with those of Adult Birds using Starch-gel Electrophoresis  

Microsoft Academic Search

IT has been established by several investigators that serum protein components of chick embryo undergo qualitative as well as quantitative changes throughout the developmental period until the time of hatching1-4. The serum protein composition of the newly hatched chick, however, resembles that of the adult bird, as revealed by moving-boundary and paper electro-phoresis. The technique of starch-gel electrophoresis suggested by

A. Amin



Tissue proteomics by one-dimensional gel electrophoresis combined with label-free protein quantification.  


Label-free methods streamline quantitative proteomics of tissues by alleviating the need for metabolic labeling of proteins with stable isotopes. Here we detail and implement solutions to common problems in label-free data processing geared toward tissue proteomics by one-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectrometry (geLC MS/MS). Our quantification pipeline showed high levels of performance in terms of duplicate reproducibility, linear dynamic range, and number of proteins identified and quantified. When applied to the liver of an adenomatous polyposis coli (APC) knockout mouse, we demonstrated an 8-fold increase in the number of statistically significant changing proteins compared to alternative approaches, including many more previously unidentified hydrophobic proteins. Better proteome coverage and quantification accuracy revealed molecular details of the perturbed energy metabolism. PMID:22671763

Vasilj, Andrej; Gentzel, Marc; Ueberham, Elke; Gebhardt, Rolf; Shevchenko, Andrej




Microsoft Academic Search

Maturation of Norway maple (Acer platanoides L.) seeds produces deep physiological dormancy and resistance to desiccation. This study used two-dimensional electrophoresis to investigate the protein products of genes activated during the complex developmental process of maturation. Qualitative and quantitative changes in protein composition during maturation were tracked in this species. The most intensive changes in protein content appeared at the




Detection of   and   Light Chain Monoclonal Proteins in Human Serum: Automated Immunoassay versus Immunofixation Electrophoresis  

Microsoft Academic Search

Recently, turbidimetric immunoassays for detecting and quantifying and free light chains (FLC) have become available and are promoted as being more sensitive than immunofixation electrophoresis (IFE) in detecting FLC monoclonal proteins. In this study, we assessed the ability of these turbidimetric assays to detect serum monoclonal proteins involving both free and heavy-chain-bound and light chains compared to standard immunofixation electrophoresis.

Troy D. Jaskowski; Christine M. Litwin; Harry R. Hill



Characterization of glutamine deamidation in a long, repetitive protein polymer via bioconjugate capillary electrophoresis.  


We describe a novel method for the determination of glutamine deamidation in a long protein polymer via bioconjugate capillary electrophoresis. Since the current best technique for detection of glutamine (or asparagine) deamidation is mass spectrometry, it is practically impossible to precisely detect the degree of deamidation (i.e., how many residues are deamidated in a polypeptide) in a large protein containing a significant number of glutamine (or asparagine) residues, because the mass difference between native and deamidated residues is just 1 atomic mass unit. However, by covalently attaching polydisperse protein polymers (337 residues) to a monodisperse DNA oligomer (22 bases), the degree of glutamine deamidation, which could not be determined accurately by mass spectrometry, was resolved by free-solution capillary electrophoresis. Electrophoretic separations were carried out after different durations of exposure of the protein to a cyanogen bromide cleavage reaction mixture, which is a general treatment for the purpose of removing an oligopeptide affinity purification tag from fusion proteins. For protein polymers with increasing extents of deamidation, the electromotive force of DNA + polypeptide conjugate molecules increases due to the introduced negative charge of deamidated glutamic acid residues, and consequently CE analysis reveals increasing differences in the electrophoretic mobilities of conjugate molecules, which qualitatively shows the degree of deamidation. Peak analysis of the electropherograms enables quantitative determination of the first four deamidations in a protein polymer. A first-order rate constant of 0.018 h(-1) was determined for the deamidation of a single glutamine residue in the protein polymer during the cyanogen bromide cleavage reaction. PMID:15003029

Won, Jong-In; Meagher, Robert J; Barron, Annelise E



Separation of basic proteins from Leishmania using a combination of Free flow electrophoresis (FFE) and 2D electrophoresis (2-DE) under basic conditions.  


Basic proteins, an important class of proteins in intracellular organisms such as Leishmania, are usually underrepresented on 2D gels. This chapter describes a method combining basic proteins fractionation using Free flow electrophoresis in isoelectric focusing mode (IEF-FFE) followed by protein separation using two-dimensional gel electrophoresis (2-DE) in basic conditions. The combination of these two techniques represents a great improvement for the visualization of Leishmania proteins with basic pI using 2D gels. PMID:25388119

Brotherton, Marie-Christine; Racine, Gina; Ouellette, Marc



Quantitative analysis of glycated proteins.  


The proposed protocol presents a comprehensive approach for large-scale qualitative and quantitative analysis of glycated proteins (GP) in complex biological samples including biological fluids and cell lysates such as plasma and red blood cells. The method, named glycation isotopic labeling (GIL), is based on the differential labeling of proteins with isotopic [(13)C6]-glucose, which supports quantitation of the resulting glycated peptides after enzymatic digestion with endoproteinase Glu-C. The key principle of the GIL approach is the detection of doublet signals for each glycated peptide in MS precursor scanning (glycated peptide with in vivo [(12)C6]- and in vitro [(13)C6]-glucose). The mass shift of the doublet signals is +6, +3 or +2 Da depending on the peptide charge state and the number of glycation sites. The intensity ratio between doublet signals generates quantitative information of glycated proteins that can be related to the glycemic state of the studied samples. Tandem mass spectrometry with high-energy collisional dissociation (HCD-MS2) and data-dependent methods with collision-induced dissociation (CID-MS3 neutral loss scan) are used for qualitative analysis. PMID:24417557

Priego-Capote, Feliciano; Ramírez-Boo, María; Finamore, Francesco; Gluck, Florent; Sanchez, Jean-Charles



Attomole quantitation of protein separations with accelerator mass spectrometry  

SciTech Connect

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 abundances 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.

Vogel, J S; Grant, P G; Buccholz, B A; Dingley, K; Turteltaub, K W



Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE)  

Microsoft Academic Search

Two-dimensional gel electrophoresis (2-D GE) is a key tool for comparative proteomics research. With its ability to separate complex protein mixtures with high resolution, 2-D GE is a technique commonly employed for protein profiling studies. Significant improvements have been made in 2-D GE technology with the development of two-dimensional fluorescence difference gel electrophoresis (2-D DIGE), where proteins are first labelled

J E Swatton; S Prabakaran; N A Karp; K S Lilley; S Bahn



Affinity capillary electrophoresis with magnetic beads for multiplex quantitative analysis of bacterial 16S rRNA  

Microsoft Academic Search

We have developed a novel method for microbial community analysis of bacterial 16S rRNAs based on affinity capillary electrophoresis using 16S rRNA-conjugated magnetic beads. We called this method magnetic beads affinity capillary electrophoresis (MB-ACE) which can be used for sequential and quantitative analysis of 16S rRNA. In this method, RNA extracted from a microbial community is biotin-modified and mixed with

Ken Adachi; Masahiro Yamaguchi; Makoto Nakashige; Takahiro Kanagawa; Masaki Torimura; Satoshi Tsuneda; Yuji Sekiguchi; Naohiro Noda



Assay of multiplex proteins from cell metabolism based on tunable aptamer and microchip electrophoresis.  


A simple and rapid method for multiplex protein assay based on tunable aptamer by microchip electrophoresis has been developed. Different lengths of aptamers can modulate the electrophoretic mobility of proteins, allowing the protein molecules to be effectively separated in hydroxyethyl cellulose buffer with 1.00 mM magnesium ion. A non-specific DNA was exploited as an internal standard to achieve the quantitative assay and to reduce the interference. A fluorescence dye SYBR gold was exploited to improve the sensitivity and to suppress the interference from sample matrix. Under optimum conditions, quantitative assay of PDGF-BB (R(2)=0.9986), VEGF165 (R(2)=0.9909), and thrombin (R(2)=0.9947) were achieved with a dynamic range in the 5.00-150.0 nM and RSDs in the 5.87-16.3% range. The recoveries were varied from 83.6% to 113.1%. Finally, the proposed method was successfully applied to analyze cell secretions, and then the concentration of PDGF-BB and VEGF165 were detected from 5.15 nM to 2.03 nM, and 3.14 to 2.53 nM, respectively, indicating the established method can be used to analyze cell secretions. PMID:25063921

Lin, Xuexia; Chen, Qiushui; Liu, Wu; Yi, Linglu; Li, Haifang; Wang, Zhihua; Lin, Jin-Ming



Free-solution electrophoresis of proteins in an improved density gradient column and by capillary electrophoresis  

Microsoft Academic Search

The electrophoretic mobilities of bovine serum albumin, ?-lactoglobulin A and B, ?-lactalbumin and myoglobin were measured in free solution using an improved version of the Boltz-Todd vertical density-gradient electrophoresis column. Dialysis membranes were used for the isolation of the side-arm electrodes from the column and large-volume electrode containers were connected to each other by a circulating buffer loop. The improvements

K. D. Cole; P. Todd; K. Srinivasan; B. K. Dutta



Compatibility of interspecific Manihot crosses presaged by protein electrophoresis .  


Cross incompatibility of wild Manihot species with cassava (M. esculenta) can impede their utilization for improving this cultigen. We tested whether compatibility could be determined based on electrophoresis results. Manihot pilosa, M. glaziovii, M. reptans, and M. cearulescens were tested. These species were allowed to hybridize with cassava to determine whether hybridization coincides with the similarity index based on electrophoresis analysis. Gene markers of leaf shape, stem surface, disk color, and fruit shape were used to confirm hybridization. Manihot pilosa and M. glaziovii successfully hybridized with cassava, while the others failed to do so under natural conditions. This result coincided with the similarity index from electrophoresis. PMID:20092040

Nassar, N M A; Bomfim, N; Chaib, A; Abreu, L F A; Gomes, P T C



Quantitative prediction of enantioseparation using ?-cyclodextrin derivatives as chiral selectors in capillary electrophoresis.  


?-Cyclodextrin derivatives as chiral selectors are becoming increasingly important for enantioseparations in capillary electrophoresis (CE). Nevertheless, there are some enormous challenges in choosing effective selectors from a variety of compounds, and up to now no systematic quantitative studies for predicting the possibility of enantiomeric separation before CE experiments have been reported. In this paper, in order to resolve previous confusions, we investigated the enantioseparations of ten chiral drugs using a method of combining experiments with theoretical calculations. MMFF, PM3, DFT and ONIOM2 methods were simultaneously utilized during the course of our computer simulations. The results indicated that a specific value of greater than or approximately equal to 6 kJ mol(-1) for the interaction energy difference (??E) between a pair of enantiomers with a selector is required in order to achieve enantiomeric separation. This discovery offers a meaningful reference to predict enantiomeric separations, so as to design and synthesize some more efficient chiral selectors. PMID:25346954

Guo, Xin; Wang, Zhiqiang; Zuo, Lihua; Zhou, Zhixu; Guo, Xingjie; Sun, Tiemin




SciTech Connect

This is the final report of a three-year, Laboratory-Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The goal of this project was to try to detect unstained, untagged, unlabeled DNA bands in real-time during gel electrophoresis using simple thermal measurements. The technical and ES&H advantages to this approach could potentially be quite significant, especially given the extreme importance of gel electrophoresis to a wide variety of practical and research fields. The project was unable to demonstrate sufficient thermal sensitivity to detect DNA bands. It is clear that we still do not understand the gel electrophoresis phenomenon very well. The temperature control techniques developed during the course of this project have other useful applications.




Distribution of the Serum Proteins of Syrian Hamster as revealed by Starch-gel Electrophoresis  

Microsoft Academic Search

ALTHOUGH considerable work has been published on the distribution of the serum proteins of various laboratory animals, little has been reported on the electrophoretic pattern of the serum proteins of hamster. Moore1 investigated the serum proteins of several species of animals, including that of the hamster, by moving boundary electrophoresis. He identified six components in the serum of hamster corresponding

Abolghassem Amin; K. D. Shamloo



Charge Shift Electrophoresis: Simple Method for Distinguishing between Amphiphilic and Hydrophilic Proteins in Detergent Solution  

Microsoft Academic Search

Seventeen hydrophilic proteins and five amphiphilic membrane proteins were subjected to agarose gel electrophoresis in the presence of a nonionic detergent (Triton X-100), a mixture of a nonionic and an anionic detergent (Triton X-100 and sodium deoxycholate), and a mixture of a nonionic and a cationic detergent (Triton X-100 and cetyltrimethylammonium bromide). The electrophoretic mobility of the hydrophilic proteins was

Ari Helenius; Kai Simons




Technology Transfer Automated Retrieval System (TEKTRAN)

High performance capillary electrophoresis (HPCE) is an analytical method that uses a voltage differential to accurately move solvents and solutes through a capillary. HPCE is a relative newcomer to the field of cereal chemistry, it utilizes small inner diameter capillaries as an anti-convective med...


Capillary electrophoresis quantitation of l-L-folinic acid in the presence of its inactive d-L-form.  


The diastereomers d-L and l-L-folinic acid were separated by capillary electrophoresis, using heptakis (2,3-di-O-methyl)-beta-cyclodextrin as a chiral active component in the background electrolyte. The method proved suitable for the quantitation of these compounds in one commercial pharmaceutical preparation. PMID:8404828

Cellai, L; Desiderio, C; Filippetti, R; Fanali, S



A simple cellulose acetate membrane-based small lanes technique for protein electrophoresis.  


Combining electrophoresis with a cellulose acetate membrane-based technique, we developed a simple and low-cost method, named cellulose acetate membrane-based small lanes (CASL), for protein electrophoresis. A home-made capillary plotter controlled by a 3D moving stage was used to create milli-to-micro channels by printing poly(dimethylsiloxane) on to a hydrophilic cellulose acetate membrane. In the hydrophilic channels, 5 nL protein mixture was separated on the basis of electro-migration under an electric field. Compared with polyacrylamide gel electrophoresis (PAGE), CASL resulted in higher protein signal intensity for separation of mixtures containing the same mass of protein. The platform was easily fabricated at low cost (approx. $0.005 for each 1-mm-wide channel), and separation of three protein mixtures was completed in 15 min. Both electrophoresis time and potential affected the separation. Rather than chromatographic separation, this method accomplished application of microchannel techniques for cellulose acetate membrane-based protein electrophoresis. It has potential in proteomic analysis, especially for rapid, low-cost, and low-volume sample analysis in clinical diagnosis. PMID:22752445

Na, Na; Liu, Tingting; Yang, Xiaojun; Sun, Binjie; Ouyang, Jenny; Ouyang, Jin



Evaluation of serum protein electrophoresis in greater rhea ( Rhea americana Linnaeus, 1758)  

Microsoft Academic Search

Serum proteins from 47 healthy greater rheas (Rhea americana; male and female) were separated by electrophoresis in order to characterize normal reference ranges. Determination of total\\u000a protein concentration was performed through biuret reaction. The mean value of total serum protein was 4.4 g\\/dL. Absolute\\u000a concentrations of serum proteins were determined by agarose gel electrophoretic fractioning. Five fractions were analyzed:\\u000a albumin, alpha,

Cybele Esteves Almeida; Barbara Charlotte Bach; Maristela Lovato Flores; Rogério Pereira Fontoura; Stefanie Dickel Segabinazi; Marta Helena Carlesso Aita



Isolation of monodisperse nanodisc-reconstituted membrane proteins using free flow electrophoresis.  


Free flow electrophoresis is used for rapid and high-recovery isolation of homogeneous preparations of functionally active membrane proteins inserted into nanodiscs. The approach enables isolation of integral and membrane anchored proteins and is also applicable following introduction of, e.g., fluorescent tags. Preparative separation of membrane protein loaded nanodiscs from empty nanodiscs and protein aggregates results in monodisperse nanodisc preparations ideal for structural and functional characterization using biophysical methods. PMID:23458128

Justesen, Bo Højen; Laursen, Tomas; Weber, Gerhard; Fuglsang, Anja Thoe; Møller, Birger Lindberg; Pomorski, Thomas Günther



Bence-Jones protein - quantitative  


Immunoglobulin light chains - urine; Urine Bence-Jones protein ... Bence-Jones proteins are a part of regular antibodies called light chains. These proteins are not normally in urine. Sometimes, when ...


Quantitative determination of serum iron in human blood by high-performance capillary electrophoresis  

Microsoft Academic Search

A capillary electrophoretic (HPCE) method that can be used to quantitatively determine trace amounts of iron has been developed and applied to determine the iron level in human serum. After precipitation of serum proteins, Fe(III) in the serum is reduced to Fe(II) with hydroxylamine hydrochloride, and a stable Fe(II)-1,10-phenanthroline complex is formed by adding 1,10-phenanthroline to the supernatant containing 2.5

Ping Che; Jie Xu; Honglan Shi; Yinfa Ma



Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein-Protein Interactions  

PubMed Central

Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound to free ratio. The method was used to screen a library of 3,443 compounds and results compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that reconfirmed in subsequent testing suggesting greater specificity. This finding was attributed to use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens but at the current stage of development it is attractive as a secondary screen to test hits found by higher throughput methods. PMID:24060167

Rauch, Jennifer N.; Nie, Jing; Buchholz, Tonia J.; Gestwicki, Jason E.; Kennedy, Robert T.



Two-dimensional gel electrophoresis mapping of proteins isolated from the hyperthermophile Pyrococcus furiosus  

Microsoft Academic Search

Two-dimensional gel electrophoresis (2DE) in polyacrylamide was used to map the proteins in lysates of the archaeon (formerly archaebacterium) Pyrococcus furiosus and to analyze enzymes purified from P. furiosus. The location of the enzymes in the 2DE maps was determined by comigration of lysate proteins with purified enzymes. A 2DE map of P. furiosus proteins with some identifications was produced,

Carol S. Giometti; Sandra L. Tollaksen; Swarnalatha Mukund; Zhi Hao Zhou; Keren Ma; Xuhong Mai; Michael W. W. Adams



Protein electrophoresis as a diagnostic and prognostic tool in raptor medicine.  


Plasma proteins of 139 healthy adult birds of prey from 10 species were separated by electrophoresis to characterize and document normal reference ranges and species-specific electrophoretic patternsand to evaluate the value of this technique for health screening, disease diagnosis, and prognostic indication. Species studied included bald eagle (Haliaeetus leucocephalus), red-tailed hawk (Buteo jamaicensis), barn owl (Tyto alba), great horned owl (Bubo virginianus), turkey vulture (Cathartes aura), Harris' hawk (Parabuteo unicinctus), Stellar's sea eagle (Haliaeetus pelagicus), barred owl (Strix varia), screech owl (Otus asio), and black vulture (Coragyps atratus). Several clinical cases show the diagnostic/therapeutic value of protein electrophoresis in raptors. This study establishes species-specific reference ranges for several birds of prey and discusses the benefit of electrophoresis as a diagnostic technique in health screens, as a diagnostic aid in conjunction with other tests, and as a prognostic indicator in clinical evaluation of raptors. PMID:11428396

Tatum, L M; Zaias, J; Mealey, B K; Cray, C; Bossart, G D



New ways in qualitative and quantitative protein analysis: Nano chromatography coupled to element mass spectrometry  

Microsoft Academic Search

The potential of inductively coupled plasma-mass spectrometry (ICP-MS), which allows element-specific detection of heteroelements (e.g. Se and S) incorporated in protein structures, is highlighted for sensitive qualitative and quantitative protein analysis. ICP-MS coupled to separation techniques such as size exclusion chromatography and gel electrophoresis (via laser ablation) can be employed at different steps in the proteomic workflow. Special emphasis is

Dirk Schaumlöffel



Concomitant Determination of Absolute Values of Cellular Protein Amounts, Synthesis Rates, and Turnover Rates by Quantitative Proteome Profiling  

Microsoft Academic Search

Two-dimensional gel electrophoresis of protein fractions isolated from 35S-radiolabeled cells provides qualitative information on intracellular amounts, 35S incorporation rates, protein modifications, and subcellular localizations of up to thousands of individual proteins. In this study we extended proteome profiling to provide quantitative data on synthesis rates of individual proteins. We combined fluorescence detection of radiolabeled proteins with SYPRO ruby™ staining and

Christopher Gerner; Susanne Vejda; Dieter Gelbmann; Editha Bayer; Josef Gotzmann; Rolf Schulte-Hermann; Wolfgang Mikulits



Total protein extraction and 2-D gel electrophoresis methods for Burkholderia species.  


The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining. PMID:24192802

Velapatiño, Billie; Zlosnik, James E A; Hird, Trevor J; Speert, David P



Targeted Quantitation of Proteins by Mass Spectrometry  

PubMed Central

Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Key advantages of MRM assays are the ability to target specific peptide sequences, including variants and modified forms, and the capacity for multiplexing that allows analysis of dozens to hundreds of peptides. Different quantitative standardization methods provide options that balance precision, sensitivity, and assay cost. Targeted protein quantitation by MRM and related mass spectrometry methods can advance biochemistry by transforming approaches to protein measurement. PMID:23517332



Seed protein electrophoresis of some cultivated and wild species of Chenopodium  

Microsoft Academic Search

Seed protein profiles of 40 cultivated and wild taxa of Chenopodium have been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The relative similarity between various taxa, estimated by Jaccard’s similarity index and clustered in UPGMA dendrogram, is generally in accordance with taxonomic position, crossability relationships and other biochemical characters. Eight accessions of C. quinoa studied are clustered together and

A. Bhargava; T. S. Rana; S. Shukla; D. Ohri



A chemometric approach to the detection of milk adulteration based on protein profiles determined by capillary electrophoresis.  


The general objective of this study was to utilize chemometrics in the interpretation of capillary electrophoresis milk protein profiles, for the detection of pasteurized milk adulteration with rehydrated milk powder or a rehydrated dairy-based milk substitute. The specific objectives were 1) to collect quantitative data on major casein and whey proteins in authentic and adulterated milks in a single CE analysis; and 2) to apply a pattern recognition procedure, Soft Independent Modeling of Class Analogies (SIMCA), on collected CE protein data, for the development of a statistical model useful in the detection of pasteurized milk adulteration. Authentic samples were fresh milk collected from various farms over a period of six months. Adulterated samples were authentic fresh milk partially or totally substituted with rehydrated milk powder or a rehydrated commercial milk substitute at different levels. Quantitative protein data obtained by capillary free zone electrophoresis for beta-lactoglobulin, alpha-lactalbumin, beta-casein, and alpha-casein of 86 samples, authentic and adulterated samples, were used as a training set to build a SIMCA multivariate statistical model. The detection of sample outliers was useful for the elimination of unusual samples and optimization of the multivariate model. From the 35 commercial pasteurized milks tested, which were treated as unknowns, a total of 14 samples (40%) were not assigned to the authentic or fresh milk group, meaning that these samples had some type of adulteration at the levels included in the training set (> 15%). Decision-making on detecting adulteration of unknown commercial pasteurized milk samples was eased since predictions were based on statistical probabilities. PMID:10797878

Vallejo-Cordoba, B



Affinity liquid chromatography and capillary electrophoresis of seminal plasma proteins.  


Interactions of boar, bull, and human seminal plasma proteins with heparin and phosphorylcholine were studied by affinity LC using heparin immobilized to a Toyopearl support. A step gradient elution from 0.15 to 1.50 M NaCl was employed to elute the seminal plasma proteins. Relative amounts of the heparin-binding fraction of seminal plasma proteins (H+) in seminal plasma of three species were determined. Further on, the fraction of seminal plasma proteins interacting with phosphorylcholine-binding proteins (P+) was evaluated. P+ proteins were not found in human seminal plasma and their highest amount was present in bull seminal plasma. A CE method was developed for separation of seminal plasma proteins. Various capillaries and separation conditions were tested; the best resolution was obtained in a bare-silica capillary, with a micellar system consisting of a 0.02 M borate buffer and 0.05 M SDS pH 10.0. The optimized conditions were applied to the identification of the components in boar plasma. PMID:16830725

Varilová, Tereza; Seménková, Hana; Horák, Pavel; Madera, Milan; Pacáková, Vera; Tichá, Marie; Stulík, Karel



Acute phase protein and protein electrophoresis values for captive Grant's zebra (Equus burchelli).  


Grant's zebra (Equus burchelli) are commonly kept in zoos and are subject to routine health monitoring and research studies. Recently, assays for acute phase proteins (APP) have been described in many wildlife species, and specific assays for serum amyloid A (SAA) have been well validated and studied in horses (Equus ferus caballus), in which it serves as a major APP. In the present study, serum samples from 26 Grant's zebra were subject to analysis by using assays for SAA, haptoglobin (HP), and protein electrophoresis. Reference intervals were calculated by using the robust method: SAA 1.8-31.4 mg/L and HP 0.37-1.58 mg/ml. Significant differences in SAA and HP were observed in clinically abnormal zebra; in some cases, these differences were marked and were noted in the absence of abnormal values for protein electrophoretic fractions. These data indicate that APP may be a valuable and sensitive tool in monitoring inflammation in this species. PMID:24450080

Cray, Carolyn; Hammond, Elizabeth; Haefele, Holly



Effects of renovascular hypertension on myocardial protein patterns: analysis by computer-assisted two-dimensional gel electrophoresis.  


Hypertensive heart disease caused by renovascular hypertension reflects the response of the heart to an increased afterload and neurohormonal stimulation. We hypothesized that in this condition the composition of the myocardial proteins of rats was altered. To identify yet unknown quantitative and qualitative differences in myocardial proteins in renovascular hypertensive heart disease, we analyzed protein patterns by computer-assisted two-dimensional polyacrylamide large gel electrophoresis. Renovascular hypertension was induced by placing a silver clip on the left renal artery in 9-week-old rat siblings. Sham-operated animals served as controls. Systolic blood pressure (197 +/- 19 mm Hg) and heart/body weight ratios (0.36 +/- 0.04%) were significantly increased in the hypertensive animals. Twenty protein patterns from the left ventricle of five hypertensive and five control rats were compared. The molecular mass and isoelectric point (pI) of proteins spots ranging from 13 to 100 kDa and from 4.5 to 8.5, respectively, were determined using marker proteins. In total, 761 +/- 88 protein spots were resolved in all twenty gels. For the quantitative data analysis a univariate (Mann-Whitney test) as well as a multivariate statistical approach (correspondence analysis) were applied. Only one myocardial protein spot (molecular mass = 41.3 kDa; pI = 6.3) was decreased by more than twofold (p < 0.05) in renovascular hypertension. The vast majority of spots did not indicate a significant alteration of intensity. Left ventricular hypertrophy in early renovascular hypertension induces a form of myocardial hypertrophy that conserves the naturally occurring protein expression pattern. PMID:9740066

Pleissner, K P; Regitz-Zagrosek, V; Krüdewagen, B; Trenkner, J; Hocher, B; Fleck, E



Human plasma protein adsorption onto dextranized surfaces: a two-dimensional electrophoresis and mass spectrometry study  

PubMed Central

Protein adsorption is fundamental to thrombosis and to the design of biocompatible materials. We report a two-dimensional electrophoresis and mass spectrometry study to characterize multiple human plasma proteins adsorbed onto four different types of model surfaces: silicon oxide, dextranized silicon, polyurethane and dextranized polyurethane. Dextran was grafted onto the surfaces of silicon and polyurethane to mimic the blood-contacting endothelial cell glycocalyx surface. Surface topography and hydrophobicity/hydrophilicity were determined and analyzed using atomic force microscopy and water contact angle measurements, respectively. Using two-dimensional electrophoresis, we show that, relative to the unmodified surfaces, dextranization significantly inhibits the adsorption of several human plasma proteins including IGHG1 protein, fibrinogen, haptoglobin, Apo A-IV, Apo A-I, immunoglobulin, serum retinal-binding protein and truncated serum albumin. We further demonstrate the selectivity of plasma protein adsorbed onto the different functionalized surfaces and the potential to control and manipulate proteins adsorption on the surfaces of medical devices, implants and microfluidic devices. This result shows that adsorption experiments using a single protein or a binary mixture of proteins are consistent with competitive protein adsorption studies. In summary, these studies indicate that coating blood-contacting biomedical applications with dextran is an effective route to reduce thrombo-inflammatory responses and to surface-direct biological activities. PMID:21277175

Tsai, Irene Y.; Tomczyk, Nancy; Eckmann, Joshua I.; Composto, Russell J.; Eckmann, David M.



Examination of the Protein Composition of the Cell Envelope of Escherichia coli by Polyacrylamide Gel Electrophoresis  

PubMed Central

An envelope preparation containing the cell wall and cytoplasmic membrane of Escherichia coli was obtained by breaking the cells with a French pressure cell and sedimentating the envelope fraction by ultracentrifugation. This fraction was prepared for polyacrylamide gel electrophoresis by dissolving the protein in an acidified N,N?-dimethylformamide, removing lipids by gel filtration in the same organic solvent and removing the solvent by dialysis against aqueous urea solutions. More than 80% of the total protein of the envelope fraction was recovered in soluble form. Electrophoresis on sodium dodecyl sulfate-containing gels yielded from 20 to 30 well-resolved bands of protein. One major protein band was observed on the gels. This protein had a molecular weight of 44,000 and accounted for as much as 40% of the total protein of the envelope fraction. A double-labeling technique was used to examine the protein composition of the envelope fraction from cells grown under different sets of conditions which result in large changes in the levels of membrane-bound oxidative enzymes. These changes in growth conditions resulted in only minor alterations in the protein profiles observed on the gels, suggesting that this organism is able to adapt to changes in growth environment with only minor modifications of the major proteins of the cell envelope. PMID:4923077

Schnaitman, Carl A.



Galactomannan assay and plasma protein electrophoresis findings in psittacine birds with aspergillosis.  


In psittacine birds, the antemortem diagnosis of aspergillosis is usually based on the clinical signalment combined with the results of diagnostic tests such as radiography, routine hematologic and biochemical analysis, and biopsy. For several years, plasma protein electrophoresis has been used as an ancillary diagnostic technique in forming a diagnosis and treatment plan in avian species. More recently, a commercially available assay to measure galactomannan, an Aspergillus species antigen, has been described for clinical use in humans, cattle, horses, dogs, and gyr falcons. This report describes several confirmed cases of aspergillosis, with accompanying clinical data, including plasma protein electrophoresis and galactomannan assay results, in addition to results of traditional evaluations by hematology, radiography, and biopsy. In clinical cases in psittacine birds, the galactomannan assay appears useful for detecting circulating Aspergillus antibody. PMID:19673459

Cray, Carolyn; Reavill, Drury; Romagnano, April; Van Sant, Fern; Champagne, Daphne; Stevenson, Rhoda; Rolfe, Vanessa; Griffin, Chris; Clubb, Susan




Microsoft Academic Search

Hematologic, protein electrophoresis, serum biochemistry, and cholinesterase values were determined in 36 free-living black stork nestlings (Ciconia nigra) between 25 and 53 days of age in order to establish normal reference values for this population. The following values were evaluated: white blood cell counts, red blood cell counts, packed cell volume, hemoglobin, heterophils, lymphocytes, monocytes, eosinophils, prealbumin, albumin, a-globulin, b-globulin,

M. Pilar Lanzarot; M. Victoria Barahona; Manuel I. San Andres; Manuel Fernandez-Garcia; Casilda Rodriguez


Characterization of discontinuous buffer junctions using pH indicators in capillary electrophoresis for protein preconcentration  

Microsoft Academic Search

An effective sample preconcentration technique for proteins and peptides was recently developed using capillary electrophoresis (CE) with discontinuous buffers [C.A. Nesbitt, J.T.-M. Lo, K.K.-C. Yeung, J. Chromatogr. A 1073 (2005) 175]. Two buffers of different pH created a junction to trap the sample molecules at their isoelectric points and resulted in over 1000-fold preconcentration for myoglobin within 30min. To study

Kristina Jurcic; Chandra A. Nesbitt; Ken K.-C. Yeung



Amino Acid Composition, Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L.) Proteins and Protein Fractionations  

PubMed Central

As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE) showed that the isoelectric point was mainly in the range of 4.8–6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa. PMID:24473146

Mao, Xiaoying; Hua, Yufei; Chen, Guogang



Two-dimensional electrophoresis analysis of proteins extracted from Alexandrium sp. LC3  

NASA Astrophysics Data System (ADS)

Two-dimensional electrophoresis(2-DE) of protein extracted and purified from Alexandrium sp. LC3 was conducted. In the SDS-PAGE study, the relative molecular weights of the proteins were mainly in the range of 14kDa-31kDa and 43kDa-66kDa, and more proteins were detected between 14kDa and 31kDa. With the improved protein preparation, the two-dimensional electrophoresis patterns indicated that the relative molecular weights of the proteins were between 14kDa and 100kDa, and most of them ranged from 14kDa to 31kDa. This was consistent with the result of the SDS-PAGE analysis. The isoelectric points were found to lie between 3.0 and 8.0, and most of them were in the range of 3.0 6.0. Better separation effect was acquired with pre-prepared immobilized gradient (IPG) strip (pH3 5.6), and about 320 protein spots could be visualized on the 2-DE map by staining. Within pH3 10 and pH3 5.6 strips, the protein samples of Alexandrium sp. LC3 could be separated well.

Li, Hao; Miao, Jinlai; Cui, Fengxia; Li, Guangyou



The effect of sample treatment on separation profiles of tear fluid proteins: Qualitative and semi-quantitative protein determination by an automated analysis system  

Microsoft Academic Search

Purpose. Qualitative and quantitative determination of tear fluid components is of increasing interest in ophthalmology. Until now, for diagnosis and course control of some diseases of the anterior parts of the eye, different methods for tear fluid protein analysis are available. Results can be obtained by polyacrylamide gel electrophoresis (PAGE), immunochemistry, and high-performance liquid chromatography (HPLC). A new method for

Otto Schmut; Jutta Horwath-Winter; Andrea Zenker; Gabriele Trummer



Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis of freshwater photosynthetic sulfur bacteria.  


Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis (SDS-PAGE) was carried out using different bacterial strains of the photosynthetic sulfur bacteria Chlorobium, Thiocapsa, Thiocystis, and Chromatium cultured in the laboratory, and the natural blooms in two karstic lakes (Lake Cisó and Lake Vilar, NE Spain) where planktonic photosynthetic bacteria (purple and green sulfur bacteria) massively developed accounting for most of the microbial biomass. Several extraction, solubilization, and electrophoresis methods were tested to develop an optimal protocol for the best resolution of the SDS-PAGE. Protein composition from different water depths and at different times of the year was visualized within a molecular mass range between 100 and 15 kDa yielding up to 20 different protein bands. Protein banding patterns were reproducible and changed in time and with depth in agreement with changes in photosynthetic bacteria composition. When a taxonomically stable community was followed in time, differences were observed in the intensity but not in the composition of the SDS-PAGE banding pattern. Three environmental variables directly related to the activity of sulfur bacteria (light, oxygen, and sulfide concentrations) had a significant effect on protein banding patterns and explained 33% of the variance. Changes in natural protein profiles of the bacterial blooms agreed with changes in species composition and in the in situ metabolic state of the populations. PMID:20524118

Osuna, M Begoña; Casamayor, Emilio O



Red wine proteins: two dimensional (2-D) electrophoresis and mass spectrometry analysis.  


The aim of the present study was to optimize protein extraction from red wine (cv. Cabernet) in order to obtain a separation by two-dimensional electrophoresis (2-DE) compatible with mass spectrometry identification. Proteins were denatured by sodium dodecyl-sulphate (SDS) and precipitated as potassium salts. The potassium-DS (KDS) protein complexes obtained were treated with different solutions in order to remove the detergent. Proteins were solubilized with different buffers and separated by different electrophoretic approaches [native, urea, acid urea PAGEs and isoelectric focusing (IEF)] as the first-dimension (1-DE). The best 2D separation was achieved by using 10% saccharose in the DS removal step, and 6-cyclohexylhexyl ?-d-maltoside detergent in the solubilisation buffer combined with the IEF approach. Several well focalized protein spots were obtained and analyzed through mass-spectrometry. PMID:24996352

Mainente, Federica; Zoccatelli, Gianni; Lorenzini, Marilinda; Cecconi, Daniela; Vincenzi, Simone; Rizzi, Corrado; Simonato, Barbara



Reference intervals for acute phase protein and serum protein electrophoresis values in captive Asian elephants (Elephas maximus).  


Acute phase protein (APP) immunoassays and serum protein electrophoresis (SPEP) are assays for evaluating the inflammatory response and have use as diagnostic tools in a variety of species. Acute phase proteins are markers of inflammation that are highly conserved across different species while SPEP separates and quantifies serum protein fractions based on their physical properties. In the current study, serum samples from 35 clinically healthy Asian elephants (Elephas maximus) were analyzed using automated assays for C-reactive protein, serum amyloid A, and haptoglobin and SPEP. Robust methods were used to generate reference intervals for the APPs: C-reactive protein (1.3-12.8 mg/l), serum amyloid A (0-47.5 mg/l), and haptoglobin (0-1.10 mg/ml). In addition, SPEP was performed on these samples to establish reference intervals for each protein fraction. A combination of APPs and SPEP measurements are valuable adjunctive diagnostic tools in elephant health care. PMID:25057161

Isaza, Ramiro; Wiedner, Ellen; Hiser, Sarah; Cray, Carolyn



High-resolution two-dimensional protein electrophoresis of pathological plasma/serum.  


The potential usefulness of an optimalized high-resolution two-dimensional gel electrophoresis (2-DGE) protocol was studied by comparative analysis of plasma/serum obtained from apparently healthy individuals and from patients with a few selected known diseases. Despite their apparent complexity, patient electrophoretograms revealed readily detectable modifications of the 'reference' protein profile for those selected diseases (listed below). Abnormal profiles were characterized by presence or absence of particular spots, by reduction or enlargement of spot size, or by alterations of spot microheterogeneity. Combinations of several modifications enabled different 'disease-associated spot pattern' to be distinguished on the protein maps of patients with: monoclonal gammopathies, hypogammaglobulinemia, hepatic failure, chronic renal failure and hemolytic anemia. This study demonstrates that identification of plasma/serum protein alterations by 2-DGE allows a few selected diseases to be diagnosed solely on the basis of protein map modifications. PMID:1932211

Tissot, J D; Schneider, P; James, R W; Daigneault, R; Hochstrasser, D F



Protein A Detection Based on Quantum Dots-Antibody Bioprobe Using Fluorescence Coupled Capillary Electrophoresis  

PubMed Central

In this report, fluorescence detection coupled capillary electrophoresis (CE-FL) was used to detect Protein A. Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs) to form QDs-antibody bioprobe. Further, we observed fluorescence resonance energy transfer (FRET) from QDs donor to Cy5 acceptor. The bioprobe was formed and brought QDs and Cy5 close enough to allow FRET to occur. After adding protein A, the FRET system was broken and caused the FRET signal to decrease. Thus, a new method for the determination of protein A was proposed based on the FRET signal changes. This study provides a new trail of thought for the detection of protein. PMID:24469315

Qiu, Lin; Bi, Yanhua; Wang, Cheli; Li, Jingyan; Guo, Peilin; Li, Jinchen; He, Weijiang; Wang, Jianhao; Jiang, Pengju



Quantitative study of protein-protein interactions by quartz nanopipettes.  


In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions. PMID:25060094

Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin



Hematologic, protein electrophoresis, biochemistry, and cholinesterase values of free-living black stork nestlings (Ciconia nigra).  


Hematologic, protein electrophoresis, serum biochemistry, and cholinesterase values were determined in 36 free-living black stork nestlings (Ciconia nigra) between 25 and 53 days of age in order to establish normal reference values for this population. The following values were evaluated: white blood cell counts, red blood cell counts, packed cell volume, hemoglobin, heterophils, lymphocytes, monocytes, eosinophils, prealbumin, albumin, alpha-globulin, beta-globulin, gamma-globulin, total protein, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatine kinase, calcium, phosphorus, iron, cholesterol, glucose, triglycerides, uric acid, urea, creatinine, total solids, bile acids, and butyrylcholinesterase. Sex-dependent differences were observed in hemoglobin, prealbumin, albumin, gamma-globulin, total protein, alkaline phosphatase, and triglycerides. Packed cell volume, butyrylcholinesterase, aspartate aminotransferase, creatine kinase, and creatinine increased with age, whereas albumin, mean cell volume, calcium, phosphorus, cholesterol, and total solids decreased with age. These hematologic and serum biochemistry values can be used as reference ranges in free-living black stork nestlings. PMID:16107673

Lanzarot, M Pilar; Barahona, M Victoria; Andrés, Manuel I San; Fernández-García, Manuel; Rodríguez, Casilda



Highly sensitive capillary electrophoresis-mass spectrometry for rapid screening and accurate quantitation of drugs of abuse in urine.  


The combination of capillary electrophoresis (CE) and mass spectrometry (MS) is particularly well adapted to bioanalysis due to its high separation efficiency, selectivity, and sensitivity; its short analytical time; and its low solvent and sample consumption. For clinical and forensic toxicology, a two-step analysis is usually performed: first, a screening step for compound identification, and second, confirmation and/or accurate quantitation in cases of presumed positive results. In this study, a fast and sensitive CE-MS workflow was developed for the screening and quantitation of drugs of abuse in urine samples. A CE with a time-of-flight MS (CE-TOF/MS) screening method was developed using a simple urine dilution and on-line sample preconcentration with pH-mediated stacking. The sample stacking allowed for a high loading capacity (20.5% of the capillary length), leading to limits of detection as low as 2 ng mL(-1) for drugs of abuse. Compound quantitation of positive samples was performed by CE-MS/MS with a triple quadrupole MS equipped with an adapted triple-tube sprayer and an electrospray ionization (ESI) source. The CE-ESI-MS/MS method was validated for two model compounds, cocaine (COC) and methadone (MTD), according to the Guidance of the Food and Drug Administration. The quantitative performance was evaluated for selectivity, response function, the lower limit of quantitation, trueness, precision, and accuracy. COC and MTD detection in urine samples was determined to be accurate over the range of 10-1000 ng mL(-1) and 21-1000 ng mL(-1), respectively. PMID:23680557

Kohler, Isabelle; Schappler, Julie; Rudaz, Serge



Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population.  


Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P < 0.001) serum amyloid A, alpha2-globulin, and haptoglobin concentrations compared with the healthy subgroup. Moreover, serum amyloid A (P = 0.020), alpha2-globulin (P < 0.001) and haptoglobin (P = 0.001) concentrations in cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non-species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease. PMID:25314816

Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine; Kjelgaard-Hansen, Mads; Christensen, Michelle; Hesta, Myriam; Tugirimana, Pierrot; Budd, Jane; Dermauw, Veronique; Janssens, Geert P J



Urine Proteins Identified by Two-Dimensional Differential Gel Electrophoresis Facilitate the Differential Diagnoses of Scrapie  

PubMed Central

The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry. PMID:23704971

Lamoureux, Lise; Simon, Sharon L. R.; Plews, Margot; Ruddat, Viola; Brunet, Simone; Graham, Catherine; Czub, Stefanie; Knox, J. David



Quantitative biophysical characterization of intrinsically disordered proteins.  


Intrinsically disordered proteins (IDPs) are broadly defined as protein regions that do not cooperatively fold into a spatially or temporally stable structure. Recent research strongly supports the hypothesis that a conserved functional role for structural disorder renders IDPs uniquely capable of functioning in biological processes such as cellular signaling and transcription. Recently, the frequency of application of rigorous mechanistic biochemistry and quantitative biophysics to disordered systems has increased dramatically. For example, the launch of the Protein Ensemble Database (pE-DB) demonstrates that the potential now exists to refine models for the native state structure of IDPs using experimental data. However, rigorous assessment of which observables place the strongest and least biased constraints on those ensembles is now needed. Most importantly, the past few years have seen strong growth in the number of biochemical and biophysical studies attempting to connect structural disorder with function. From the perspective of equilibrium thermodynamics, there is a clear need to assess the relative significance of hydrophobic versus electrostatic forces in IDP interactions, if it is possible to generalize at all. Finally, kinetic mechanisms that invoke conformational selection and/or induced fit are often used to characterize coupled IDP folding and binding, although application of these models is typically built upon thermodynamic observations. Recently, the reaction rates and kinetic mechanisms of more intrinsically disordered systems have been tested through rigorous kinetic experiments. Motivated by these exciting advances, here we provide a review and prospectus for the quantitative study of IDP structure, thermodynamics, and kinetics. PMID:25631161

Gibbs, Eric B; Showalter, Scott A



Field-amplified sample stacking ?-cyclodextrin modified capillary electrophoresis for quantitative determination of diastereomeric saponins.  


Successful simultaneous diastereomeric separation and sensitive determination of two pairs of triterpenoidal saponins have been achieved by capillary electrophoresis (CE) using ?-cyclodextrin (?-CD) as a stereoselective agent to cooperate with borate complexation. A usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to the determination of individual saponins by CE. Soyasaponin I ( S1: ), azukisaponin V ( S2: ), bersimoside I ( S3: ) and bersimoside II ( S4: ) could be well separated within 14 min in a fused-silica capillary (60 cm long to the detector with an additional 10 cm to the cathode; 75 µm i.d.). The background electrolyte was borate buffer (80 mM, pH 10), containing 24 mM ?-CD. The separation voltage was 14 kV with a detection wavelength of 195 nm. The sample was electrokinetically injected using a voltage of 16 kV for 12 s. Methanol (70%) was used as the diluent for field-amplified sample stacking after hydrodynamic injection of short water plug (5 cm, 4 s). The method was partially validated for linearity, repeatability, reproducibility, limits of detection and limits of quantification. The correlation coefficients of the calibration curves were all >0.998, and the recoveries were from 98.23 to 96.21%. PMID:24248558

Emara, Samy; Masujima, Tsutomu; Zarad, Walaa; Mohamed, Khaled; Kamal, Maha; Fouad, Marwa; EL-Bagary, Ramzia



Quantitative separation of oxytocin, norfloxacin and diclofenac sodium in milk samples using capillary electrophoresis.  


A simple, sensitive and rapid method has been developed for simultaneous separation and quantification of three different drugs: oxytocin (OT), norfloxacin (NOR) and diclofenac (DIC) sodium in milk samples using capillary electrophoresis (CE) with UV detection at 220 nm. Factors affecting the separation were pH, concentration of buffer and applied voltage. Separation was obtained in less than 9 min with sodium tetraborate buffer of pH 10.0 and applied voltage 30 kV. The separation was carried out from uncoated fused silica capillary with effective length of 50 cm with 75 microm i.d. The carrier electrolyte gave reproducible separation with calibration plots linear over 0.15-4.0 microg/mL for OT, 5-1000 microg/mL for NOR and 3-125 microg/mL for DIC. The lower limits of detection (LOD) were found to be 50 ng/mL for OT, and 1 microg/mL for NOR and DIC. The method was validated for the analysis of drugs in milk samples and pharmaceutical preparations with recovery of drugs within the range 96-100% with RSD 0.9-2.8%. PMID:19402177

Solangi, Amber R; Memon, Saima Q; Mallah, Arfana; Khuhawar, M Y; Bhanger, M I



Quantitative study of protein-protein interactions by quartz nanopipettes  

NASA Astrophysics Data System (ADS)

In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions. Electronic supplementary information (ESI) available: Determination of nanopipette diameter; surface modification scheme; numerical simulation; noise analysis; SPR experiments. See DOI: 10.1039/c4nr02964j

Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin



Serum protein electrophoresis by using high-resolution agarose gel in clinically healthy and Aspergillus species-infected falcons.  


Serum protein electrophoresis has gained importance in avian medicine during the past decade. Interpretation of electrophoretic patterns should be based on species-specific reference intervals and the electrophoresis gel system. In this study, serum protein electrophoresis by using high-resolution agarose gels was performed on blood samples collected from 105 falcons, including peregrine falcons (Falco peregrinus), gyrfalcons (Falco rusticolus), saker falcons (Falco cherrug), red-naped shaheens (Falco pelegrinoides babylonicus), and hybrid falcons, that were submitted to the Dubai Falcon Hospital (Dubai, United Arab Emirates) between 2003 and 2006. Reference values were established in clinically healthy birds and compared with values from falcons infected with Aspergillus species (n = 32). Falcons with confirmed aspergillosis showed significantly lower prealbumin values, which is a novel finding. Prealbumin has been documented in many avian species, but further investigation is required to illuminate the diagnostic significance of this negative acute-phase protein. PMID:23409432

Kummrow, Maya; Silvanose, Christudas; Di Somma, Antonio; Bailey, Thomas A; Vorbrüggen, Susanne



Suitability of two-dimensional electrophoretic protein separations for quantitative detection of mutations  

SciTech Connect

Separation of proteins by two-dimensional electrophoresis (2DE) provides a powerful method for mutagenesis studies, since hundreds of proteins can be monitored simultaneously. In previous mutation studies in which 2DE has been used, only qualitative protein differences were monitored; quantitative protein variations were not evaluated. Although significant differences in protein abundance can be detected by eye, the large number of protein spots present in 2DE patterns together with the large number of individual patterns required for a mutagenesis study would necessitate the use of a computerized analysis system to detect the rare quantitative protein changes indicative of gene deletions or inactivation of genes by point mutations in regulatory genes. A pilot study to search for heritable mutations induced by treatment of mice with either ethylnitrosourea or gamma radiation is underway. Samples are being monitored for quantitative changes that reduce the amount of protein by about 50%. The results of this study indicate that the key methods to improve the application of 2DE to mutation screening are to increase the number of measurable spots (i.e., improve stain sensitivity) and to decrease the spread of values for the volume measurements. Even small improvements in these areas could greatly increase the number of monitorable spots. 9 refs., 4 figs.

Taylor, J.; Anderson, N.L.; Anderson, N.G.; Gemmell, A.; Giometti, C.S.; Nance, S.L.; Tollaksen, S.L.



High resolution two-dimensional gel electrophoresis of human erythrocyte membrane proteins.  

PubMed Central

Three different two-dimensional (2-D) gel electrophoretic techniques have been modified to provide high resolution of human erythrocyte membrane proteins. The resulting gels were referenced to the established one-dimensional (1-D) sodium dodecylsulfate (SDS) gel electrophoretic profile, and the effects of endogenous proteolysis and cytosolic contamination were studied. It is concluded that in vitro proteolysis and cytosolic contamination do not contribute significantly to the patterns observed on the 2-D gels, under the conditions used for erythrocyte ghost preparation. The procedures require only small quantities of blood; as many as twenty 2-D gel profiles can be obtained from 5 ml of blood. The combination of nonequilibrium isoelectric focusing (IEF) in the first dimension, SDS electrophoresis in the second dimension, and very sensitive silver staining techniques resolves more than 250 individual protein spots. This appears to be the most useful single procedure for the analysis of red cell membrane proteins. Membrane protein profiles from patients with Duchenne muscular dystrophy, Wernicke-Korsakoff syndrome, and acanthocytosis with degeneration of the basal ganglia were compared with normal controls. The patterns for Duchenne muscular dystrophy and Wernicke-Korsakoff syndrome were not different from normal patterns. The pattern for the patient with acanthocytosis and degeneration of the basal ganglia consistently showed a high level for one protein in the 100,000 mol. wt. range. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7081216

Copeland, B R; Todd, S A; Furlong, C E



Difference gel electrophoresis (DiGE) identifies differentially expressed proteins in endoscopically-collected pancreatic fluid  

PubMed Central

Alterations in the pancreatic fluid proteome of individuals with chronic pancreatitis may offer insights into the development and progression of the disease. The endoscopic pancreas function test (ePFT) can safely collect large volumes of pancreatic fluid that are potentially amenable to proteomic analyses using difference gel electrophoresis (DiGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pancreatic fluid was collected endoscopically using the ePFT method following secretin stimulation from three individuals with severe chronic pancreatitis and three chronic abdominal pain controls. The fluid was processed to minimize protein degradation and the protein profiles of each cohort, as determined by DiGE and LC-MS/MS, were compared. This DiGE-LC-MS/MS analysis reveals proteins that are differentially expressed in chronic pancreatitis compared to chronic abdominal pain controls. Proteins with higher abundance in pancreatic fluid from chronic pancreatitis individuals include: actin, desmoplankin, alpha-1-antitrypsin, SNC73, and serotransferrin. Those of relatively lower abundance include carboxypeptidase B, lipase, alpha-1-antichymotrypsin, alpha-2-macroglobulin, Arp2/3 subunit 4, glyceraldehyde-3-phosphate dehydrogenase, and protein disulfide isomerase. Endoscopic collection (ePFT) in tandem with DiGE-LC-MS/MS is a suitable approach for pancreatic fluid proteome analysis, however, further optimization of our protocol, as outlined herein, may improve proteome coverage in future analyses. PMID:21792986

Paulo, Joao A.; Lee, Linda S.; Banks, Peter A.; Steen, Hanno; Conwell, Darwin L.



Effective Electrophoretic Mobilities and Charges of Anti-VEGF Proteins Determined by Capillary Zone Electrophoresis  

PubMed Central

Macromolecules such as therapeutic proteins currently serve an important role in the treatment of eye diseases such as wet age-related macular degeneration and diabetic retinopathy. Particularly, bevacizumab and ranibizumab have been shown to be effective in the treatment of these diseases. Iontophoresis can be employed to enhance ocular delivery of these macromolecules, but the lack of information on the properties of these macromolecules has hindered its development. The objectives of the present study were to determine the effective electrophoretic mobilities and charges of bevacizumab, ranibizumab, and model compound polystyrene sulfonate (PSS) using capillary zone electrophoresis. Salicylate, lidocaine, and bovine serum albumin (BSA), which have known electrophoretic mobilities in the literature, were also studied to validate the present technique. The hydrodynamic radii and diffusion coefficients of BSA, bevacizumab, ranibizumab, and PSS were measured by dynamic light scattering. The effective charges were calculated using the Einstein relation between diffusion coefficient and electrophoretic mobility and the Henry equation. The results show that bevacizumab and ranibizumab have low electrophoretic mobilities and are net negatively charged in phosphate buffered saline (PBS) of pH 7.4 and 0.16 M ionic strength. PSS has high negative charge but the electrophoretic mobility in PBS is lower than that expected from the polymer structure. The present study demonstrated that capillary electrophoresis could be used to characterize the mobility and charge properties of drug candidates in the development of iontophoretic drug delivery. PMID:21269789

Li, S. Kevin; Liddell, Mark R.; Wen, He



Separation of proteins by sodium dodecylsulfate capillary electrophoresis in hydroxypropylcellulose sieving matrix with laser-induced fluorescence detection  

Microsoft Academic Search

Sodium dodecyl sulfate capillary electrophoresis by using hydroxypropylcellulose as the sieving matrix was developed for separation of proteins. 3-(2-furoyl)quinoline-2-carboxaldehyde, a fluorogenic dye, was used as the pre-column reagent to label proteins, which allows the use of laser-induced fluorescence to improve the detection sensitivity. Five standard proteins within the molecular mass range of 14?000–97?000 were used to test this method and

Shen Hu; Zheru Zhang; Lillian M Cook; Eric J Carpenter; Norman J Dovichi



The application of whole-cell protein electrophoresis for the classification and identification of basidiomycetous yeast species  

Microsoft Academic Search

The relationships among 65 basidiomycetous yeast strains were determined by one-dimensional electrophoresis of SDS-solubilized whole-cell proteins. Protein profiles were compared by the Pearson product moment correlation coefficient (r). The strains investigated represented species from the generaCystofilobasidium, Filobasidium, Filobasidiella, Kondoa, Leucosporidium, Mrakia andRhodosporidium. Except for the genusMrakia, all species constituted separate protein electrophoretic clusters. The species of the genusMrakia (M. frigida,

Marc Vancanneyt; Eddy Van Lerberge; Jean-Francois Berny; Gregoire L. Hennebert; Karel Kersters



Performing Isoelectric Focusing and Simultaneous Fractionation of Proteins on A Rotary Valve Followed by Sodium Dodecyl – Polyacrylamide Gel Electrophoresis  

PubMed Central

In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl – polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl – polyacrylamide gel electrophoresis (SDS-PAGE, the 2nd-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed. PMID:23819755

Wang, Wei; Lu, Joann J.; Gu, Congying; Zhou, Lei; Liu, Shaorong



Two dimensional non-equilibrium pH gel electrophoresis mapping of cytosolic protein changes caused by dietary protein depletion in mouse liver  

Microsoft Academic Search

Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate the effects of dietary protein depletion on the protein composition of mouse liver cytosol. Analysing the cytosol from both normal and protein depleted liver, the position in gels of more than three hundred protein spots was determined. After 5 days of protein depletion, about 20% of the spots either

Pedro Mariano Sanllorenti; Jorge Rosenfeld; Virginia Paola Ronchi; Pascual Ferrara; Rubén Danilo Conde



Analysis of secondary modifications of mouse mammary tumor virus proteins by two-dimensional gel electrophoresis.  

PubMed Central

The structural proteins of mouse mammary tumor virus (MMTV) were analyzed by two-dimensional electrophoresis on isoelectric focusing and sodium dodecyl sulfate gels. Many of the viral proteins displayed heterogeneity in charge due to variable contents of carbohydrates (in particular, sialic acid) and phosphate residues. Neuraminidase treatment of the virions influenced the isoelectric pattern of the envelope glycoproteins. The glycoproteins of an MMTV variant which was attenuated by replication in feline kidney cells had different isoelectric points. This suggested that the acquisition of an altered carbohydrate configuration had changed the host range of the virus. The major MMTV structural core protein, p27, consisted of two species, which had identical iodinated tryptic peptide compositions but differed in phosphate contents. Another MMTV phosphoprotein, p21, was separated into four different phosphorylated species. Phosphorylation of p21 could be performed in vitro by the MMTV virion-associated protein kinase. This enzyme also has a high affinity for MMTV p30 as a substrate. Possible functions of this enzyme are discussed. Images PMID:6255175

Nusse, R; Janssen, H; de Vries, L; Michalides, R



Detection of the end point temperature of thermal denatured protein in fish and chicken meat through SDS-PAGE electrophoresis  

NASA Astrophysics Data System (ADS)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied in the detection of the end point temperature (EPT) of thermal denatured protein in fish and meat in this study. It was also used in studying the thermal denatured temperature range of proteins in salmon and chicken meat. The results show that the temperature ranges of denatured proteins were from 65°C to 75°C, and these temperature ranges were influenced by the processing methods. Through SDS-PAGE, the features of repeated heating thermal denatured proteins under the same temperature and processing time were studied. The electrophoresis patterns of thermal denatured proteins determined through repeated heating at the same temperature did not exhibit any change. For the detection of cooked fish and meat samples, they were subjected to applying the SDS-PAGE method, which revealed an EPT ranging from 60°C to 80°C.

Gao, Hongwei; Mao, Mao; Liang, Chengzhu; Lin, Chao; Xiang, Jianhai



Hematology, Serum Chemistry, and Serum Protein Electrophoresis Ranges for Free-ranging Roe Deer (Capreolus capreolus) in Sweden.  


Abstract We present the first reference ranges for hematology (n?=?35 animals), serum biochemistry (n?=?62), and serum protein electrophoresis (n?=?32) in physically restrained free-ranging roe deer (Capreolus capreolus). Animals were captured in box traps and physically restrained for blood sampling during the winter in Sweden, 2011-13. No clinically significant sex or age differences were found. PMID:25375949

Küker, Susanne; Huber, Nikolaus; Evans, Alina; Kjellander, Petter; Bergvall, Ulrika A; Jones, Krista L; Arnemo, Jon M



Detection of iron-containing proteins contributing to the cellular labile iron pool by a native electrophoresis metal blotting technique  

Microsoft Academic Search

The labile iron pool (LIP) plays a role in generation of free radicals and is thus the target of chelators used for the treatment of iron overload. We have previously shown that the LIP is bound mostly to high molecular weight carriers (MW>5000). However, the iron does not remain associated with these proteins during native gel electrophoresis. In this study

Ji??? Petrák; Daniel Vyoral



Capillary electrophoresis of hair proteins modified by alcohol intake in laboratory rats.  


A capillary zone electrophoretic method was used to obtain profiles of solubilized rat hair keratin proteins. The same methodology was used to reveal the presence of additional protein peaks in alcohol-consuming rats. Two types of separation were investigated. Alkali-solubilized keratins from hair of rats treated for 5 weeks with 5% ethanol and 2 weeks with 10% ethanol (instead of drinking water) and from controls were analysed. Whereas under alkaline conditions (pH 9.2, 50 mM borate) an additional fraction of "low-sulphur" keratins with the highest anodic mobility of this keratin category was shown in alcohol-treated animals, acid electrophoresis carried out at pH 3.5 in phosphate buffer (50 mM) revealed the presence of two sharp peaks absent in the controls. These findings were confirmed by two-dimensional separations of carboxymethylated keratin samples. An attempt was made to identify further one of the newly occurring fractions in alcohol-consuming animals. It was revealed that the tryptic hydrolysate of "low-sulphur" proteins obtained from alcohol-consuming animals contained a peptide not found in controls. PMID:7581840

Jelínková, D; Deyl, Z; Miksík, I; Tagliaro, F




Technology Transfer Automated Retrieval System (TEKTRAN)

Two-dimensional difference gel electrophoresis (2-D DIGE) is the most effective method utilized to carry out gel-based quantitative proteomics. Unfortunately, the most popular image analysis software used to process DIGE images (DeCyder) produces picking coordinates in a format that is incompatible...


Characterization of low viscosity polymer solutions for microchip electrophoresis of non-denatured proteins on plastic chips.  


In this paper, we study characteristics of polymers (methylcellulose, hypromellose ((hydroxypropyl)methyl cellulose), poly(vinylpyrrolidone), and poly(vinyl alcohol)) with different chemical structures for microchip electrophoresis of non-denatured protein samples in a plastic microchip made of poly(methyl methacrylate) (PMMA). Coating efficiency of these polymers for controlling protein adsorption onto the channel surface of the plastic microchip, wettability of the PMMA surface, and electroosmotic flow in the PMMA microchannels in the presence of these polymers were compared. Also relative electrophoretic mobility of protein samples in solutions of these polymers was studied. We showed that when using low polymer concentrations (lower than the polymer entanglement point) where the sieving effect is substantially negligible, the interaction of the samples with the polymer affected the electrophoretic mobility of the samples. This effect can be used for achieving better resolution in microchip electrophoresis of protein samples. PMID:22685502

Yasui, Takao; Reza Mohamadi, Mohamad; Kaji, Noritada; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu



A multichannel gel electrophoresis and continuous fraction collection apparatus for high-throughput protein separation and characterization.  


To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "Counter Free-Flow" elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of approximately 10-150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 microL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 microg per channel and reduced resolution. PMID:20119951

Choi, Megan; Nordmeyer, Robert A; Cornell, Earl; Dong, Ming; Biggin, Mark D; Jin, Jian



A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization  

SciTech Connect

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.

Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl; Dong, Ming; Biggin, Mark D.; Jin, Jian



Studies on proteinograms in dermatorphytes by disc electrophoresis. Part 2: Protein bands of keratinophilic fungi  

NASA Technical Reports Server (NTRS)

Disc electrophoresis studies on keratinophili fungi demonstrated corresponding proteinograms in morphologically homogeneous strains of the same species, but different in different species of one and the same genus.

Danev, P.; Balabanov, V.; Friedrich, E.



Effect of antifungal agents on protein composition of Candida albicans studied by capillary electrophoresis and chip technology.  


In the present study protein profile of a Candida albicans strain had been examined by chip technology and conventional capillary electrophoresis (CE). Profiles could be characterised by the presence of ten dominating protein peaks. These proteins could be distinguished by both techniques, but their quantity showed significant differences in the electropherograms obtained by CE and chip method. Changes in the protein profile were induced by administration of different antifungal agents. Fluconazole and amphotericin B treatment was able to induce similar changes in the pattern, appearance of a 40-kDa protein and up-regulation of a 60-kDa protein was observed by chip technology. Increase in the quantity of these proteins under stress effect (antifungal treatment) might refer to their stress function in the fungal cell. Treatment of C. albicans cells with MK 94 (fused cyclic Mannich ketone) antifungal compound induced not only the previously mentioned changes, but further specific alterations, appearance of a 19-kDa protein and up-regulation of the low molecular weight proteins. This might refer to the different mode of action of this agent on the fungal cells. Conventional capillary electrophoresis was suitable to detect the appearance of the 19-kDa peak, and up-regulation of the 60 kDa protein, but the other changes could not be detected by this technique. Shorter running time, more effective and baseline separation of proteins refer to the advantages of microchip-based method in the analysis of complex biological samples. PMID:16556464

Kustos, Ildikó; Nyul, Adrienn; Lóránd, Tamás; Kocsis, Béla; Kilár, Ferenc



Determination of vegetal proteins in milk powder by sodium dodecyl sulfate-capillary gel electrophoresis: interlaboratory study.  


An interlaboratory study, with the participation of 8 laboratories, was conducted to evaluate a sodium dodecyl sulfate-capillary gel electrophoresis method for determination of adulteration of milk powder with soy and pea proteins. Calibration standards (0-8%, w/w, soy and pea protein in total protein) and adulterated skim milk powders (0-5%, w/w, soy and pea proteins in total protein) were produced. Vegetal proteins were determined after removal of milk proteins by pretreatment of the samples with tetraborate-EDTA buffer, pH 8.3. Repeatability standard deviations ranged from 9 to 15% and reproducibility standard deviations ranged from 25 to 30% in the samples containing 5% vegetal protein in total protein. PMID:12374408

Manso, María A; Cattaneo, Tiziana Maria; Barzaghi, Stefania; Olieman, Cornelis; López-Fandiño, Rosina



Characterization of discontinuous buffer junctions using pH indicators in capillary electrophoresis for protein preconcentration.  


An effective sample preconcentration technique for proteins and peptides was recently developed using capillary electrophoresis (CE) with discontinuous buffers [C.A. Nesbitt, J.T.-M. Lo, K.K.-C. Yeung, J. Chromatogr. A 1073 (2005) 175]. Two buffers of different pH created a junction to trap the sample molecules at their isoelectric points and resulted in over 1000-fold preconcentration for myoglobin within 30 min. To study the formation of pH junctions in CE, a pH indicator, bromothymol blue, is used in this work to reveal the pH changes at the discontinuous buffer boundary. Bromothymol blue (BTB) exhibits a drastic change in its visible absorption spectrum (300-600 nm) going from the acidic to basic pH conditions, and is therefore ideal for visualizing the changes in pH at the junctions created by various buffer combinations. Preconcentration of myoglobin was performed in discontinuous buffers containing BTB. Major differences in the BTB absorption profiles were identified from buffer systems that differ significantly in preconcentration performance, which in turn, allowed for the identification of ideal buffers for sample preconcentration. Up to 2000-fold preconcentrations of myoglobin were achieved in the buffer systems studied in this work. In addition, the role of the electroosmotic flow (EOF) on the preconcentration performance was investigated. A low EOF was found to be desirable, as the pH junction could stay longer in the capillary for accumulation of proteins. The pH junction also displayed characteristics to resist bandbroadening. Potential laminar flow resulted from the mismatched residual EOFs under the two pH conditions within the discontinuous buffers appeared to have minimal effect on the preconcentration. In fact, external applied pressure can be used to control the migration of the pH junction without compromising the protein preconcentration. PMID:17022988

Jurcic, Kristina; Nesbitt, Chandra A; Yeung, Ken K-C



Two-dimensional electrophoresis of Cereus peruvianus (Cactaceae) callus tissue proteins.  


Two-dimensional electrophoresis of Cereus peruvianus callus tissues grown in culture media containing two different 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin combinations was used to identify minor differences in polypeptide composition of these cell clones. Altered expression during growth in the two 2,4-D and kinetin combinations was apparent for 13 polypeptides when calluses in the two media were compared. The number of proteins with differential expression (presence or absence of specific spots) was higher in callus tissues cultured in the 4.0 mg/L 2,4-D and 8.0 mg/L kinetin combination than in callus tissues cultured in the 4.0 mg/L 2,4-D and 4.0 mg/L kinetin combination. The present results show that the callus tissues maintained at 4.0 mg/L 2,4-D and 8.0 mg/L kinetin can be used as a matrix for in vitro selection programs. PMID:10217179

Mangolin, C A; Ottoboni, L M; Machado, M F



Separation of proteins by zone electrophoresis on-line coupled with isotachophoresis on a column-coupling chip with conductivity detection.  


This feasibility study deals with the separations of proteins by an on-line combination of zone electrophoresis (ZE) with isotachophoresis (ITP) on a poly(methylmethacrylate) column-coupling (CC) chip with integrated conductivity detection. ITP and ZE provided specific analytical functions while performing the cationic mode of the separation. ITP served, mainly, for concentrations of proteins and its concentrating power was beneficial in reaching a low dispersion transfer (injection) of the proteinous constituents, loaded on the CC chip in a 960 nL volume, into the ZE separation stage. This was complemented by an electrophoretically driven removal of the sample constituents migrating in front of the focused proteins from the separation system before the ZE separation. On the other hand, ZE served as a final separation (destacking) method and it was used under the separating conditions providing the resolutions and sensitive conductivity detections of the test proteins. In this way, ITP and ZE cooperatively contributed to low- or sub-microg/mL concentration detectabilities of proteins and their quantitations at 1-5 microg/mL concentrations. However, a full benefit in concentration detectabilities of proteins, expected from the use of the ITP-ZE combination, was not reached in this work. Small adsorption losses of proteins and detection disturbances in the ZE stage of separation, very likely due to trace constituents concentrated by ITP, appear to set limits in the detection of proteins in our experiments. The ITP-ZE separations were carried out in a hydrodynamically closed separation compartment of the chip with suppressed hydrodynamic and electroosmotic flows of the electrolyte solutions. Such transport conditions, minimizing fluctuations of the migration velocities of the separated constituents, undoubtedly contributed to highly reproducible migrations of the separated proteins (fluctuations of the migration time of a particular protein were typically 0.5% RSD in repeated ITP-ZE runs). PMID:15565671

Olvecká, Eva; Kaniansky, Dusan; Pollák, Branislav; Stanislawski, Bernd



Structural analysis of protein complexes with sodium alkyl sulfates by small-angle scattering and polyacrylamide gel electrophoresis.  


Small-angle X-ray (SAXS) and neutron (SANS) scattering is used to probe the structure of protein-surfactant complexes in solution and to correlate this information with their performance in gel electrophoresis. Proteins with sizes between 6.5 to 116 kDa are denatured with sodium alkyl sulfates (SC(x)S) of variable tail lengths. Several combinations of proteins and surfactants are analyzed to measure micelle radii, the distance between micelles, the extension of the complex, the radius of gyration, and the electrophoretic mobility. The structural characterization shows that most protein-surfactant complexes can be accurately described as pearl-necklace structures with spherical micelles. However, protein complexes with short surfactants (SC(8)S) bind with micelles that deviate significantly from spherical shape. Sodium decyl (SC(10)S) and dodecyl (SC(12)S, more commonly abbreviated as SDS) sulfates result in the best protein separations in standard gel electrophoresis. Particularly, SC(10)S shows higher resolutions for complexes of low molecular weight. The systematic characterization of alkyl sulfate surfactants demonstrates that changes in the chain architecture can significantly affect electrophoretic migration so that protein-surfactant structures could be optimized for high resolution protein separations. PMID:21182321

Ospinal-Jiménez, Mónica; Pozzo, Danilo C



The application of whole-cell protein electrophoresis for the classification and identification of basidiomycetous yeast species.  


The relationships among 65 basidiomycetous yeast strains were determined by one-dimensional electrophoresis of SDS-solubilized whole-cell proteins. Protein profiles were compared by the Pearson product moment correlation coefficient (r). The strains investigated represented species from the genera Cystofilobasidium, Filobasidium, Filobasidiella, Kondoa, Leucosporidium, Mrakia and Rhodosporidium. Except for the genus Mrakia, all species constituted separate protein electrophoretic clusters. The species of the genus Mrakia (M. frigida, M. gelida, M. nivalis and M. stokesii) show highly similar protein patterns, suggesting that these four species may be synonymous. Strains of two varieties of Filobasidiella neoformans, F. neoformans var. neoformans and F. neoformans var. bacillispora, could not be differentiated by protein electrophoresis. For the delineation of the protein electrophoretic clusters of the yeasts studied, literature data relying on other criteria, such as DNA base composition, carbon source utilization patterns, enzymatic protein electrophoregrams, ubiquinone systems, DNA-DNA homology and rRNA sequence data were used. It was demonstrated that a database of SDS-protein patterns provides a valuable tool for the identification of yeasts. PMID:1575470

Vancanneyt, M; Van Lerberge, E; Berny, J F; Hennebert, G L; Kersters, K



Plasma protein electrophoresis in birds: comparison of a semiautomated agarose gel system with an automated capillary system.  


Plasma agarose gel electrophoresis (AGE) is recognized as a very reliable diagnostic tool in avian medicine. Within the last 10 years, new electrophoresis techniques such as capillary zone electrophoresis (CZE) have emerged in human laboratory medicine but have never been investigated in birds. To investigate the use of CZE in birds and to compare it with AGE, plasma samples from 30 roosters (Gallus gallus), 20 black kites (Milvus migrans), and 10 racing pigeons (Columba livia) were analyzed by both AGE and CZE. For the 3 species studied, values determined by AGE and CZE were well correlated for albumin and beta and gamma fractions whereas other values differed significantly. Values for alpha-3 fraction in the rooster, alpha-1 fraction in the black kite, and alpha fractions in the pigeon obtained by AGE were very well correlated with the prealbumin fraction values obtained by CZE. Repeatability and reproducibility appeared higher with CZE than with AGE. Although the interpretation of CZE electrophoresis patterns seems to produce results similar to those obtained with AGE, some proteins present in the alpha fraction measured with AGE migrated to the prealbumin fraction found with CZE. Although CZE requires the use of specific reference intervals and a much higher sample volume, this method has many advantages when compared with AGE, including better repeatability and reproducibility and higher analysis output. PMID:23971218

Roman, Yannick; Bomsel-Demontoy, Marie-Claude; Levrier, Julie; Chaste-Duvernoy, Daniel; Saint Jalme, Michel



Use of polyacrylamide gel moving boundary electrophoresis to enable low-power protein analysis in a compact microdevice.  


In designing a protein electrophoresis platform composed of a single-inlet, single-outlet microchannel powered solely by voltage control (no pumps, values, injectors), we adapted the original protein electrophoresis format-moving boundary electrophoresis (MBE)-to a high-performance, compact microfluidic format. Key to the microfluidic adaptation is minimization of injection dispersion during sample injection. To reduce injection dispersion, we utilize a photopatterned free-solution-polyacrylamide gel (PAG) stacking interface at the head of the MBE microchannel. The nanoporous PAG molecular sieve physically induces a mobility shift that acts to enrich and sharpen protein fronts as proteins enter the microchannel. Various PAG configurations are characterized, with injection dispersion reduced by up to 85%. When employed for analysis of a model protein sample, microfluidic PAG MBE baseline-resolved species in 5 s and in a separation distance of less than 1 mm. PAG MBE thus demonstrates electrophoretic assays with minimal interfacing and sample handling, while maintaining separation performance. Owing to the short separation lengths needed in PAG MBE, we reduced the separation channel length to demonstrate an electrophoretic immunoassay powered with an off-the-shelf 9 V battery. The electrophoretic immunoassay consumed less than 3 ?W of power and was completed in 30 s. To our knowledge, this is the lowest voltage and lowest power electrophoretic protein separation reported. Looking forward, we see the low-power PAG MBE as a basis for highly multiplexed protein separations (mobility shift screening assays) as well as for portable low-power diagnostic assays. PMID:22971048

Duncombe, Todd A; Herr, Amy E



A comparison of three serological assays, protein gel electrophoresis and the polymerase chain reaction for the detection of Chlomydia psittici infections in pet birds  

E-print Network

Three serological assays, protein gel electrophoresis hics. and the polymerase chain reaction assays were evaluated in psittacine birds. Birds suspected of Chlamydia psittaci infections were identified and tested with the following assays...

Hofle, Michael David



Proteomic Analysis of Methylarginine-Containing Proteins in HeLa Cells by Two-Dimensional Gel Electrophoresis and Immunoblotting with a Methylarginine-Specific Antibody  

Microsoft Academic Search

Protein arginine methylation is found in many nucleic acid binding proteins affecting numerous cellular functions. In this\\u000a study we identified methylarginine-containing proteins in HeLa cell extracts by two-dimensional electrophoresis and immunoblotting\\u000a with a methylarginine-specific antibody. Protein spots with matched protein stain and blotting signals were analyzed by mass\\u000a spectrometry. The identities of 12 protein spots as 11 different proteins were

Chien-Jen Hung; Yu-Jen Lee; Da-Huang Chen; Chuan Li



Mining Quantitative Association Rules in Protein Sequences  

E-print Network

acid sequences of proteins are not random, and thus the patterns of amino acids that we observe of associations between different amino acids that are present in a protein. This very basic analysis pro- vides insights into the co-occurrence of certain amino acids in a protein. Such association rules are desirable

Mitra, Pabitra


Capillary electrophoresis as a routine industrial tool for quantitative analytical testing Determination of sodium dimethyldithiocarbamate in effluents  

Microsoft Academic Search

The determination of specific anionic species (sodium dimethyldithiocarbamate) in waste water was performed by capillary electrophoresis with direct UV detection. The application of new technology sulfonic acid polymer-coated capillary columns achieved sensitive analysis with robust electroosmotic flow (EOF), where other coated columns and conventional fused-silica had failed due to analyte adsorption problems. Optimum conditions for the separation of cationic, neutral,

Albert J. Nitowski; Areej A. Nitowski; Jennifer A. Lent; David W. Bairley; Deborah Van Valkenburg



Quantitative thermodynamic model for globular protein folding  

NASA Astrophysics Data System (ADS)

We present a statistical mechanics formalism for theoretical description of the process of protein folding ? unfolding transition in water environment. The formalism is based on the construction of the partition function of a protein obeying two-stage-like folding kinetics. Using the statistical mechanics model of solvation of hydrophobic hydrocarbons we obtain the partition function of infinitely diluted solution of proteins in water environment. The calculated dependencies of the protein heat capacities upon temperature are compared with the corresponding results of experimental measurements for staphylococcal nuclease and metmyoglobin.

Yakubovich, Alexander V.; Solov'yov, Andrey V.



Determination of free acidic and alkaline residues of protein via moving reaction boundary titration in microdevice electrophoresis.  


As two important physico-chemical parameters, the acidic and alkaline residues of protein are of evident significance for the evaluation of protein properties and the design of relevant separation and analysis. However, there is still no electrophoretic method used for the direct detection of free acidic and alkaline residues of protein. Herein, we developed the concepts of moving reaction boundary (MRB) and MRB titration, relevant MRB titration theory, and the method of microdevice electrophoresis for the determination of free acidic and alkaline residues of protein. In the MRB titration, the boundary was created with acid or alkali and target protein immobilized via highly cross-linked polyacrylamide gel (PAG). It was theoretically revealed that the number of free acidic or alkaline residues of protein was as a function of MRB displacement in the electrophoretic titration system. As a proof of concept, seven model proteins were chosen for the determination of acidic or alkaline residues of protein via MRB titration. The results showed that the numbers of free acidic and alkaline residues of proteins detected were in good agreement with those obtained from the relevant amino sequences in the NCBI database, demonstrating the feasibility of the developed concept, theory and technique. The general methodology of MRB titration has potential application for inexpensive, facilitative and informative protein structure analysis of free acidic or alkaline residues of protein. PMID:23671907

Wang, Hou-yu; Li, Si; Tang, Yun-yun; Dong, Jing-yu; Fan, Liu-yin; Cao, Cheng-xi



Definition of Mycobacterium tuberculosis Culture Filtrate Proteins by Two-Dimensional Polyacrylamide Gel Electrophoresis, N-Terminal Amino Acid Sequencing, and Electrospray Mass Spectrometry  

Microsoft Academic Search

A number of the culture filtrate proteins secreted by Mycobacterium tuberculosis are known to contribute to the immunology of tuberculosis and to possess enzymatic activities associated with pathogenicity. However, a complete analysis of the protein composition of this fraction has been lacking. By using two-dimensional polyacrylamide gel electrophoresis, detailed maps of the culture filtrate proteins of M. tuberculosis H37Rv were




Broad-spectrum Four-dimensional Orthogonal Electrophoresis: A Novel Comprehensively Feasible System for Protein Complexomics Investigation*  

PubMed Central

The major challenge of “protein complexomics” is to separate intact protein complexes or interactional proteins without dissociation or denaturation from complex biological samples and to characterize structural subunits of protein complexes. To address these issues, we developed a novel approach termed “broad-spectrum four-dimensional orthogonal electrophoresis (BS4-DE) system,” which is composed of a nondenaturing part I and denaturing part II. Here we developed a mild acidic-native-PAGE to constitute part I, together with native-thin-layer-IEF and basic-native-PAGE, widening the range of BS4-DE system application for extremely basic proteins with the range of pI from about 8 to 11 (there are obviously 1000 kinds of proteins in this interval), and also speculated on the mechanism of separating. We first proposed ammonium hydroxide-ultrasonic protein extractive strategy as a seamless connection between part I and part II, and also speculated on the extractive mechanism. More than 4000 protein complexes could be theoretically solved by this system. Using this approach, we focus on blood rich in protein complexes which make it challenging to sera/plasma proteome study. Our results indicated that the BS4-DE system could be applied to blood protein complexomics investigation, providing a comprehensively feasible approach for disease proteomics. PMID:22375076

Wang, Xiaodong; Li, Fenjie; Song, Gaoguang; Guo, Shuai; Liu, Hui; Chen, Guoqiang; Li, Zhili



Colorful Electrophoresis  

NSDL National Science Digital Library

In this activity, learners follow step-by-step instructions to build a gel electrophoresis chamber using inexpensive materials from local hardware and electronic stores. Then, learners follow instructions to simulate DNA electrophoresis using food colors from the kitchen pantry.

Utah, University O.



MP 33200 EZQ Protein Quantitation Kit  

E-print Network

for future use. 1.2 Prepare dilutions of the ovalbumin stock solution. Prepare standards by making serial Standards 1.1 Make a stock solution of ovalbumin. The ovalbumin (Com- ponent D) supplied with the kit can be used to make protein standards for the assay. To make a 10 mg/mL stock solution, add 200 µL of buffer

Lebendiker, Mario


Heterogeneity of a labeled tumor surface protein from a murine lung carcinoma demonstrated by two-dimensional electrophoresis  

SciTech Connect

Heterogeneity of a tumor surface protein (designated TSP-180) has been demonstrated by two-dimensional electrophoresis. Line 1 carcinoma cells derived from a spontaneous alveolar carcinoma of BALB/c mice were labeled externally with /sup 125/I by use of lactoperoxidase or metabolically with (/sup 3/H)-leucine before cell proteins were solubilized with Triton X-100 detergent. Immunoprecipitates prepared with heterologous antisera allowed comparison of two-dimensional patterns of line 1 surface proteins labeled with /sup 125/I or /sup 3/H. The isoelectric point of /sup 125/I-labeled TSP-180 was heterogeneous and varied between 6.1 and 6.3. Treatment with neuraminidase shifted the pI values to between 5.9 and 6.1 and reduced, but did not eliminate, the banding heterogeneity. These data show that charge heterogeneity due to sialization, as well as other factors, exists in TSP-180.

Eisinger, R.W. (Univ. of Tennessee, Oak Ridge); Kennel, S.J.



Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis  

SciTech Connect

The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various time postinfection were also analyzed. At least 13 proteins labeled with (/sup 3/H)glucosamine were detected in vaccinia-infected HeLa cells.

Carrasco, L.; Bravo, R.



Quantitation, networking, and function of protein phosphorylation in plant cell.  


Protein phosphorylation is one of the most important post-translational modifications (PTMs) as it participates in regulating various cellular processes and biological functions. It is therefore crucial to identify phosphorylated proteins to construct a phosphor-relay network, and eventually to understand the underlying molecular regulatory mechanism in response to both internal and external stimuli. The changes in phosphorylation status at these novel phosphosites can be accurately measured using a (15)N-stable isotopic labeling in Arabidopsis (SILIA) quantitative proteomic approach in a high-throughput manner. One of the unique characteristics of the SILIA quantitative phosphoproteomic approach is the preservation of native PTM status on protein during the entire peptide preparation procedure. Evolved from SILIA is another quantitative PTM proteomic approach, AQUIP (absolute quantitation of isoforms of post-translationally modified proteins), which was developed by combining the advantages of targeted proteomics with SILIA. Bioinformatics-based phosphorylation site prediction coupled with an MS-based in vitro kinase assay is an additional way to extend the capability of phosphosite identification from the total cellular protein. The combined use of SILIA and AQUIP provides a novel strategy for molecular systems biological study and for investigation of in vivo biological functions of these phosphoprotein isoforms and combinatorial codes of PTMs. PMID:23316209

Zhu, Lin; Li, Ning



New ways in qualitative and quantitative protein analysis: nano chromatography coupled to element mass spectrometry.  


The potential of inductively coupled plasma-mass spectrometry (ICP-MS), which allows element-specific detection of heteroelements (e.g. Se and S) incorporated in protein structures, is highlighted for sensitive qualitative and quantitative protein analysis. ICP-MS coupled to separation techniques such as size exclusion chromatography and gel electrophoresis (via laser ablation) can be employed at different steps in the proteomic workflow. Special emphasis is made on the couplings of capillary and nanoHPLC to ICP-MS that required the development of dedicated interfaces. Element-specific peptide mapping by nanoHPLC-ICP-MS has turned out to be a key technique in combination with peptide sequencing via nanoHPLC-electrospray MS. This could impressively be demonstrated for the identification of selenium-containing proteins in selenium-rich yeast. Furthermore the potential of sulfur isotope dilution analysis in nanoHPLC-ICP-MS is presented as generic tool for highly accurate, absolute protein quantification. PMID:18039489

Schaumlöffel, Dirk



Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

In cooperation with the RAS Initiative, the NCI's Clinical Proteomic Tumor Analysis Consortium (CPTAC) has launched a project to develop quantitative assays for proteins and phosphopeptides involved in RAS signaling. Within the next 1-2 years these assays should allow the amounts and phosphorylation states of tens of RAS and RAS-related proteins to be determined in tumor samples, cell lines, or cancer models in a single run.


The contribution of genetic and environmental factors to quantitative variability of erythrocyte membrane proteins in primary hypotension.  


Our previous studies have shown that, compared with healthy individuals, patients with primary arterial hypotension (PAH) have significant quantitative changes in erythrocyte membrane proteins. The purpose of the present study was to evaluate the contribution made by genetic and environmental factors to quantitative variation of erythrocyte membrane proteins in PAH. We studied 109 hypotensive patients, 124 normotensive subjects, 222 of their first-degree relatives and 24 twin pairs by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. The decomposition of total phenotypic variance of erythrocyte membrane proteins to genetic and environmental components was performed on the basis of correlations among first-degree relatives by the least squares method. The genetic dominance and shared environmental factors were found to influence the variability of cytoskeletal membrane proteins whose contents were changed in PAH. Furthermore, variations in alpha-spectrin, actin and anion exchanger in hypotensives were substantially influenced by major gene and maternal effects. Ankyrin 2.1 and actin content was under the control of common underlying genes. Variations in membrane-associated glutathione-S-transferase and tropomyosin were predominantly affected by polygenes. These findings suggest that the putative major genes with pleiotropic effects appear to be involved in the control of quantitative disorders of erythrocyte membrane proteins in primary hypotension. PMID:15638825

Ivanov, V P; Polonikov, A V; Solodilova, M A



Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.  


Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots. PMID:25062492

Bertolde, F Z; Almeida, A-A F; Silva, F A C; Oliveira, T M; Pirovani, C P



Electrolytic Reduction: Modification of Proteins Occurring in Isoelectric Focusing Electrophoresis and in Electrolytic Reactions in the Presence of High Salts  

PubMed Central

Artifacts in two-dimensional electrophoresis (2-DE) caused by the presence of salts in isoelectric focusing (IEF) have been previously described as a result of increasing conductivity and inducing electroosmosis. However, electrolysis induced by the presence of salts should not be disregarded. In this study, electrolytic reduction?oxidation reaction (redox) was found to be enhanced in the presence of salts in IEF. The consequence of the electrolytic redox leads to acidification of the low-pH region and alkalization of the high-pH region within the immobilized pH gradient (IPG) strip. As a result, a breakdown of immobilized pH buffer near the high pH region of IPG strips along with reduction of basic proteins resulted in uncharacterized artifacts in 2-DE. Electrolytic reduction in the presence of alkali and alkaline metal ions was demonstrated to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protein disulfide bonds, and protein carboxylic acids. Importantly, semipreparative electrolytic reduction of proteins can be carried out in the presence of sodium ions in a homemade electrolytic apparatus. These findings give additional explanations to the observed artifacts in 2-DE and reveal the unknown effects of salts in IEF. Moreover, we have provided a method with the potential to convert proteins or peptides to corresponding modified products containing aldehyde groups that can be used for conjugation with amine-containing compounds. PMID:19438264



Quantitative analysis of tryptophan analogue incorporation in recombinant proteins.  


Three different methods to quantitate tryptophan (Trp) analogue incorporation into recombinant proteins are described: first, spectroscopic analysis based on a linear combination of the absorption spectra of the aromatic residues in the denatured Trp-containing or analogue-substituted protein; second, chromatographic separation of analogue-substituted and Trp-containing proteins by HPLC; and third, mass spectrum analysis of the mixture of analogue-substituted and Trp-containing proteins. An accurate estimate of analogue incorporation in single-Trp proteins can be obtained directly by either analysis of the absorption spectrum or HPLC chromatography. While analysis of the absorption spectrum or HPLC chromatogram can provide an assessment of the average level of analogue incorporation for proteins that contain two or more Trp residues, mass spectroscopy analysis of peptides generated by protease digestion and separated by HPLC provides a general method for a complete quantitative description of the distribution of analogue incorporation. The more complex analysis by mass spectroscopy becomes important for multi-Trp proteins because the distribution of analogue versus Trp-containing polypeptide chains may not be the same as that predicted on the basis of average level of analogue incorporation. PMID:11743694

Senear, Donald F; Mendelson, Robert A; Stone, Deborah B; Luck, Linda A; Rusinova, Elena; Ross, J B Alexander



Correlation between the charge of proteins in solution and in the gas phase investigated by protein charge ladders, capillary electrophoresis, and electrospray ionization mass spectrometry  

SciTech Connect

Charge ladders of bovine carbonic anhydrase II, hen egg-white lysozyme, and bovine pancreatic trypsin inhibitor, prepared by partial acetylation of primary amino groups on the surface of the protein, have been analyzed by capillary electrophoresis (CE) and on-line electrospray ionization mass spectrometry (ESIMS) using solution conditions that maintain the native structure of the protein. CE was used to separate the proteins that constitute the charge ladder into individual rungs-protein derivatives that have the same number of acetylated amino groups and approximately the same net charge in solution. ESI was used to produce ions i the gas phase of the proteins that constitute each rung of the charge ladder; the mass spectra of these ions were obtained and analyzed. The distributions in charge states observed in the gas phase for the groups of proteins comprising each rung of the charge ladders were narrow, consistent with the retention of a compact structure of the proteins in the gas phase, and substantially independent of the number of acetylated amino groups. The ions observed in the gas phase had surface charge densities in a relatively narrow range of {approximately}0.9--1.5 units of charge per 10{sup 3}{angstrom}{sup 2} of surface area (as estimated from crystallographic structures). These results demonstrate that the distribution of charge states for proteins produced in the gas phase by ESI do not necessarily reflect the net charge of the protein in solution or the number of amino groups on the protein.

Carbeck, J.D.; Gao, J.; Smith, R.D.; Whitesides, G.M. [Harvard Univ., Cambridge, MA (United States). Dept. of Chemistry and Chemical Biology] [Harvard Univ., Cambridge, MA (United States). Dept. of Chemistry and Chemical Biology; Severs, J.C.; Wu, Q. [Pacific Northwest National Lab., Richland, WA (United States). Environmental Molecular Science Lab.] [Pacific Northwest National Lab., Richland, WA (United States). Environmental Molecular Science Lab.



Quantitative Protein Localization Signatures Reveal an Association between Spatial and Functional Divergences of Proteins  

PubMed Central

Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein functions and how these functions were acquired in cells from different organisms or species. A public web interface of PLAST is available at PMID:24603469

Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling



Detection and quantitation of a bioactive compound in Vicia narbonensis L. seeds by capillary electrophoresis-mass spectrometry: a comparative study with UV detection.  


Capillary zone electrophoresis with mass spectrometry (CE-MS) and UV detection (CE-UV) was applied to the quantitative determination of gamma-glutamyl-S-ethenyl-cysteine (GEC), a bioactive and unstable compound present in Vicia narbonensis L. seeds. This compound is responsible for, among other negative effects, palatability reduction and grain toxicity. In order to carry out the quantitative analysis of GEC, different conditions (such as composition, concentration and pH of the background electrolyte, and type and time of extraction) were studied. Also, adequate conditions for electrospray-mass spectrometry of this bioactive compound were investigated. The best extraction conditions of GEC from V. narbonensis L. seeds flour were obtained using ethanol-water (70:30 v/v) for 45 min. The use of a 20 m ammonium hydrogen carbonate at pH 7 provided adequate analytical conditions compatible with the unstable nature of GEC as well as with the requirements of CE-UV and CE-MS analysis. A comparative study was carried out between the different figures of merit of CE-UV and CE-MS for quantitative purposes. Both techniques provided similar limit of detection and can be applied with confidence within the same linear dynamic range. However, reproducibility and speed of analysis were better using CE-UV. The developed methods were readily applied to quantify GEC in seeds of 21 genotypes of V. narbonensis L. A good agreement between CE-MS and CE-UV results was observed corroborating the usefulness of both approaches for quantitative purposes. PMID:15966020

Arias, Marina; Simó, Carolina; Ortiz, Luis T; de Ios Mozos-Pascual, Marcelino; Barbas, Coral; Cifuentes, Alejandro



Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry  

Technology Transfer Automated Retrieval System (TEKTRAN)

Wheat flour is one of the world's major food ingredients, but it is difficult to distinguish and identify the many proteins in a flour sample. The abundant glutamine and proline rich gluten proteins are responsible for many of the unique end-use qualities of wheat flour but it is challenging to dis...


Protein quantitation using various modes of high performance liquid chromatography.  


Pharmaceuticals based on proteins (biologicals), such as monoclonal antibodies (mAb), attain more and more relevance since they were established as potent drugs in anticancer therapy or for the treatment of autoimmune based diseases. Due to their high efficiency it is essential to have accurate and precise methods for protein quantitation and the detection of protein aggregates, which in some cases may lead to adverse effects after application. Selectivity and precision of traditional protein quantification methods such as the Bradford assay or SDS-PAGE are insufficient for quality control (QC) purposes. In this work several HPLC separation modes, which can significantly improve these important parameters, were compared for their application in this field. High performance size exclusion (HP-SEC), strong anion exchange (SAX), weak cation exchange (WCX) as well as reversed phase chromatography are all already successfully applied in protein analysis. Good precision (SEC: <1.9%, SAX: <5%, RP: <2% and WCX: <3.5% - RSD% for peak areas day-to-day), high selectivity and low quantitation limits (<15?g/ml) for the model proteins ovalbumin, myoglobin and bovine serum albumin (BSA), respectively cytochrome c and lysozyme in the cation exchange mode, could be achieved. Consecutively, the four separation modes were compared to each other and to electrophoretic techniques in terms of precision, selectivity, analysis time, effort of sample and mobile phase preparation as well as separating capacity. Moreover, the analysis of an IgG1-type antibody was included in this study. PMID:22980318

Grotefend, Sandra; Kaminski, Lukas; Wroblewitz, Stefanie; Deeb, Sami El; Kühn, Nancy; Reichl, Stephan; Limberger, Markus; Watt, Steven; Wätzig, Hermann



Molecular phylogeny of the hominoid primates as indicated by two-dimensional protein electrophoresis  

SciTech Connect

A molecular phylogeny for the hominoid primates was constructed by using genetic distances from a survey of 383 radiolabeled fibroblast polypeptides resolved by two-dimensional electrophoresis (2DE). An internally consistent matrix of Nei genetic distances was generated on the basis of variants in electrophoretic position. The derived phylogenetic tree indicated a branching sequence, from oldest to most recent, of cercopithecoids (Macaca fascicularis), gibbon-siamang, orangutan, gorilla, and human-chimpanzee. A cladistic analysis of 240 electrophoretic characters that varied between ape species produced an identical tree. Genetic distance measures obtained by 2DE are largely consistent with those generated by other molecular procedures. In addition, the 2DE data set appears to resolve the human-chimpanzee-gorilla trichotomy in favor of a more recent association of chimpanzees and humans.

Goldman, D.; Giri, P.R.; O'Brien, J.O.



Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry  

PubMed Central

Background Wheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use MS/MS to distinguish the many proteins in a flour sample and relate them to gene sequences. Results Grain from the extensively characterized spring wheat cultivar Triticum aestivum 'Butte 86' was milled to white flour from which proteins were extracted, then separated and quantified by 2-DE. Protein spots were identified by separate digestions with three proteases, followed by tandem mass spectrometry analysis of the peptides. The spectra were used to interrogate an improved protein sequence database and results were integrated using the Scaffold program. Inclusion of cultivar specific sequences in the database greatly improved the results, and 233 spots were identified, accounting for 93.1% of normalized spot volume. Identified proteins were assigned to 157 wheat sequences, many for proteins unique to wheat and nearly 40% from Butte 86. Alpha-gliadins accounted for 20.4% of flour protein, low molecular weight glutenin subunits 18.0%, high molecular weight glutenin subunits 17.1%, gamma-gliadins 12.2%, omega-gliadins 10.5%, amylase/protease inhibitors 4.1%, triticins 1.6%, serpins 1.6%, purinins 0.9%, farinins 0.8%, beta-amylase 0.5%, globulins 0.4%, other enzymes and factors 1.9%, and all other 3%. Conclusions This is the first successful effort to identify the majority of abundant flour proteins for a single wheat cultivar, relate them to individual gene sequences and estimate their relative levels. Many genes for wheat flour proteins are not expressed, so this study represents further progress in describing the expressed wheat genome. Use of cultivar-specific contigs helped to overcome the difficulties of matching peptides to gene sequences for members of highly similar, rapidly evolving storage protein families. Prospects for simplifying this process for routine analyses are discussed. The ability to measure expression levels for individual flour protein genes complements information gained from efforts to sequence the wheat genome and is essential for studies of effects of environment on gene expression. PMID:21314956



Protein expression dynamics during replicative senescence of endothelial cells studied by 2-D difference in-gel electrophoresis.  


Endothelial senescence contributes to endothelium dysfunctionality and is thereby linked to vascular aging. A dynamic proteomic study on human umbilical vein endothelial cells, isolated from three umbilical cords, was performed. The cells were cultured towards replicative senescence and whole cell lysates were subjected to 2-D difference gel electrophoresis (DIGE). Despite the biological variability of the three independent isolations, a set of proteins was found that showed senescence-dependent expression patterns in all isolations. We focused on those proteins that showed significant changes, with a paired analysis of variance (RM-ANOVA) p-value of < or =0.05. Thirty-five proteins were identified with LC-Fourier transform MS, and functional annotation revealed that endothelial replicative senescence is accompanied by increased cellular stress, protein biosynthesis and reduction in DNA repair and maintenance. Nuclear integrity becomes affected and cytoskeletal structure is also changed. Such important changes in the cell infrastructure might accelerate endothelium dysfunctionality. This study provides biological information that will initiate studies to further unravel endothelial senescence and gain more knowledge about the consequences of this process in the in vivo situation. PMID:16609940

Eman, Michael R; Regan-Klapisz, Elsa; Pinkse, Martijn W H; Koop, Inge M; Haverkamp, Johan; Heck, Albert J R; Verkleij, Arie J; Post, Jan A



Stress Responsive Proteins Are Actively Regulated during Rice (Oryza sativa) Embryogenesis as Indicated by Quantitative Proteomics Analysis  

PubMed Central

Embryogenesis is the initial step in a plant’s life, and the molecular changes that occur during embryonic development are largely unknown. To explore the relevant molecular events, we used the isobaric tags for relative and absolute quantification (iTRAQ) coupled with the shotgun proteomics technique (iTRAQ/Shotgun) to study the proteomic changes of rice embryos during embryogenesis. For the first time, a total of 2 165 unique proteins were identified in rice embryos, and the abundances of 867 proteins were actively changed based on the statistical evaluation of the quantitative MS/MS signals. The quantitative data were then confirmed using multiple reactions monitoring (MRM) and were also supported by our previous study based on two-dimensional gel electrophoresis (2 DE). Using the proteome at 6 days after pollination (DAP) as a reference, cluster analysis of these differential proteins throughout rice embryogenesis revealed that 25% were up-regulated and 75% were down-regulated. Gene Ontology (GO) analysis implicated that most of the up-regulated proteins were functionally categorized as stress responsive, mainly including heat shock-, lipid transfer-, and reactive oxygen species-related proteins. The stress-responsive proteins were thus postulated to play an important role during seed maturation. PMID:24058531

Zi, Jin; Zhang, Jiyuan; Wang, Quanhui; Zhou, Baojin; Zhong, Junyan; Zhang, Chaoliang; Qiu, Xuemei; Wen, Bo; Zhang, Shenyan; Fu, Xiqin; Lin, Liang; Liu, Siqi



Study of Early Leaf Senescence in Arabidopsis thaliana by Quantitative Proteomics Using Reciprocal 14N\\/15N Labeling and Difference Gel Electrophoresis  

Microsoft Academic Search

Leaf senescence represents the final stage of leaf develop- ment and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senes- cence, we designed an elaborate double and reverse label- ing strategy simultaneously employing fluorescent two-di- mensional DIGE as well as metabolic 15N labeling followed

Romano Hebeler; Silke Oeljeklaus; Kai A. Reidegeld; Martin Eisenacher; Christian Stephan; Barbara Sitek; Kai Stuhler; Helmut E. Meyer; Marcel J. G. Sturre; Paul P. Dijkwel; Bettina Warscheid



Towards quantitative prediction of proteasomal digestion patterns of proteins  

E-print Network

We discuss the problem of proteasomal degradation of proteins. Though proteasomes are important for all aspects of the cellular metabolism, some details of the physical mechanism of the process remain unknown. We introduce a stochastic model of the proteasomal degradation of proteins, which accounts for the protein translocation and the topology of the positioning of cleavage centers of a proteasome from first principles. For this model we develop the mathematical description based on a master-equation and techniques for reconstruction of the cleavage specificity inherent to proteins and the proteasomal translocation rates, which are a property of the proteasome specie, from mass spectroscopy data on digestion patterns. With these properties determined, one can quantitatively predict digestion patterns for new experimental set-ups. Additionally we design an experimental set-up for a synthetic polypeptide with a periodic sequence of amino acids, which enables especially reliable determination of translocation ...

Goldobin, Denis S



Towards quantitative prediction of proteasomal digestion patterns of proteins  

E-print Network

We discuss the problem of proteasomal degradation of proteins. Though proteasomes are important for all aspects of the cellular metabolism, some details of the physical mechanism of the process remain unknown. We introduce a stochastic model of the proteasomal degradation of proteins, which accounts for the protein translocation and the topology of the positioning of cleavage centers of a proteasome from first principles. For this model we develop the mathematical description based on a master-equation and techniques for reconstruction of the cleavage specificity inherent to proteins and the proteasomal translocation rates, which are a property of the proteasome specie, from mass spectroscopy data on digestion patterns. With these properties determined, one can quantitatively predict digestion patterns for new experimental set-ups. Additionally we design an experimental set-up for a synthetic polypeptide with a periodic sequence of amino acids, which enables especially reliable determination of translocation rates.

Denis S. Goldobin; Alexey Zaikin



Quantitating protein synthesis, degradation, and endogenous antigen processing.  


Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate. PMID:12648452

Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W



Biochemical and quantitative analysis of Tamm Horsfall protein in rats.  


The involvement of Tamm Horsfall protein (THP) in nephrolithiasis is currently under investigation in several laboratories. Although rat is a commonly used species as an in vivo model for such studies, there is only limited information available about the biochemical properties and excretion profile of THP in normal rats. In order to characterize rat THP, we purified and analyzed normal male rat THP, and compared it with normal human male urinary THP by gel electrophoresis. Both THPs migrated at approximately 90 KDa, and stained similarly for protein (Coomassie blue) as well as carbohydrates (periodic acid Schiff reagent). Compositional analysis revealed that rat THP was largely similar to human THP in amino acid and carbohydrate contents but showed differences in the individual sugar components from other mammals. There was considerable variation in the day-to-day urinary excretion of THP in normal rats, with values ranging from 552.96 micrograms to 2865.60 micrograms and a mean value of 1679.54 micrograms per 24 h. It was concluded from this study that rat THP did not contain any unusual biochemical components and was primarily similar to human THP in composition and mean urinary concentration. PMID:9373916

Gokhale, J A; Glenton, P A; Khan, S R



Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics*  

PubMed Central

Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ?5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution. PMID:23242552

Larance, Mark; Ahmad, Yasmeen; Kirkwood, Kathryn J.; Ly, Tony; Lamond, Angus I.



Kinetic capillary electrophoresis with mass-spectrometry detection (KCE-MS) facilitates label-free solution-based kinetic analysis of protein-small molecule binding.  


Tandem tracker: Here we introduce a method for studying the kinetics of protein-small-molecule interactions based on kinetic capillary electrophoresis (KCE) separation and MS detection. Due to the variety of KCE methods and MS modes available, the KCE-MS tandem is a highly versatile platform for label-free, solution-based kinetic studies of affinity interactions. PMID:22012742

Bao, Jiayin; Krylova, Svetlana M; Wilson, Derek J; Reinstein, Oren; Johnson, Philip E; Krylov, Sergey N



Evaluation of a novel hydrophilic derivatized capillary for protein analysis by capillary electrophoresis-electrospray mass spectrometry.  


A new type of hydrophilic derivatized capillary has been used to enable the on-line capillary electrophoresis separation and electrospray mass spectrometric detection of a mixture of proteins containing bovine cytochrome c, tuna cytochrome c and horse heart myoglobin. Less than 40 fmol of each compound were loaded into the capillary. Baseline resolution of components was achieved, as were accurate assignments of molecular masses. The hydrophilic derivatized capillaries were taken through extensive testing procedures to characterize their performance and capabilities for protein analysis. A mixture of six proteins (cytochrome c, ribonuclease A, alpha-chymotrypsinogen, myoglobin, carbonic anhydrase II and alpha-lactalbumin) in acetic acid-sodium acetate buffer was used to delineate the relationships between migration time and pH, along with migration time and buffer concentration for each protein. The variations in capillary efficiency as a function of pH and as a function of buffer concentration were also characterized for the same six proteins in the acetic acid-sodium acetate system. A pH of 4.8 was found to offer an excellent compromise between separation efficiency (up to 500,000 theoretical plates) and analysis time. Capillary efficiencies were also found to be very good when employing a Tris.HCl electrolyte adjusted to pH 4.8. Lastly, electropherogram reproducibility and capillary durability were examined with the finding that little deterioration of the capillary occurred over the course of 400 injections (200 h run time). This represents a notable improvement over previously documented derivatization procedures designed to reduce protein adsorption to fused-silica capillary walls. PMID:7981821

Cole, R B; Varghese, J; McCormick, R M; Kadlecek, D



Analysis of differentially expressed mitochondrial proteins in chromophobe renal cell carcinomas and renal oncocytomas by 2-D gel electrophoresis  

PubMed Central

Renal oncocytomas (RO) and chromophobe renal cell carcinomas (RCC) display morphological and functional alterations of the mitochondria. Previous studies showed that accumulation of mitochondria in ROs is associated with somatic mutations of mitochondrial DNA (mtDNA) resulting in decreased activity of the respiratory chain complex I, whereas in chromophobe RCC only heteroplasmic mtDNA mutations were found. To identify proteins associated with these changes, for the first time we have compared the mitochondrial proteomes of mitochondria isolated from ROs and chromophobe RCCs as well as from normal kidney tissues by two-dimensional polyacrylamide gel electrophoresis. The proteome profiles were reproducible within the same group of tissues in subsequent experiments. The expression patterns within each group of samples were compared and 81 in-gel digested spots were subjected to nanoLC-MS/MS-based identification of proteins. Although the list of mitochondrial proteins identified in this study is incomplete, we identified the downregulation of NDUFS3 from complex I of the respiratory chain and upregulation of COX5A, COX5B, and ATP5H from complex IV and V in ROs. In chromophobe RCCs downregulation of ATP5A1, the alpha subunit of complex V, has been observed, but no changes in expression of other complexes of the respiratory chain were detected. To confirm the role of respiratory chain complex alterations in the morphological and/or functional changes in chromophobe RCCs and ROs, further studies will be necessary. PMID:20440404

Yusenko, Maria V.; Ruppert, Thomas; Kovacs, Gyula



Capillary electrophoresis-mass spectrometry of basic proteins using a new physically adsorbed polymer coating. Some applications in food analysis.  


A new physically adsorbed capillary coating for capillary electrophoresis-mass spectrometry (CE-MS) of basic proteins is presented, which is easily obtained by flushing the capillary with a polymer aqueous solution for two min. This coating significantly reduces the electrostatic adsorption of a group of basic proteins (i.e., cytochrome c, lysozyme, and ribonuclease A) onto the capillary wall allowing their analysis by CE-MS. The coating protocol is compatible with electrospray inonization (ESI)-MS via the reproducible separation of the standard basic proteins (%RSD values (n = 5) < 1% for analysis time reproducibility and < 5% for peak heights, measured from the total ion electropherograms (TIEs) within the same day). The LODs determined using cytochrome c with total ion current and extracted ion current defection were 24.5 and 2.9 fmol, respectively. Using this new coating lysozymes from chicken and turkey egg white could be easily distinguished by CE-MS, demonstrating the usefulness of this method to differentiate animal species. Even after sterilization at 120 degrees C for 30 min, lysozyme could be detected, as well as in wines at concentrations much lower than the limit marked by the EC Commission Regulation. Adulteration of minced meat with 5% of egg-white could also be analysed by our CE-MS protocol. PMID:15237406

Simó, Carolina; Elvira, Carlos; González, Nieves; San Román, J; Barbas, Coral; Cifuentes, Alejandro



Immunoreactive Coxiella burnetii Nine Mile proteins separated by 2D electrophoresis and identified by tandem mass spectrometry.  


Coxiella burnetii is a Gram-negative obligate intracellular pathogen and the causative agent of Q fever in humans. Q fever causes acute flu-like symptoms and may develop into a chronic disease leading to endocarditis. Its potential as a bioweapon has led to its classification as a category B select agent. An effective inactivated whole-cell vaccine (WCV) currently exists but causes severe granulomatous/necrotizing reactions in individuals with prior exposure, and is not licensed for use in most countries. Current efforts to reduce or eliminate the deleterious reactions associated with WCVs have focused on identifying potential subunit vaccine candidates. Both humoral and T cell-mediated responses are required for protection in animal models. In this study, nine novel immunogenic C. burnetii proteins were identified in extracted whole-cell lysates using 2D electrophoresis, immunoblotting with immune guinea pig sera, and tandem MS. The immunogenic C. burnetii proteins elicited antigen-specific IgG in guinea pigs vaccinated with whole-cell killed Nine Mile phase I vaccine, suggesting a T cell-dependent response. Eleven additional proteins previously shown to react with immune human sera were also antigenic in guinea pigs, showing the relevance of the guinea pig immunization model for antigen discovery. The antigens described here warrant further investigation to validate their potential use as subunit vaccine candidates. PMID:21030434

Deringer, James R; Chen, Chen; Samuel, James E; Brown, Wendy C



Assessment of the quality of dairy products by capillary electrophoresis of milk proteins.  


This paper presents an overview of existing capillary electrophoretic methods for the study of milk proteins. The main methods of analysis of caseins, whey proteins and peptides are examined with particular attention to their application to the evaluation of the quality of dairy products. Aspects such as the study of protein polymorphism, evaluation of heat treatments, detection of adulteration and assessment of proteolysis are considered in detail. PMID:9342674

Recio, I; Amigo, L; López-Fandiño, R



Capillary electrophoresis separation of neutral organic compounds, pharmaceutical drugs, proteins and peptides, enantiomers, and anions  

SciTech Connect

Addition of a novel anionic surfactant, namely lauryl polyoxyethylene sulfate, to an aqueous-acetonitrile electrolyte makes it possible to separate nonionic organic compounds by capillary electrophoresis. Separation is based on differences in the association between analytes and the surfactant. Highly hydrophobic compounds such as polyaromatic hydrocarbons are well separated by this new surfactant. Migration times of analytes can be readily changed over an unusually large range by varying the additive concentration and the proportion of acetonitrile in the electrolyte. Several examples are given, including the separation of four methylbenz[a]anthracene isomers and the separation of normal and deuterated acetophenone. The effect of adding this new surfactant to the acidic electrolyte was also investigated. Incorporation of cetyltrimethylammonium bromide in the electrolyte is shown to dynamically coat the capillary and reverse electroosmotic flow. Chiral recognition mechanism is studied using novel synthetic surfactants as chiral selectors, which are made from amino acids reacting with alkyl chloroformates. A satisfactory separation of both inorganic and organic anions is obtained using electrolyte solutions as high as 5 M sodium chloride using direct photometric detection. The effect of various salts on electrophoretic and electroosmotic mobility is further discussed. Several examples are given under high-salt conditions.

Ding, W.L.



Use of capillary electrophoresis-sodium dodecyl sulfate to monitor disulfide scrambled forms of an Fc fusion protein during purification process.  


Overexpression of recombinant Fc fusion proteins in Escherichia coli frequently results in the production of inclusion bodies that are subsequently used to produce fully functional protein by an in vitro refolding process. During the refolding step, misfolded proteins such as disulfide scrambled forms can be formed, and purification steps are used to remove these product-related impurities to produce highly purified therapeutic proteins. A variety of analytical methods are commonly used to monitor protein variants throughout the purification process. Capillary electrophoresis (CE)-based techniques are gaining popularity for such applications. In this work, we used a nonreduced capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS) method for the analysis of disulfide scrambled forms in a fusion protein. Under denatured nonreduced conditions, an extra post-shoulder peak was observed at all purification steps. Detailed characterization revealed that the peak was related to the disulfide scrambled forms and was isobaric with the correctly folded product. In addition, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used during the CE-SDS peak characterization, we observed that the migration order of scrambled forms is reversed on CE-SDS versus SDS-PAGE. This illustrates the importance of establishing proper correlation of these two techniques when they are used interchangeably to guide the purification process and to characterize proteins. PMID:21420378

Hapuarachchi, Suminda; Fodor, Szilan; Apostol, Izydor; Huang, Gang



Electrophoresis and isoelectric focusing of whole cell and membrane proteins from the extremely halophilic archaebacteria  

NASA Technical Reports Server (NTRS)

The subunits from two purified halobacterial membrane enzymes (ATPase and nitrate reductase) behaved differently with respect to isoelectric focusing, silver staining and interaction with ampholytes. Differential behavior was also observed in whole cell proteins from Halobacterium saccharovorum regarding resolution in two-dimensional gels and silver staining. It is proposed that these differences reflect the existence of two classes of halobacterial proteins.

Stan-Lotter, Helga; Lang, Frank J., Jr.; Hochstein, Lawrence I.



Optimizing capillary electrophoresis for top-down proteomics of 30-80 kDa proteins.  


The direct analysis of intact proteins via MS offers compelling advantages in comparison to alternative methods due to the direct and unambiguous identification and characterization of protein sequences it provides. The inability to efficiently analyze proteins in the "middle mass range," defined here as proteins from 30 to 80 kDa, in a robust fashion has limited the adoption of these "top-down" methods. Largely, a result of poor liquid chromatographic performance, the limitations in this mass range may be addressed by alternative separations that replace chromatography. Herein, the short migration times of CZE-ESI-MS/MS have been extended to size-sorted whole proteins in complex mixtures from Pseudomonas aeruginosa PA01. An electrokinetically pumped nanospray interface, a coated capillary, and a stacking method for on-column sample concentration were developed to achieve high-loading capacity and separation resolution. We achieved full width at half maximum of 8-16 s for model proteins up to 29 kDa and identified 30 proteins in the mass range of 30-80 kDa from P. aeruginosa PA01 whole cell lysate. These results suggest that CZE-ESI-MS/MS is capable of identifying proteins in the middle mass range in top-down proteomics. PMID:24596178

Li, Yihan; Compton, Philip D; Tran, John C; Ntai, Ioanna; Kelleher, Neil L



Quantitative Characterization of Protein Associations in Highly Concentrated Solution  

NASA Astrophysics Data System (ADS)

With few exceptions, one cannot reliably predict the behavior of a protein at high concentration on the basis of knowledge obtained from experiments carried out at low concentration. Detection and quantitative characterization of protein-protein interactions in the high concentration regime (> 50 g/L) therefore presents both experimental and theoretical challenges to the investigator. Two experimental methods devised in our laboratory specifically for this purpose are described. (1) Non-ideal tracer sedimentation equilibrium. Instrumentation and theory for measuring and interpreting the equilibrium gradient of a labeled dilute tracer protein in a solution containing an arbitrary concentration of one or more unlabeled macromolecules are outlined. The composition dependence of the equilibrium gradient of several proteins, including ribonuclease at concentrations up to 200 g/L and immunoglobulin G at concentrations up to 125 g/L, will be presented and interpreted in the context of models taking into account both equilibrium self-association, and nonspecific repulsive steric or electrostatic repulsion. (2) Non-ideal light scattering. Recently developed instrumentation and theory for rapid measurement and interpretation of the light scattering of a protein solution over a broad range of concentration are outlined. The concentration-dependent light scattering of chymotrypsin A at three different pH values at concentrations up to 60 g/L, and the concentration-dependent light scattering of two monoclonal antibodies at concentrations up to over 200 g/L in solutions of varying ionic strength, are quantitatively accounted for by models that take into account both nonideal repulsion between protein molecules and specific modes of equilibrium self-association.

Minton, Allen



Changes in muscle protein composition induced by disuse atrophy - Analysis by two-dimensional electrophoresis  

NASA Technical Reports Server (NTRS)

Using 320 g rats, a two-dimensional electrophoretic analysis of muscle proteins in the soleus and EDL muscles from hindlimbs maintained load-free for 10 days is performed. Statistical analysis of the two-dimensional patterns of control and suspended groups reveals more protein alteration in the soleus muscle, with 25 protein differences, than the EDL muscle, with 9 protein differences, as a result of atrophy. Most of the soleus differences reside in minor components. It is suggested that the EDL may also show alteration in its two-dimensional protein map, even though no significant atrophy occurred in muscle wet weight. It is cautioned that strict interpretation of data must take into account possible endocrine perturbations.

Ellis, S.; Giometti, C. S.; Riley, D. A.



Microfluidic Electrophoresis Assays for Rapid Characterization of Protein in Research and Development  

NSDL National Science Digital Library

This presentation discussed the use of microfluidic-CE platforms for characterization assays such as purity assessment of monoclonal antibodies under reducing and nonreducing conditions, N-glycan profiling, and determination of protein charge heterogeneity.

Sean Sanders (Science/AAAS;); Bahram Fathollahi (PerkinElmer;); Timothy Blanc (ImClone Systems;); Joey Studts (Boehringer Ingelheim Pharma GmbH & Co. KG;)



Gel Electrophoresis  

NSDL National Science Digital Library

This interactive activity from the Dolan DNA Learning Center illustrates the process of gel electrophoresis, in which DNA fragments are separated by size as they migrate at different rates through a gel matrix.

Foundation, Wgbh E.



Identification of Leguminosae gums and evaluation of carob-guar mixtures by capillary zone electrophoresis of protein extracts.  


A procedure for the extraction and capillary zone electrophoresis (CZE) separation of proteins from carob, guar and tara gums in a background electrolyte (BGE) of pH 9 containing 0.1% polyvinyl alcohol is described. The CZE protein profiles exhibit characteristic peaks for each one of the Leguminosae gums, which can be used to construct models capable of identifying samples of carob, guar and tara gums, and predicting the guar content in binary carob-guar mixtures of different geographical origin and harvested in different years. The classification and prediction models are constructed by using linear discriminant analysis (LDA) and multiple linear regression (MLR), respectively. An excellent resolution between the three categories is obtained with LDA, the model being capable of classifying samples with recognition and prediction capabilities of 100%. For MLR models constructed with carob-guar mixtures with and without a common history, the average of the calibration residuals are +/- 0.50 and +/- 0.90%, respectively (average values for the 2-20% guar range). For the later model, the detection limit was 3.2% guar (from the standard deviation of 18 mixtures with 2-4% guar, and for alpha = beta = 0.05). PMID:12179992

Ruiz-Angel, María J; Simó-Alfonso, Ernesto F; Mongay-Fernández, Carlos; Ramis-Ramos, Guillermo



Separation of metalloproteins using a novel metal ion contaminant sweeping technique and detection of protein-bound copper by a metal ion probe in polyacrylamide gel electrophoresis: distribution of copper in human serum.  


A polyacrylamide gel electrophoresis (PAGE)-based method has been developed, consisting of two types of gel electrophoresis, to obtain an accurate distribution of protein-bound metal ions in biological samples. First, proteins are separated by PAGE without the uptake of contaminant metal ions in the separation field and dissociation of metal ions from the proteins. This is followed by another PAGE for the separation and detection of protein-bound metal ions in small volume samples with high sensitivity in the ppt range using a fluorescent metal probe. The former is a new technique using blue-native (BN) PAGE to electrophoretically sweep all metal contaminants by employing two kinds of chelating agents. These agents form complexes with contaminants in the gel and the separation buffer solution, which migrate towards opposite pole directions, thus lowering the contaminants to below the ppt level during separation. This is termed "Metal Ion Contaminant Sweeping BN-PAGE (MICS-BN-PAGE)". After the separation of proteins under these first metal-free conditions, the metal ions in the gel fractions are eluted, followed by derivatization of copper ions into the metal probe complexes to be separated and determined by fluorescence detection in the second PAGE. In this PAGE-based method, the copper ions bound to ceruloplasmin and superoxide dismutase were quantitatively determined, in addition to the exchangeable albumin-bound copper ions. This system successfully provided distribution maps of protein-copper in human serum. The precise distribution of copper in human serum was investigated, and found to be different from that which is widely accepted. PMID:23964357

Saito, Shingo; Kawashima, Mitsuyoshi; Ohshima, Hiroki; Enomoto, Kazuki; Sato, Makoto; Yoshimura, Hajime; Yoshimoto, Keitaro; Maeda, Mizuo; Shibukawa, Masami



Quantitative analysis of pyoluteorin in anti-fungal fermentation liquor of Pseudomonas species by capillary zone electrophoresis with UV-vis detector.  


This paper investigated potential utility of capillary zone electrophoresis (CZE) for very succinct but robust quantitative analysis of pyoluteorin (Plt) in anti-fungal fermentation liquor of Pseudomonas species. The experimental conditions for the separation and quantification of Plt were optimized at first. The optimized conditions are: 80 mmol/L pH 8.40 Gly-NaOH buffer, 51 cm total length (42 cm effective) and 75 microm I.D. capillary, 230 nm wavelength, 25 kV, 13 mbar 10s pressure sample injection and 24 degrees C air-cooling. Under the optimized conditions, the migration times of Plt and the internal standard phenobarbital are 2.09 and 2.49 min, respectively, the linear response of Plt concentration ranges from 5.0 to 1000 microg/mL with high correlation coefficient (r=0.99977, n=9), the limits of detection (LOD) and quantification (LOQ) for Plt are 0.66 and 2.2 microg/mL, the precision values (expressed as R.S.D.) of intra- and inter-day are 1.19-1.94% and 1.55-6.21%, respectively, the recoveries of Plt at three concentration levels of 750, 250 and 50 microg/mL range from 90.31% to 97.85% and to 98.96%, respectively. The developed method can be well used for the quantification of Plt in the fermentation liquor. PMID:16140044

Wang, Qiu-Ling; Zhang, Xue-Hong; Fan, Liu-Yin; Zhang, Wei; Xu, Yu-Qian; Hu, Hong-Bo; Cao, Cheng-Xi



Disease proteomics of high-molecular-mass proteins by two-dimensional gel electrophoresis with agarose gels in the first dimension (Agarose 2-DE).  


Agarose gel is the preferred electrophoretic medium currently used for separating high molecular mass (HMM) proteins (MW>100 kDa). Agarose gels are widely used for both SDS-agarose gel electrophoresis and agarose isoelectric focusing (IEF). A two-dimensional gel electrophoresis method employing agarose gels in the first dimension (agarose 2-DE) that is sufficiently good at separating up to 1.5mg of HMM proteins with molecular masses as large as 500 kDa has been used to separate proteins from various diseased tissues and cells. Although resolution of the agarose 2-DE pattern always depends on the tissue being analyzed, sample preparation procedures including (i) protein extraction with an SDS sample buffer; (ii) ultracentrifugation of a tissue homogenate; and (iii) 1% SDS in both stacking and separation gels of the second-dimension SDS-PAGE gel, are generally effective for HMM protein detection. In a comprehensive prostate cancer proteome study using agarose 2-DE, the HMM region of the gel was rich in proteins of particular gene/protein expression groups (39.1% of the HMM proteins but only 28.4% of the LMM ones were classified as transcription/translation-related proteins). Examples include transcription factors, DNA or RNA binding proteins, and ribosomal proteins. To understand oxidative stress-induced cellular damage at the protein level, a novel proteomic method, in which protein carbonyls were derivatized with biotin hydrazide followed by agarose 2-DE, was useful for detecting HMM protein carbonyls in tissues of both a diabetes model Ostuka Long-Evans Tokushima Fatty (OLETF) rat and a control Long-Evans Tokushima Otsuka (LETO) rat. In this paper, we review the use of agarose gels for separation of HMM proteins and disease proteomics of HMM proteins in general, with particular attention paid to our proteome analyzes based on the use of agarose 2-DE for protein separation followed by the use of mass spectrometry for protein identification. PMID:17141588

Oh-Ishi, Masamichi; Maeda, Tadakazu



Binary Oscillatory Crossflow Electrophoresis  

NASA Technical Reports Server (NTRS)

Electrophoresis has long been recognized as an effective analytic technique for the separation of proteins and other charged species, however attempts at scaling up to accommodate commercial volumes have met with limited success. In this report we describe a novel electrophoretic separation technique - Binary Oscillatory Crossflow Electrophoresis (BOCE). Numerical simulations indicate that the technique has the potential for preparative scale throughputs with high resolution, while simultaneously avoiding many problems common to conventional electrophoresis. The technique utilizes the interaction of an oscillatory electric field and a transverse oscillatory shear flow to create an active binary filter for the separation of charged protein species. An oscillatory electric field is applied across the narrow gap of a rectangular channel inducing a periodic motion of charged protein species. The amplitude of this motion depends on the dimensionless electrophoretic mobility, alpha = E(sub o)mu/(omega)d, where E(sub o) is the amplitude of the electric field oscillations, mu is the dimensional mobility, omega is the angular frequency of oscillation and d is the channel gap width. An oscillatory shear flow is induced along the length of the channel resulting in the separation of species with different mobilities. We present a model that predicts the oscillatory behavior of charged species and allows estimation of both the magnitude of the induced convective velocity and the effective diffusivity as a function of a in infinitely long channels. Numerical results indicate that in addition to the mobility dependence, the steady state behavior of solute species may be strongly affected by oscillating fluid into and out of the active electric field region at the ends of the cell. The effect is most pronounced using time dependent shear flows of the same frequency (cos((omega)t)) flow mode) as the electric field oscillations. Under such conditions, experiments indicate that solute is drawn into the cell from reservoirs at both ends of the cell leading to a large mass build up. As a consequence, any initially induced mass flux will vanish after short times. This effect was not captured by the infinite channel model and hence numerical and experimental results deviated significantly. The revised model including finite cell lengths and reservoir volumes allowed quantitative predictions of the time history of the concentration profile throughout the system. This latter model accurately describes the fluxes observed for both oscillatory flow modes in experiments using single protein species. Based on the results obtained from research funded under NASA grant NAG-8-1080.S, we conclude that binary separations are not possible using purely oscillatory flow modes because of end effects associated with the cos((omega)t) mode. Our research shows, however, that a combination of cos(2(omega)t) and steady flow should lead to efficient separation free of end effects. This possibility is currently under investigation.

Molloy, Richard F.; Gallagher, Christopher T.; Leighton, David T., Jr.



Studies of proteinograms in dermatophytes by disc electrophoresis. 1. Protein bands in relation to growth phase  

NASA Technical Reports Server (NTRS)

Homogenates were prepared from various growth phases of Microsporum gypseum grown on different amino acids as the nitrogen source. When analyzed on 7.5% polyacrylamide disc gels, the water-soluble proteins in these homogenates gave essentially identical banding patterns.

Danev, P.; Friedrich, E.; Balabanov, V.



Imaging metals in proteins by combining electrophoresis with rapid x-ray fluorescence mapping.  

SciTech Connect

Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal?protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis.

Finney, L.; Chishti, Y.; Khare, T.; Giometti, C.; Levina, A.; Lay, P. A.; Vogt, S.; Univ. of Sydney; Northwestern Univ.



Protein analysis by capillary zone electrophoresis utilizing a trifunctional diamine for silica coating.  


A novel method is here reported for the analysis of mixture of proteins with pI ranging from pH 3-9.5 in an ample pH interval (pH 2.5-9.0) without adsorption onto the naked silica wall. It consists of treating the capillary surface at alkaline pH, typically 9.0, with small amounts (2-4 mM) of a quaternarized piperazine derivative: (N-methyl-N-omega-iodobutyl)-N'-methylpiperazine (Q-PzI). It appears that this compound is able to dock onto the wall via trifunctional links: a salt bridge via the quaternary nitrogen, a hydrogen bond via the tertiary nitrogen, and finally, a covalent link via the terminal iodine in the butyl chain and a neighboring ionized silanol. This last reaction seems to be completed in a few minutes of incubation of the capillary at room temperature. Because the compound is permanently affixed to the wall, its presence is not needed during protein/peptide separations. By properly dosing the level of Q-PzI in the preconditioning step, it is possible to strongly reduce the electroendoosmotic flow (EOF), zero it, or reverse it. Unlike dynamic coatings with oligoamines, which are most effective only at acidic pH values and are required as additives during separations, Q-PzI is effective in an ample pH interval (pH 2.5-9.0) and is not needed during the CZE analysis. A broad pI (pH 3-10) protein mix can be separated according to protein mobility in free phase, suggesting a strong modulating capacity of the functionalized wall. The same separation is not obtained in capillaries permanently coated with neutral, hydrophilic polymers (such as polyacrylamide), even if the quality of a single protein/peptide profile in Q-PzI-conditioned capillaries is equivalent to those obtained in capillaries permanently coated. Although there is strong indirect evidence of the ability of Q-PzI to alkylate the silica wall, to which it is then irreversibly bound, such an alkylation event does not occur with proteins on potentially reacting sites, such as the free -SH of Cys or the -OH group of Tyr, as demonstrated by incubating them overnight in a large molar excess at strongly alkaline pH values and analyzing such proteins by MALDI-TOF mass spectrometry. PMID:11534708

Gelfi, C; Viganò, A; Ripamonti, M; Righetti, P G; Sebastiano, R; Citterio, A



Electrophoresis technology  

NASA Technical Reports Server (NTRS)

A new high resolution apparatus designed for space was built as a laboratory prototype. Using a moving wall with a low zeta potential coating, the major sources of flow distortion for an electrophoretic sample stream are removed. Highly resolved fractions, however, will only be produced in space because of the sensitivity of this chamber to buoyancy-induced convection in the laboratory. The second and third flights of the McDonnell Douglas Astronautics Corporation continuous flow electrophoresis system carried samples developed at MSFC intended to evaluate the broad capabilities of free flow electrophoresis in a reduced gravity environment. Biological model materials, hemoglobin and polystyrene latex microspheres, were selected because of their past use as electrophoresis standards and as visible markers for fluid flow due to electroosmosis, spacecraft acceleration or other factors. The dependence of the separation resolution on the properties of the sample and its suspension solution was assessed.

Snyder, R. S.



Quantitative Characterization of Local Protein Solvation To Predict Solvent Effects on Protein Structure  

PubMed Central

Characterization of solvent preferences of proteins is essential to the understanding of solvent effects on protein structure and stability. Although it is generally believed that solvent preferences at distinct loci of a protein surface may differ, quantitative characterization of local protein solvation has remained elusive. In this study, we show that local solvation preferences can be quantified over the entire protein surface from extended molecular dynamics simulations. By subjecting microsecond trajectories of two proteins (lysozyme and antibody fragment D1.3) in 4 M glycerol to rigorous statistical analyses, solvent preferences of individual protein residues are quantified by local preferential interaction coefficients. Local solvent preferences for glycerol vary widely from residue to residue and may change as a result of protein side-chain motions that are slower than the longest intrinsic solvation timescale of ?10 ns. Differences of local solvent preferences between distinct protein side-chain conformations predict solvent effects on local protein structure in good agreement with experiment. This study extends the application scope of preferential interaction theory and enables molecular understanding of solvent effects on protein structure through comprehensive characterization of local protein solvation. PMID:22995508

Vagenende, Vincent; Trout, Bernhardt L.



Single-step quantitation of DNA in microchip electrophoresis with linear imaging UV detection and fluorescence detection through comigration with a digest.  


We demonstrate a convenient single-step quantitation technique for double-stranded DNA (dsDNA) fragments in polymerase chain reaction (PCR) products based on microchip capillary electrophoresis (micro-CE)/UV or fluorescence detection. PCR products of polymorphisms on the human Y-chromosome related to spermatogenic failure did not need purification. They were premixed and comigrated with a DNA digest whose concentration was known. Hydroxyethyl cellulose (HEC) dissolved in 5x Tris-borate-EDTA (5x TBE, pH 8.3) was used as a separation matrix in a linear polyacrylamide-coated quartz microchip, while mixed poly(ethyl oxides) (PEOs) of different molar-masses dissolved in 1 x TBE (pH 8.3) containing 1 ng/microl ethidium bromide was used as a separation matrix in an uncoated poly(methyl methacrylate) (PMMA) microchip. Elution profiles were monitored under either real-time linear imaging UV detection in the snapshot mode where the total separation time is fixed, or light-emitting diode (LED) confocal fluorescence detection in the finishline mode where solutes migrate over the same separation length. It is found that, in both modes, a linear relation exists between the peak areas (A) and the multiplication of the digest concentrations (C) and the fragment sizes (L) in a DNA restrictive digest. Using the comigration electropherogram of a single-step experiment, the concentrations of PCR products were directly determined using the A versus LC linear relationship. The sole condition to obey is that the chosen digest has different fragment sizes with the PCR products of interest. This condition is easy to obey, because micro-CE owns high separation ability, and many digests are commercially available. The recovery of the technique was between 98 and 105%. The R.S.D. for chip-to-chip concentration measurements was less than 6.0% (n = 6). Hence, the technique was accurate and reliable for DNA assays. PMID:15532567

Xu, Feng; Jabasini, Mohammad; Zhu, Bingmei; Ying, Ling; Cui, Xuezhi; Arai, Akihiro; Baba, Yoshinobu



An effective protein extraction method for two-dimensional electrophoresis in the anticancer herb Andrographis paniculata Nees.  


Proteomic analysis of plants relies on high yields of pure protein. In plants, protein extraction and purification present a great challenge due to accumulation of a large amount of interfering substances, including polysaccharides, polyphenols, and secondary metabolites. Therefore, it is necessary to modify the extraction protocols. A study was conducted to compare four protein extraction and precipitation methods for proteomic analysis. The results showed significant differences in protein content among the four methods. The chloroform-trichloroacetic acid-acetone method using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer provided the best results in terms of protein content, pellets, spot resolution, and intensity of unique spots detected. An overall of 83 qualitative or quantitative significant differential spots were found among the four methods. Based on the 2-DE gel map, the method is expected to benefit the development of high-level proteomic and biochemical studies of Andrographis paniculata, which may also be applied to other recalcitrant medicinal plant tissues. PMID:23725097

Talei, Daryush; Valdiani, Alireza; Puad, Mohd Abdullah



Gel Electrophoresis  

NSDL National Science Digital Library

In this activity, learners simulate the process of DNA fingerprinting by using electricity to separate colored dyes. Learners use simple materials to assemble a comb (electrophoresis chamber) to hold the samples, make a 0.2% sodium bicarbonate buffer and 1% gel solution, connect a high voltage power supply, and prepare 5 different samples. Then learners test their model and observe each sample.

Yu, Julie



The development of simple and sensitive small-molecule fluorescent probes for the detection of serum proteins after native polyacrylamide gel electrophoresis.  


In this paper, a simple and sensitive small-molecule fluorescent probe, 2,5-dihydroxy-4'-dimethylaminochalcone (DHDMAC), was designed and synthesized for the detection of human serum proteins via hydrophobic interactions after polyacrylamide gel electrophoresis (PAGE). This probe produced lower fluorescence emission in the absence of proteins, and the emission intensity was significantly increased after the interaction with serum proteins. To demonstrate the imaging performance of this probe as a fluorescent dye, a series of experiments was conducted that included sensitivity comparison and 2D-PAGE. The results indicated that the sensitivity of DHDMAC staining is comparable to that of the most widely used fluorescent dye, SYPRO Ruby, and more protein spots (including thyroxine-binding globulin, angiotensinogen, afamin, zinc-?-2-glycoprotein and ?-1-antichymotrypsin) were detected after 2D-PAGE. Therefore, DHDMAC is a good protein reporter due to its fast staining procedure, low detection limits and high resolution. PMID:22475746

Wang, Fangfang; Huang, Lingyun; Na, Na; He, Dacheng; Sun, Dezhi; Ouyang, Jin



Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients.  


A modified procedure for the two-dimensional electrophoretic analysis of basic polypeptides is described. This method uses isoelectric focusing with carrier ampholytes in the first dimension, and sodium dodecyl sulfate-electrophoresis in the second dimension. Counteraction of the cathodic drift is achieved by glass tube treatment (silanization), electrolyte modification (use of weak bases and acids), protection of the catholyte from carbon dioxide, and the addition of glycerol to the gel mix. Better resolution and reproducibility are obtained than with nonequilibrium pH gradient electrophoresis, since quasi equilibrium focusing can be obtained. PMID:8026444

Rabilloud, T



A replaceable microreactor for on-line protein digestion in a two-dimensional capillary electrophoresis system with tandem mass spectrometry detection.  


We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and ?-casein as model proteins. PMID:21030030

Li, Yihan; Wojcik, Roza; Dovichi, Norman J



Quantitative analysis of pheromone-binding protein specificity  

PubMed Central

Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using ?-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between ?-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the ?-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ~100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ~200 nM for the silk moth pheromone bombykol and ~90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the pheromone receptor model proposed by Laughlin et al. (Cell 133: 1255–65, 2008) are discussed. PMID:23121132

Katti, S.; Lokhande, N.; González, D.; Cassill, A.; Renthal, R.



Graft copolymer composed of cationic backbone and bottle brush-like side chains as a physically adsorbed coating for protein separation by capillary electrophoresis.  


To stabilize electroosmotic flow (EOF) and suppress protein adsorption onto the silica capillary inner wall, a cationic hydroxyethylcellulose-graft-poly (poly(ethylene glycol) methyl ether methacrylate) (cat-HEC-g-PPEGMA) graft copolymer composed of cationic backbone and bottle brush-like side chains was synthesized for the first time and used as a novel physically adsorbed coating for protein separation by capillary electrophoresis. Reversed (anodal) and very stable EOF was obtained in cat-HEC-g-PPEGMA-coated capillary at pH 2.2-7.8. The effects of degree of cationization, PEGMA grafting ratio, PEGMA molecular mass, and buffer pH on the separation of basic proteins were investigated. A systematic comparative study of protein separation in bare and HEC-coated capillaries and in cat-HEC-g-PPEGMA-coated capillary was also performed. The basic proteins can be well separated in cat-HEC-g-PPEGMA-coated capillary over the pH range of 2.8-6.8 with good repeatability and high separation efficiency, because the coating combines good protein-resistant property of bottle brush-like PPEGMA side chains with excellent coating ability of cat-HEC backbone. Besides its success in separation of basic proteins, the cat-HEC-g-PPEGMA coating was also superior in the fast separation of other protein samples, such as protein mixture, egg white, and saliva, which indicates that it is a promising coating for further proteomics analysis. PMID:22038787

Zhou, Dan; Xiang, Lina; Zeng, Rongju; Cao, Fuhu; Zhu, Xiaoxi; Wang, Yanmei



Identification of low-abundance proteins of bovine colostral and mature milk using two-dimensional electrophoresis followed by microsequencing and mass spectrometry.  


We identified several low-abundance proteins of bovine colostrum and mature milk using the immunoabsorption technique and two-dimensional electrophoresis (2-DE) followed by microsequencing and mass spectrometry. Two major milk proteins, beta-casein and immunoglobulin G (IgG), were effectively removed from the milk using immunoabsorbents. Milk samples before and after immunoabsorption were separated by 2-DE. Protein identification of the spots on 2-DE was performed by either gel comparison, microsequencing, matrix-assisted laser desorption/ionization-time of flight mass-spectrometry (MALDI-TOF-MS), peptide mass fingerprinting or peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight-MS (Q-TOF). Significant differences in protein patterns were observed between the low-abundance proteins of colostrum and mature milk. In addition, several low-abundance proteins including fibrinogen beta-chain, chitinase 3-like 1, alpha-antitrypsin, complement C3 alpha-chain, gelsolin and apolipoprotein H were observed only in colostrum. However, the level of beta-casein fragments increased significantly during this lactation period. alpha-Lactalbumin and beta-lactoglobulin as well as some low-abundance proteins including bovine serum albumin, serotransferrin and lactoferrin were identified in both colostral and mature milk. Low-abundance proteins in bovine colostrum may have special physiologic relevance to the health and development of calves early in lactation. PMID:11981865

Yamada, Masamichi; Murakami, Kouki; Wallingford, John C; Yuki, Yoshikazu



Evaluation of two-dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples  

SciTech Connect

Laser microdissection (LM) allows the collection of homogeneous tissue- and cell specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there is as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryosectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.

Schad, Martina; Lipton, Mary S.; Giavalisco, Patrick; Smith, Richard D.; Kehr, Julia



Affinity chromatography, two-dimensional electrophoresis, adapted immunodepletion and mass spectrometry used for detection of porcine and piscine heparin-binding human plasma proteins.  


Heparin-binding proteins in human plasma were studied using affinity chromatography columns with porcine (2mL, 10.7mg capacity) and piscine heparin (5mL, 2.7mg capacity). Two-dimensional electrophoresis (Bio-Rad Protean II gel system with 16cm×16cm gels using isoelectric focusing (IEF) and nonequilibrium pH-gradient gel electrophoresis (NEPHGE)), Bruker Ultraflex MALDI-TOF mass spectrometry and immunoblotting (NovaBlot semidry discontinuous blotting) were used for unfractionated plasma. This revealed electropherograms with differences between porcine and piscine heparin-binding and totally 17 different fibrinogen variants from all 3 chains. Immunodepletion was used to remove fibrinogen (42.1mg anti-human fibrinogen in 8.4mL resin) and serum albumin (0.42mg binding capacity in 14mL resin) and porcine and piscine heparin-binding proteins were identified using liquid chromatography-mass spectrometry (Ultimate 3000 NanoLC with Acclaim PepMap 100 column (50cm×75?m)-LTQ Orbitrap Mass XL). In total, the binding of 76 putative or acknowledged biomarkers are shown. Of the identified proteins, 14 are not previously shown to be heparin-binding, such as the low concentration proteins lipocalin-1 and tropomyosin and a hitherto not detected protein in plasma, zinc finger protein 483. The putative heparin-binding sequences were analyzed. The results suggest that the combination of group specific affinity and adapted immunodepletion chromatography could be useful in the study of the plasma proteome. PMID:24316520

Bjarnadóttir, Stefanía Guðrún; Flengsrud, Ragnar



Quantitation of the ribosomal protein autoregulatory network using mass spectrometry  

PubMed Central

Relative levels of ribosomal proteins were quantified in crude cell lysate using mass spectrometry. A method for quantifying cellular protein levels using macromolecular standards is presented that does not require complex sample separation, identification of high-responding peptides, affinity purification or post-growth modifications. Perturbations in ribosomal protein levels by overexpression of individual proteins correlate to known autoregulatory mechanisms and extend the network of ribosomal protein regulation. PMID:20481440

Sykes, Michael T.; Sperling, Edit; Chen, Stephen S.; Williamson, James R.



Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelity approach to identify constituents of protein complexes  

PubMed Central

Background Here we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophila melanogaster, PINCH has two known high-affinity binding partners—Integrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5—but has been postulated to bind additional proteins as well. Results To purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry. Conclusions The iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co-purifications inherent in iGEO allow for rapid and unambiguous identification of the constituents of protein complexes, without the need for extensive follow-up experiments. PMID:23663728



Semi-quantitative measurement of specific proteins in human cumulus cells using reverse phase protein array  

PubMed Central

Background The ability to predict the developmental and implantation ability of embryos remains a major goal in human assisted-reproductive technology (ART) and most ART laboratories use morphological criteria to evaluate the oocyte competence despite the poor predictive value of this analysis. Transcriptomic and proteomic approaches on somatic cells surrounding the oocyte (granulosa cells, cumulus cells [CCs]) have been proposed for the identification of biomarkers of oocyte competence. We propose to use a Reverse Phase Protein Array (RPPA) approach to investigate new potential biomarkers of oocyte competence in human CCs at the protein level, an approach that is already used in cancer research to identify biomarkers in clinical diagnostics. Methods Antibodies targeting proteins of interest were validated for their utilisation in RPPA by measuring siRNA-mediated knockdown efficiency in HEK293 cells in parallel with Western blotting (WB) and RPPA from the same lysates. The proteins of interests were measured by RPPA across 13 individual human CCs from four patients undergoing intracytoplasmic sperm injection procedure. Results The knockdown efficiency of VCL, RGS2 and SRC were measured in HEK293 cells by WB and by RPPA and were acceptable for VCL and SRC proteins. The antibodies targeting these proteins were used for their detection in human CCs by RPPA. The detection of protein VCL, SRC and ERK2 (by using an antibody already validated for RPPA) was then carried out on individual CCs and signals were detected for each individual sample. After normalisation by VCL, we showed that the level of expression of ERK2 was almost the same across the 13 individual CCs while the level of expression of SRC was different between the 13 individual CCs of the four patients and between the CCs from one individual patient. Conclusions The exquisite sensitivity of RPPA allowed detection of specific proteins in individual CCs. Although the validation of antibodies for RPPA is labour intensive, RRPA is a sensitive and quantitative technique allowing the detection of specific proteins from very small quantities of biological samples. RPPA may be of great interest in clinical diagnostics to predict the oocyte competence prior to transfer of the embryo using robust protein biomarkers expressed by CCs. PMID:24148967



The structure and thermodynamics of protein—SDS complexes in solution and the mechanism of their transports in gel electrophoresis process  

NASA Astrophysics Data System (ADS)

A unified exposition of the structure, thermodynamics and transport of complexes formed by association of a surfactant sodium dodecyl sulfate (SDS) with water-soluble globular proteins in solution is presented. From small angle neutron scattering experiments we have determined the structure of the complex to be a polymer-like object consisting of a string of SDS micelles decorating the hydrophobic patches of the unfolded polypeptide chain in solution. Based on this structural model we have predicted and observed a polymer-like phase separation of the denatured protein solution at suitable pH. This model can also successfully predict the mobility of the complexes in polyacrylamide gel during an electrophoresis process, if we adapt the well known reptation concept of diffusion of a single chain of polymer in gels.

Guo, Xuan-Hui; Chen, Sow-Hsin



Proteins pattern alteration in AZT-treated K562 cells detected by two-dimensional gel electrophoresis and peptide mass fingerprinting  

PubMed Central

In this study we report the effect of AZT on the whole protein expression profile both in the control and the AZT-treated K562 cells, evidenced by two-dimensional gel electrophoresis and peptide mass fingerprinting analysis. Two-dimensional gels computer digital image analysis showed two spots that appeared up-regulated in AZT-treated cells and one spot present only in the drug exposed samples. Upon extraction and analysis by peptide mass fingerprinting, the first two spots were identified as PDI-A3 and stathmin, while the third one was proved to be NDPK-A. Conversely, two protein spots were present only in the untreated K562 cells, and were identified as SOD1 and HSP-60, respectively. PMID:16571109

D'Andrea, Gabriele; Lizzi, Anna R; Venditti, Sara; Di Francesco, Laura; Giorgi, Alessandra; Mignogna, Giuseppina; Oratore, Arduino; Bozzi, Argante



Variation and Genomic Localization of Genes Encoding DROSOPHILA MELANOGASTER Male Accessory Gland Proteins Separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis  

PubMed Central

Accessory gland proteins from Drosophila melanogaster males have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into nine major bands. When individual males from 175 strains were examined, considerable polymorphism for nearly one-half of the major protein bands was seen, including null alleles for three bands. Variation was observed not only among long-established laboratory strains but also among stocks recently derived from natural populations. There was little difference in the amount of variation between P and M strains, indicating that P element mutagenesis is not a factor producing the variation. Codominant expression of variants for each of five bands was found in heterozygotes, suggesting structural gene variation and not posttranslational modification variation. Stocks carrying electrophoretic variants of four of the major proteins were used to map the presumed structural genes for these proteins; the loci were found to be dispersed on the second chromosome. Since males homozygous for variant proteins were fertile, the polymorphism seems to have little immediate effect on successful sperm transfer. We propose that a high degree of polymorphism can be tolerated because these proteins play a nutritive rather than enzymatic role in Drosophila reproduction. PMID:3095182

Whalen, Michael; Wilson, Thomas G.



Gel Electrophoresis on a Budget to Dye For  

NSDL National Science Digital Library

Gel electrophoresis is one of the most important tools used in molecular biology and has facilitated the entire field of genetic engineering by enabling the separation of nucleic acids and proteins. However, commercial electrophoresis kits can cost up to

Yu, Julie H.



In vitro assay of the interaction between Rnc1 protein and Pmp1 mRNA by affinity capillary electrophoresis with a carboxylated capillary.  


The interaction between Rnc1, an RNA interactive protein, and a Pmp1 mRNA was investigated by affinity capillary electrophoresis (ACE). Prior to the ACE experiments, the column performances of three capillaries (an untreated fused silica capillary, a polybrene-polyacrylic acid (PB-PAA) double layer coating capillary, and a carboxylated capillary with a covalent modification) were studied with model proteins including ribonuclease B (RNase B) and bovine serum albumin (BSA). Using an untreated fused silica and a PB-PAA double layer coating capillaries, both of the protein peaks were broad and tailing. However, using a carboxylated capillary, the protein peaks were sharp and symmetric, and migration times were repeatable (RSD<0.4%). Further, the proteins in human serum also gave sharp peaks and its repeatability was kept at a high level by pre-treatment of a capillary inner wall with 1M sodium chloride solution before each run. An Rnc1 protein was analyzed by ACE with background electrolytes containing various concentrations of Pmp1 sense mRNA using a carboxylated capillary. Increase in the concentration of the mRNA was found to delay the migration time of the protein. But the migration time of the protein was kept constant with increasing Pmp1 anti-sense mRNA instead of Pmp1 sense mRNA. A straight line (r=0.987) was obtained by plotting 1/(migration time shift) versus 1/(Pmp1 sense mRNA concentration) and the association constant of Rnc1 protein with Pmp1 sense mRNA could be estimated to be 4.15x10(6)M(-1). These results suggest that the association constants of proteins with mRNAs as ligands were easily determined by the proposed method. PMID:20692117

Taga, Atsushi; Satoh, Ryosuke; Ishiwata, Shunji; Kodama, Shuji; Sato, Atsushi; Suzuki, Kentaro; Sugiura, Reiko



Quantitative studies in effects of additives on protein aggregation  

E-print Network

Rational design of protein additives has been limited by the understanding of mechanism of protein and additive interaction. In this work we have applied molecular dynamics with all atom potentials in order to study the ...

Shinde, Chetan (Chetan Ulhas)



Detection of metals in proteins by means of polyacrylamide gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry: application to selenium.  


The capabilities of laser ablation-inductively coupled plasma-mass spectrometry for the detection of trace elements in a gel after gel electrophoresis were systematically studied. Figures of merit, such as limit of detection, linearity, and repeatability, were evaluated for various elements (Li, V, Cr, Mn, Ni, Cu, Zn, As, Se, Mo, Pd, Ag, Cd, Pt, Tl, Pb). Two ablation strategies were followed: single hole drilling, relevant for ablation of spots after two-dimensional (2-D) separations, and ablation with translation, i.e., on a line, relevant for one-dimensional (1-D) separations. This technique was applied to the detection of selenoproteins in red blood cells extracts after a 1-D separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the detection of selenium-containing proteins in yeast after 2-D electrophoresis (2-DE). The detection procedure was further improved by using the dynamic reaction cell technology, which allowed the removal of the Ar_2(+) interference and hence the use of the most abundant Se isotope, (80)Se. Reaction gases were compared (methane, carbon monoxide, ammonia, oxygen and the combination of argon (collision gas) and hydrogen (reaction gas)). In each instance, the reaction cell parameters were optimized in order to obtain the lowest detection limit for Se (as (80)Se(+), (82)Se(+) or (77)Se(+); and as (80)Se(16)O(+), (82)Se(16)O(+) or (77)Se(16)O(+) with O(2) as the reaction gas). Carbon monoxide was found to offer the best performance. The detection limit with the use of DRC and He as transport gas was 0.07 microg Se g(-1) gel with single hole drilling and 0.15 microg Se g(-1) gel for ablation with translation. PMID:14595676

Chéry, Cyrille C; Günther, Detlef; Cornelis, Rita; Vanhaecke, Frank; Moens, Luc



Interferences of suspended clay fraction in protein quantitation by several determination methods  

Microsoft Academic Search

Seven current methods of protein quantitation, Bradford (standard, micro, and 590\\/450nm ratio), Lowry, bicinchoninic acid (BCA), UV spectrophotometry at 280nm, and Quant-iT fluorescence-based determination, were compared with regard to their susceptibility to interferences due to the presence of suspended and not easily detectable clay particles. Bovine serum albumin (BSA) and Na-Wyoming montmorillonite were selected as model protein and reference clay,

I. Lozzi; A. Pucci; O. L. Pantani; L. P. D’Acqui; L. Calamai



A microfluidic platform for high-throughput multiplexed protein quantitation  

E-print Network

We present a high-throughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. Samples are quantitated in each unit cell by four independent MITOMI detection areas, allowing four samples to be analyzed in parallel for a total of 1,536 assays per device. We show that the device can be pre-assembled and stored for weeks at elevated temperature and we performed proof-of-concept experiments simultaneously quantitating IL-6, IL-1\\b{eta}, TNF-{\\alpha}, PSA, and GFP. Finally, we show that the platform can be used to identify functional antibody combinations by screening 64 antibody combinations requiring up to 384 unique assays per device.

Volpetti, Francesca; Maerkl, Sebastian



Comparison of radial immunodiffusion and alkaline cellulose acetate electrophoresis for quantitating elevated levels of fetal hemoglobin (HbF): application to evaluating patients with sickle cell disease treated with hydroxyurea.  


Radial immunodiffusion (RID), alkaline cellulose acetate electrophoresis, and high-performance liquid chromatography (HPLC) were compared for quantitating the elevated (> 10%) level of fetal hemoglobin (HbF) found in the red blood cells of sickle cell disease patients undergoing treatment with hydroxyurea. HPLC- and electrophoresis-determined values were comparable. The RID-determined values were higher, in many cases twofold higher. False high HbF values would be misleading in assessing the effectiveness of hydroxyurea therapy in sickle cell disease patients. We subsequently initiated an examination of the variation in HbF values due to the use of different HbF radial immunodiffusion QUIPlates and different positions within a single plate in an attempt to determine the cause of these discrepancies. Within-run precision studies indicated that significantly different size precipitin rings were obtained depending upon which area of the plate the hemolysate containing antigen (HbF) was applied. A common feature associated with poor precision plates was a marked difference in degree of coloration of gel throughout the plate. Spuriously high HF concentrations were obtained with antigen (HbF) placed in wells located in the lighter colored gel area while antigen placed in wells in the darker colored area of the agarose gel bed were more in agreement with the electrophoretically determined HbF concentrations. The variation in HbF values was significantly greater in the diluted (HbF QUIPlate Diluent) samples than in the neat samples even on plates of uniform gel coloration. As a result of this study, we will continue to monitor high HbF levels by densitometry following alkaline cellulose acetate electrophoresis. PMID:10102137

Schultz, J C



Analytical biotechnology: Capillary electrophoresis and chromatography  

SciTech Connect

The papers describe the separation, characterization, and equipment required for the electrophoresis or chromatography of cyclic nucleotides, pharmaceuticals, therapeutic proteins, recombinant DNA products, pheromones, peptides, and other biological materials. One paper, On-column radioisotope detection for capillary electrophoresis, has been indexed separately for inclusion on the data base.

Horvath, C.; Nikelly, J.G. (eds.)



Getting the Most out of Electrophoresis Units  

ERIC Educational Resources Information Center

At Oklahoma City Community College, they have developed gel electrophoresis activities that support active learning of many scientific concepts, including: pH, electrolysis, oxidation reduction, electrical currents, potentials, conductivity, molarity, gel electrophoresis, DNA and protein separation, and DNA fingerprinting. This article presents…

Mulvihill, Charlotte



A combined radiolabelling and silver staining technique for improved visualisation, localisation, and identification of proteins separated by two-dimensional gel electrophoresis.  


Two-dimensional gel electrophoresis (2-DE) remains the method of choice for the Separation of protein mixtures whilst mass spectrometry (MS) is rapidly becoming the premier tool for protein identification. When combined, 2-DE and MS form the current operating paradigm for classical proteomics. One of the key challenges of proteome research is that of detecting and identifying all of the elements (proteins) of a proteome. Silver staining and radiolabelling, e.g. with 35S-methionine ([35S]-met), represent two sensitive methods used to visualise many of the constitutive and synthesised elements of a proteome, respectively. The latter method allows a very low total protein loading on a two-dimensional (2-D) gel and challenges protein identification using current MS-based technology. Therefore, it is necessary to refer to and locate a radiolabelled spot's cognate on a preparatively loaded stained gel, or Western blot, and use that protein spot for identification. Unfortunately, the images of autoradiographs and preparative gels or blots, even of the same sample, often do not correspond making it difficult to accurately locate and select spots of interest by visual comparison. We have established a technique that permits the unambiguous localisation of radiolabelled proteins on the same silver stained 2-D gel. Protein identification of superimposed spots is described by peptide mass fingerprinting and database searching using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and by peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight MS (Q-TOF). PMID:11680883

Westbrook, J A; Yan, J X; Wait, R; Dunn, M J




EPA Science Inventory

An immunoassay method is described for the quantitative determination of synapsin I (protein I) and of a 36,000-dalton membrane protein from rat brain synaptic vesicles. The samples are spotted on nitrocellulose membrane filters, incubated sequentially with specific antibodies an...


Quantitative determination of curcuminoids in Curcuma rhizomes and rapid differentiation of Curcuma domestica Val. and Curcuma xanthorrhiza Roxb. by capillary electrophoresis.  


The three major curcuminoids, curcumin, demethoxycurcumin and bis-demethoxycurcumin, from Curcuma domestica Val. (Curcuma longa L.) and Curcuma xanthorrhiza Roxb. (Zingiberaceae) were fully separated and quantified in less than 5 min using a capillary zone electrophoresis method with standard fused-silica capillaries and photodiode array detection. An electrolyte solution of 20 mM phosphate, 50 mM sodium hydroxide and 14 mM beta-cyclodextrin was found to be appropriate. Quantification was performed with 3,4-dimethoxy-trans-cinnamic acid as internal standard, and the limit of detection was 0.01 mg/mL. Extraction, stabilisation during sample storage and quantification procedures were optimised and carried out with drugs and commercial curry powder from different provenances. The results were compared with the photometric method of the monograph Curcumae xanthorrhizae rhizoma of the European Pharmacopoeia. PMID:15202598

Lechtenberg, Matthias; Quandt, Bettina; Nahrstedt, Adolf



A quantitative study of antigen—antibody combination during disk electrophoresis in acrylamide gel using Iodine-131 labelled human growth hormone  

PubMed Central

An adaptation of the technique of disk electrophoresis is described. It allows combination of antigen and antibody, as well as the separation of the free antigen from the complex, in a single electrophoretic step. An Iodine-131 labelled human growth hormone: antihuman growth hormone system was used to demonstrate this combination which was expressed as per cent radioactivity bound to immune ?-globulin. Dilution of antiserum gave reproducible titration curves when run at three different temperatures: 10°, 20°, 35°. The curves obtained at 35° showed increased binding. The reduction of bound radioactivity on addition of unlabelled growth hormone to the sample was a linear function of the logarithm of unlabelled growth hormone. The standard curves obtained are suitable for a highly sensitive assay of human growth hormone. ImagesFIG. 1FIG. 3 PMID:14169113

Fitschen, W.




EPA Science Inventory

Acute exposure of humans to 0.4 ppm ozone is known to cause production of components which mediate inflammation and damage in the lung. he contribution of alveolar macrophages to this process is not well understood. n addition, ozone may cause more extensive cellular changes than...


The effect of sample salt additives on capillary electrophoresis analysis of intact proteins using surface modified capillaries.  


The effect of adding alkali salts to protein samples for capillary electrophoretic (CE) analysis of intact proteins was studied. A high degree of peak stacking, even for large proteins, was found to occur when alkali salts were added to the sample. The addition of salt to the protein sample promotes a strong improvement in the peak efficiency of individual proteins giving up to 2.1x 10(6)apparent plates/m. The concentration of salt required in the sample to reach optimal peak efficiency show dependency on both the molecular weight and molar concentration of the protein. However, adding salt will, at a sufficiently high concentration, cause a mixture of proteins to co-migrate to one very sharp peak. The observed sample stacking effect was obtained with a number of different surface modified silica capillaries indicating a general phenomenon and not surface coating specific. PMID:19150070

Elhamili, Anisa; Wetterhall, Magnus; Puerta, Angel; Westerlund, Douglas; Bergquist, Jonas



A comparison of extracted proteins of isolates of Dermatophilus congolensis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting.  


Antigenic diversity within a collection of 18 isolates of Dermatophilus congolensis from different Continents was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting with sera from cattle with clinical dermatophilosis using whole cell extracts obtained by three methods and one extract of extracellular products of D. congolensis. One of the methods involving the release of a lysostaphin-solubilized protein (LSP) of whole cells of D. congolensis revealed a number of discrete and easily-identifiable bands in SDS-PAGE which were found suitable for characterizing protein patterns and was, therefore, subsequently used for a comparative analysis of the proteins of all the D. congolensis isolates. Six electropherotypes (ET) of D. congolensis were identified among the 18 isolates using the protein profiles based on the presence of four protein bands at Molecular weights (MW) 62, 28, 17.4 and 16.4 kDa. The ETs were found among isolates from different animal species and from different sources with ET1 consisting of three bovine and two equine isolates; ET2, two bovine and three ovine isolates; ET3, two bovine isolates; ET4, two bovine isolates; ET5, one bovine and one ovine isolates and ET6, two bovine isolates. Immunoblotting of the extracts of D. congolensis isolates with sera from cattle with clinical dermatophilosis infection demonstrated protein bands of MW ranging from 9 kDa to 188 kDa. Sera from chronic dermatophilosis infection demonstrated a 28 kDa protein which was immunodominant in the LSP extracts of all the 18 isolates of D. congolensis tested while sera from mild infections demonstrated mainly the 62 kDa protein in the same extracts. However, many protein bands were demonstrated in surface membrane (TSMP) and extracellular protein extracts with sera from only mildly infected animals. The protein patterns observed in all isolates of D. congolensis revealed global antigenic similarities and distinct differences among isolates which could not be associated with either geographic, climatic or host factors. Also sera from infected animals from endemic regions of dermatophilosis could not differentiate isolates of D. congolensis. This suggests the possibility that such sera must have come from animals that had been infected by a multitude of D. congolensis strains present in the herd environment and strains an animal could have come across during the 'ritual' annual cross-country migration of the cattle herds. PMID:10466501

Makinde, A A; Gyles, C L



Dendritic glycopolymers as dynamic and covalent coating in capillary electrophoresis: View on protein separation processes and detection of nanogram-scaled albumin in biological samples.  


Unique properties of dendritic polymers make them promising candidates for application as additives in various analytical methods. From this point, we investigated the potential use of maltose-modified hyperbranched poly(ethylene imine) (PEI-Mal) as a dynamically or covalently bound coating and a pseudostationary phase in capillary electrophoresis. The EOF mobilities were measured at different pH values (2.2, 8.5, and 10.2) using PEI-Mal in the background electrolyte (BGE) and desirable repeatability of the EOF with % RSD (n=50) ?3.3 was obtained. The influence of pH, polymer concentration, and density of maltose shell on the separation properties of a model mixture of four proteins (albumin, lysozyme, myoglobin, insulin) were investigated. Applying PEI-Mal as a dynamic coating, improved separation of the protein mixture with a high repeatability was achieved. Applying PEI-Mal as a covalent coating for concentrating proteins in the large volume sample stacking (LVSS) combined with the field-enhanced sample injection (FESI), up to 1320-fold enhancement of sensitivity was achieved. The detection limit of 100-500ng/ml allowed successful analysis of albumin level both in blood and urine samples without additional preconcentration. PMID:25555410

Polikarpov, Nikita; Potolytsyna, Vera; Bessonova, Elena; Tripp, Sandra; Appelhans, Dietmar; Voit, Brigitte; Kartsova, Ludmilla



Large-gel two-dimensional electrophoresis-matrix assisted laser desorption/ionization-time of flight-mass spectrometry: an analytical challenge for studying complex protein mixtures.  


The large-gel two-dimensional electrophoresis (2-DE) technique, developed by Klose and co-workers over the past 25 years, provides the resolving power necessary to separate crude proteome extracts of higher eukaryotes. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) provides the sample throughput necessary to identify thousands of different protein species in an adequate time period. Spot excision, in situ proteolysis, and extraction of the cleavage products from the gel matrix, peptide purification and concentration as well as the mass spectrometric sample preparation are the crucial steps that interface the two analytical techniques. Today, these routines and not the mass spectrometric instrumentation determine how many protein digests can be analyzed per day per instrument. The present paper focuses on this analytical interface and reports on an integrated protocol and technology developed in our laboratory. Automated identification of proteins in sequence databases by mass spectrometric peptide mapping requires a powerful search engine that makes full use of the information contained in the experimental data, and scores the search results accordingly. This challenge is heading a second part of the paper. PMID:11565779

Nordhoff, E; Egelhofer, V; Giavalisco, P; Eickhoff, H; Horn, M; Przewieslik, T; Theiss, D; Schneider, U; Lehrach, H; Gobom, J



Stage-specific analysis of plasma protein profiles in ovarian cancer: Difference in-gel electrophoresis analysis of pooled clinical samples  

PubMed Central

Introduction: Ovarian cancer is the leading cause of death from gynecological cancer. Non-specific symptoms early in disease and the lack of specific biomarkers hinder early diagnosis. Multi-marker blood screening tests have shown promise for improving identification of early stage disease; however, available tests lack sensitivity, and specificity. Materials and Methods: In this study, pooled deeply-depleted plasma from women with Stage 1, 2 or 3 ovarian cancer and healthy controls were used to compare the 2-dimensional gel electrophoresis (2-DE) protein profiles and identify potential novel markers of ovarian cancer progression. Results/Discussion: Stage-specific variation in biomarker expression was observed. For example, apolipoprotein A1 expression is relatively low in control and Stage 1, but shows a substantial increase in Stage 2 and 3, thus, potential of utility for disease confirmation rather than early detection. A better marker for early stage disease was tropomyosin 4 (TPM4). The expression of TPM4 increased by 2-fold in Stage 2 before returning to “normal” levels in Stage 3 disease. Multiple isoforms were also identified for some proteins and in some cases, displayed stage-specific expression. An interesting example was fibrinogen alpha, for which 8 isoforms were identified. Four displayed a moderate increase at Stage 1 and a substantial increase for Stages 2 and 3 while the other 4 showed only moderate increases. Conclusion: Herein is provided an improved summary of blood protein profiles for women with ovarian cancer stratified by stage. PMID:23858298

Bailey, Mark J.; Shield-Artin, Kristy L.; Oliva, Karen; Ayhan, Mustafa; Reisman, Simone; Rice, Gregory E.



Microfabricated Channel Array Electrophoresis for Rapid Characterization and Screening of Enzymes using RGS-G Protein Interactions as a Model System  

PubMed Central

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 × 7.62 cm glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial CCD camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menton constants. A chip with 36 channels was used for screening for modulators of the G protein: RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. 36 electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4,320 assays per hour with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11% respectively in the 16 and 36-channel designs. PMID:18465881

Pei, Jian; Dishinger, John F.; Roman, David L.; Rungwanitcha, Chetwana; Neubig, Richard R.; Kennedy, Robert T.



A simple technique to improve the resolution of membrane acidic proteins of the haloarchaeon Haloferax volcanii by 2D electrophoresis.  


Proteins present in the archaeal cell envelope play key roles in a variety of processes necessary for survival in extreme environments. The haloarchaeon Haloferax volcanii is a good model for membrane proteomic studies because its genome sequence is known, it can be genetically manipulated, and a number of studies at the "omics" level have been performed in this organism. This work reports an easy strategy to improve the resolution of acidic membrane proteins from H. volcanii by 2DE. The method is based on the solubilization, delipidation, and salt removal from membrane proteins. Due to the abundance of the S-layer glycoprotein (SLG) in membrane protein extracts, other proteins from the envelope are consequently underrepresented. Thus, a protocol to reduce the amount of the SLG by EDTA treatment was applied and 11 cm narrow range pH (3.9-5.1) IPG strips were used to fractionate the remaining proteins. Using this method, horizontal streaking was substantially decreased and at least 75 defined spots (20% of the predicted membrane proteome within this pI/Mw range) were reproducibly detected. Two of these spots were identified as thermosome subunit 1 and NADH dehydrogenase from H. volcanii, confirming that proteins from the membrane fraction were enriched. Removal of the SLG from membrane protein extracts can be applied to increase protein load for 2DE as well as for other proteomic methods. PMID:25224925

Paggi, Roberto A; Giménez, María Inés; De Castro, Rosana E; Cesari, Andreina



Determination of cow's milk and ripening time in nonbovine cheese by capillary electrophoresis of the ethanol-water protein fraction.  


A novel method is reported for analyzing adulteration of goat and ewe cheeses with cow's milk: capillary zone electrophoresis (CZE) in isoelectric, acidic buffers (50 mM imino diacetic acid, IDA, pH = pI 2.3). The cheese samples were extracted with a 20:80 v/v ethanol-water mixture in presence of 3 M urea and 1% beta-mercaptoethanol for 1 h. After centrifugation and lipid extraction, the samples were dissolved in 50 mM IDA, 6 M urea and 0.5% hydroxyethyl cellulose and analyzed by CZE at 700 V/cm. A total of 18 characteristic peaks were resolved among the three types of cheeses and 18 variables were defined as their respective areas. There was excellent similarity among the electrophoretic patterns obtained with cheeses of a given type of milk, while cheeses made with different types of milk were easily distinguishable. Most peaks were common to all cheeses, but the profile differed depending on the type of milk used. Principal component analysis, linear discriminant analysis, and partial least squares regression (PLS) were used for statistical analysis of the data obtained by CZE. In particular, by using PLS multivariate regression, the contents of cow's milk in presumably pure goat and ewe cheeses, as well as in binary and ternary mixtures, could be predicted with relative standard deviations of ca. 6-7%. In addition, the ripening time in goat and ewe cheeses could also be predicted. PMID:10726770

Herrero-Martínez, J M; Simó-Alfonso, E F; Ramis-Ramos, G; Gelfi, C; Righetti, P G



High-Sensitivity Detection and Quantitative Analysis of Native Protein-Protein Interactions and Multiprotein Complexes by Flow Cytometry  

PubMed Central

Most mechanisms of cell development, physiology, and signal transduction are controlled by protein-protein interactions. Immunoprecipitation of multiprotein complexes detected by flow cytometry (IP-FCM) is a means to quantitatively measure these interactions. The high sensitivity of this method makes it useful even when very little biomaterial is available for analysis, as in the case of rare primary cell subsets or patient samples. Detection of the T cell antigen receptor associated with the CD3 multiprotein complex from as few as 300 primary murine T cells is presented as an example. The method is compatible with quantitative flow cytometry techniques, making it possible to estimate the number of coimmunoprecipitated molecules. Both constitutive and inducible protein-protein interactions can be analyzed, as illustrated in related methodology using glutathione S-transferase–fusion protein pull-down experiments. IP-FCM represents a robust, quantitative, biochemical technique to assess native protein-protein interactions, without requiring genetic engineering or large sample sizes. PMID:17551170

Schrum, Adam G.; Gil, Diana; Dopfer, Elaine P.; Wiest, David L.; Turka, Laurence A.; Schamel, Wolfgang W. A.; Palmer, Ed



Role of Escherichia coli histone-like nucleoid-structuring protein in bacterial metabolism and stress response--identification of targets by two-dimensional electrophoresis.  


The histone-like nucleoid-structuring protein, H-NS, is a major bacterial chromatin component which influences DNA structure and gene expression. Mutations in hns, the structural gene of H-NS protein, have been shown to result in highly pleiotropic effects in Escherichia coli cells. In this study, we have initiated an index of the proteins whose synthesis is, directly or indirectly regulated by H-NS. Using two-dimensional gel electrophoresis, we have examined the global changes in gene expression which occured in an hns background compared with its wild-type parent. In addition, we analysed the effects of mutations in two other genes i.e. lrp and pta, which are also involved in global regulatory pathways. Although these comparative analyses revealed several common differences, thus suggesting possible interactions between these regulatory mechanisms, i.e. H-NS, Lrp (leucine-responsive regulatory protein) and acetylphosphate, the most extensive modifications occurred in an hns mutant. Among the polypeptides whose level of synthesis was specifically altered in an hns mutant, several corresponded to H-NS targets previously identified by classical selection methods. Moreover, the present study allows us to characterize several H-NS targets, which were identified either by comparison with the E. coli two-dimensional reference maps or by microsequencing procedure. Many of these newly identified polypeptides are involved in adaptation of E. coli cells to environmental challenges, and one of them could be involved in bacterial virulence. Finally, synthesis of several proteins belonging to the heat-shock regulon, more particularly molecular chaperones, was induced in an hns mutant. PMID:9108246

Laurent-Winter, C; Ngo, S; Danchin, A; Bertin, P



Kidney cell electrophoresis  

NASA Technical Reports Server (NTRS)

A kidney cell electrophoresis technique is described in four parts: (1) the development and testing of electrophoresis solutions; (2) optimization of freezing and thawing; (3) procedures for evaluation of separated kidney cells; and (4) electrophoretic mobility characteristics of kidney cells.

Todd, P.



Accurate Label-Free Protein Quantitation with High- and Low-Resolution Mass Spectrometers  

PubMed Central

Label-free quantitation of proteins analyzed by tandem mass spectrometry uses either integrated peak intensity from the parent-ion mass analysis (MS1) or features from fragment-ion analysis (MS2), such as spectral counts or summed fragment-ion intensity. We directly compared MS1 and MS2 quantitation by analyzing human protein standards diluted into Escherichia coli extracts on an Orbitrap mass spectrometer. We found that summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity. We compared these results to those obtained from two low-resolution ion-trap mass spectrometers; summed MS2 intensities from LTQ and LTQ Velos instruments were similar in accuracy to those from the Orbitrap. Data from all three instruments are available via ProteomeXchange with identifier PXD000602. Abundance measurements using MS1 or MS2 intensities had limitations, however. While measured protein concentration was on average well correlated with the known concentration, there was considerable protein-to-protein variation. Moreover, not all human proteins diluted to a mole fraction of 10?3 or lower were detected, with a strong fall-off below 10?4 mole fraction. These results show that MS1 and MS2 intensities are simple measures of protein abundance that are on average accurate, but should be limited to quantitation of proteins of intermediate to higher fractional abundance. PMID:24295401

Shin, Jung-Bum; Klimek, John; Sherman, Nicholas E.; Jeffery, Erin D.; Choi, Dongseok; David, Larry L.; Barr-Gillespie, Peter G.



Comprehensive multiplexed protein quantitation delineates eosinophilic and neutrophilic experimental asthma  

PubMed Central

Background Improvements in asthma diagnosis and management require deeper understanding of the heterogeneity of the complex airway inflammation. We hypothesise that differences in the two major inflammatory phenotypes of asthma; eosinophilic and neutrophilic asthma, will be reflected in the lung protein expression profile of murine asthma models and can be delineated using proteomics of bronchoalveolar lavage (BAL). Methods BAL from mice challenged with ovalbumin (OVA/OVA) alone (standard model of asthma, here considered eosinophilic) or OVA in combination with endotoxin (OVA/LPS, model of neutrophilic asthma) was analysed using liquid chromatography coupled to high resolution mass spectrometry, and compared with steroid-treated animals and healthy controls. In addition, conventional inflammatory markers were analysed using multiplexed ELISA (Bio-Plex™ assay). Multivariate statistics was performed on integrative proteomic fingerprints using principal component analysis. Proteomic data were complemented with lung mechanics and BAL cell counts. Results Several of the analysed proteins displayed significant differences between the controls and either or both of the two models reflecting eosinophilic and neutrophilic asthma. Most of the proteins found with mass spectrometry analysis displayed a considerable increase in neutrophilic asthma compared with the other groups. Conversely, the larger number of the inflammatory markers analysed with Bio-Plex™ analysis were found to be increased in the eosinophilic model. In addition, major inflammation markers were correlated to peripheral airway closure, while commonly used asthma biomarkers only reflect central inflammation. Conclusion Our data suggest that the commercial markers we are currently relying on to diagnose asthma subtypes are not giving us comprehensive or specific enough information. The analysed protein profiles allowed to discriminate the two models and may add useful information for characterization of different asthma phenotypes. PMID:24993465



Quantitation of E. coli protein impurities in recombinant human interferon-?  

Microsoft Academic Search

A multiple antigen ELISA forE. coli proteins (ECPs) that may be present in purified recombinant human interferon-? (rIFN-?) was developed. SDS-PAGE and Western blotting analyses\\u000a showed that the assay antibodies reacted with a wide spectrum of ECPs in the standard and with ECPs in a production run. In\\u000a spike recovery studies, rIFN-? at concentrations of 0.05 mg\\/mL and higher augmented

A. B. Chen; A. A. Championsmith; J. Blanchard; J. Gorrell; B. A. Niepelt; M. M. Federici; J. Formento; D. V. Sinicropi



Quantitative approaches to monitor protein–nucleic acid interactions using fluorescent probes  

PubMed Central

Sequence-specific recognition of nucleic acids by proteins is required for nearly every aspect of gene expression. Quantitative binding experiments are a useful tool to measure the ability of a protein to distinguish between multiple sequences. Here, we describe the use of fluorophore-labeled oligonucleotide probes to quantitatively monitor protein/nucleic acid interactions. We review two complementary experimental methods, fluorescence polarization and fluorescence electrophoretic mobility shift assays, that enable the quantitative measurement of binding affinity. We also present two strategies for post-synthetic end-labeling of DNA or RNA oligonucleotides with fluorescent dyes. The approaches discussed here are efficient and sensitive, providing a safe and accessible alternative to the more commonly used radio-isotopic methods. PMID:21098142

Pagano, John M.; Clingman, Carina C.; Ryder, Sean P.



Secondary Reactions and Strategies to Improve Quantitative Protein Footprinting  

SciTech Connect

Hydroxyl radical-mediated footprinting permits detailed examination of structure and dynamic processes of proteins and large biological assemblies, as changes in the rate of reaction of radicals with target peptides are governed by changes in the solvent accessibility of the side-chain probe residues. The precise and accurate determination of peptide reaction rates is essential to successfully probing protein structure using footprinting. In this study, we specifically examine the magnitude and mechanisms of secondary oxidation occurring after radiolytic exposure and prior to mass spectrometric analysis. Secondary oxidation results from hydrogen peroxide and other oxidative species generated during radiolysis, significantly impacting the oxidation of Met and Cys but not aromatic or other reactive residues. Secondary oxidation of Met with formation of sulfoxide degrades data reproducibility and inflates the perceived solvent accessibility of Met-containing peptides. It can be suppressed by adding trace amounts of catalase or millimolar Met-NH{sub 2} (or Met-OH) buffer immediately after irradiation; this leads to greatly improved adherence to first-order kinetics and more precise observed oxidation rates. The strategy is shown to suppress secondary oxidation in model peptides and improve data quality in examining the reactivity of peptides within the Arp2/3 protein complex. Cysteine is also subject to secondary oxidation generating disulfide as the principal product. The disulfides can be reduced before mass spectrometric analysis by reducing agents such as TCEP, while methionine sulfoxide is refractory to reduction by this reagent under typical reducing conditions.

Xu,G.; Kiselar, J.; He, Q.; Chance, M.



Attomole level capillary electrophoresis-mass spectrometric protein analysis using 5-[mu]m-i. d. capillaries  

SciTech Connect

In this correspondence the authors report the use of chemically modified 5-[mu]-i.d. capillaries for CE-MS of proteins. The authors have found that the use of small inner diameter capillaries results in greatly improved sensitivity in CE-ESI/MS, an approximately 25-50-fold improvement, for the detection of attomole quantities of injected protein. Previously, Moseley et al. have demonstrated femtomole level detection for peptide separation using approximately 15-[mu]m-i.d. capillaries where a continuous-flow fast atom bombardment interface was utilized, but the choice of capillary i.d. was largely dictated by their interface design, no particular sensitivity advantage related to capillary i.d. was suggested, and the ionization method used is inappropriate for proteins. To the authors knowledge this report demonstrates the first attomole range CE-MS results for proteins and, in particular, the first obtained with scanning MS detection. 19 refs., 2 figs.

Wahl, J.H.; Goodlett, D.R.; Udseth, H.R.; Smith, R.D. (Pacific Northwest Lab., Richland, WA (United States))



A rapid method of species identification of wild chironomids (Diptera: Chironomidae) via electrophoresis of hemoglobin proteins in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE).  


Studying aquatic benthic macroinvertebrates (BMIs) in the field requires accurate taxonomic identification, which can be difficult and time consuming. Conventionally, head capsule morphology has been used to identify wild larvae of Chironomidae. However, due to the number of species and possible damage and/or deformity of their head capsules, another supporting approach for identification is needed. Here, we provide hemoglobin (Hb) protein in hemolymph of chironomids as a new biomarker that may help resolve some of the ambiguities and difficulties encountered during taxonomic identification. Chironomids collected from two locations in Maine and New Jersey, USA were identified to the genus level and in some cases to the species-level using head capsule and body morphologies. The head capsule for a particular individual was then associated with a corresponding Hb protein profile generated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Distinct Hb profiles were observed from one group (Thienemannimyia) and four genera (Chironomus, Cricotopus, Dicrotendipes, and Glyptotendipes) of chironomids. Several species were polymorphic, having more than one Hb profile and/or having bands of the same size as those of other species. However, major bands and the combination of bands could distinguish individuals at the genus and sometimes species-level. Overall, this study showed that Hb profiles can be used in combination with head capsule morphology to identify wild chironomids. PMID:24923437

Oh, J T; Epler, J H; Bentivegna, C S



Emulsion PCR Significantly Improves Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures-Based Aptamer Selection: Allowing for Efficient and Rapid Selection of Aptamer to Unmodified ABH2 Protein.  


Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), a homogeneous approach to select DNA aptamers, is among the most efficient partitioning techniques. In contrast with surface-based systematic evolution of ligands by exponential enrichment (SELEX) approaches, the ability of NECEEM to select aptamers to unmodified proteins in solution is preferable for identifying aptamers for eventual in vivo use. The high stringency and low sample volumes of NECEEM, although generally beneficial, can result in binding of very few aptamers, requiring highly efficient amplification to propagate them. When amplified with standard PCR, detectable library enrichment can fail due to the fast conversion of the aptamers into byproducts and preferential amplification of nonbinders. As an alternative, we proposed the use of emulsion PCR (ePCR), which is known to reduce byproduct formation, as a PCR mode for coupling with NECEEM partitioning. For the first time, we tested the advantages of ePCR in NECEEM-based aptamer selection to a medically relevant DNA repair enzyme, ABH2. We report that the combination of ePCR with NECEEM allowed for the selection of aptamers in the first three rounds of SELEX, while SELEX with conventional PCR failed in a number of attempts. Selected aptamers to an unmodified ABH2 protein have potential use in diagnostics and as leads for anticancer cotherapies, used as enhancements of alkylating agents in chemotherapy. PMID:25495441

Yufa, Roman; Krylova, Svetlana M; Bruce, Christine; Bagg, Eleanor A; Schofield, Christopher J; Krylov, Sergey N



Optimization of an Efficient Protein Extraction Protocol Compatible with Two-Dimensional Electrophoresis and Mass Spectrometry from Recalcitrant Phenolic Rich Roots of Chickpea (Cicer arietinum L.)  

PubMed Central

Two-dimensional electrophoresis and mass spectrometry are undoubtedly two essential tools popularly used in proteomic analyses. Utilization of these techniques however largely depends on efficient and optimized sample preparation, regarded as one of the most crucial steps for recovering maximum amount of reliable information. The present study highlights the optimization of an effective and efficient protocol, capable of extraction of root proteins from recalcitrant phenolic rich tissues of chickpea. The widely applicable TCA-acetone and phenol-based methods have been comparatively evaluated, amongst which the latter appeared to be better suited for the sample. The phenol extraction-based method further complemented with sodium dodecyl sulphate (SDS) and pulsatory treatments proved to be the most suitable method represented by greatest spot number, good resolution, and spot intensities. All the randomly selected spots showed successful identification when subjected to further downstream MALDI-TOF and MS/MS analyses. Hence, the information obtained collectively proposes the present protein extraction protocol to be an effective one that could be applicable for recalcitrant leguminous root samples. PMID:23193474

Chatterjee, Moniya; Gupta, Sumanti; Bhar, Anirban; Das, Sampa



Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) N-terminal sequencing of proteins and proteolytic cleavage sites by quantitative mass spectrometry.  


Edman degradation is a long-established technique for N-terminal sequencing of proteins and cleavage fragments. However, for accurate data analysis and amino acid assignments, Edman sequencing proceeds on samples of single proteins only and so lacks high-throughput capabilities. We describe a new method for the high-throughput determination of N-terminal sequences of multiple protein fragments in solution. Proteolytic processing can change the activity of bioactive proteins and also reveal cryptic binding sites and generate proteins with new functions (neoproteins) not found in the parent molecule. For example, extracellular matrix (ECM) protein processing often produces multiple proteolytic fragments with the generation of cryptic binding sites and neoproteins by ECM protein processing being well documented. The exact proteolytic cleavage sites need to be identified to fully understand the functions of the cleavage fragments and biological roles of proteases in vivo. However, the identification of cleavage sites in complex high molecular proteins such as those composing the ECM is not trivial. N-terminal microsequencing of proteolytic fragments is the usual method employed, but it suffers from poor resolution of sodium dodecylsulfate-polyacrylamide gel electrophoresis gels and is inefficient at identifying multiple cleavages, requiring preparation of numerous gels or membrane slices for analysis. We recently developed Amino-Terminal Oriented Mass spectrometry of Substrates (ATOMS) to overcome these limitations as a complement for N-terminal sequencing. ATOMS employs isotopic labeling and quantitative tandem mass spectrometry to identify cleavage sites in a fast and accurate manner. We successfully used ATOMS to identify nearly 100 cleavage sites in the ECM proteins laminin and fibronectin. Presented herein is the detailed step-by-step protocol for ATOMS. PMID:22078539

Doucet, Alain; Overall, Christopher M



Quantitation of dopamine, serotonin and adenosine content in a tissue punch from a brain slice using capillary electrophoresis with fast-scan cyclic voltammetry detection  

PubMed Central

Methods to determine neurochemical concentrations in small samples of tissue are needed to map interactions among neurotransmitters. In particular, correlating physiological measurements of neurotransmitter release and the tissue content in a small region would be valuable. HPLC is the standard method for tissue content analysis but it requires microliter samples and the detector often varies by the class of compound being quantified; thus detecting molecules from different classes can be difficult. In this paper, we develop capillary electrophoresis with fast-scan cyclic voltammetry detection (CE-FSCV) for analysis of dopamine, serotonin, and adenosine content in tissue punches from rat brain slices. Using field-amplified sample stacking, the limit of detection was 5 nM for dopamine, 10 nM for serotonin, and 50 nM for adenosine. Neurotransmitters could be measured from a tissue punch as small as 7 µg (7 nL) of tissue, three orders of magnitude smaller than a typical HPLC sample. Tissue content analysis of punches in successive slices through the striatum revealed higher dopamine but lower adenosine content in the anterior striatum. Stimulated dopamine release was measured in a brain slice, then a tissue punch collected from the recording region. Dopamine content and release had a correlation coefficient of 0.71, which indicates much of the variance in stimulated release is due to variance in tissue content. CE-FSCV should facilitate measurements of tissue content in nanoliter samples, leading to a better understanding of how diseases or drugs affect dopamine, serotonin, and adenosine content. PMID:23795210

Fang, Huaifang; Pajski, Megan L.; Ross, Ashley E.; Venton, B. Jill



Laboratory and field validation of a Cry1Ab protein quantitation method for water.  


The widespread planting of crops expressing insecticidal proteins derived from the soil bacterium Bacillus thuringiensis (Bt) has given rise to concerns regarding potential exposure to non-target species. These proteins are released from the plant throughout the growing season into soil and surface runoff and may enter adjacent waterways as runoff, erosion, aerial deposition of particulates, or plant debris. It is crucial to be able to accurately quantify Bt protein concentrations in the environment to aid in risk analyses and decision making. Enzyme-linked immunosorbent assay (ELISA) is commonly used for quantitation of Bt proteins in the environment; however, there are no published methods detailing and validating the extraction and quantitation of Bt proteins in water. The objective of the current study was to optimize the extraction of a Bt protein, Cry1Ab, from three water matrices and validate the ELISA method for specificity, precision, accuracy, stability, and sensitivity. Recovery of the Cry1Ab protein was matrix-dependent and ranged from 40 to 88% in the validated matrices, with an overall method detection limit of 2.1 ng/L. Precision among two plates and within a single plate was confirmed with a coefficient of variation less than 20%. The ELISA method was verified in field and laboratory samples, demonstrating the utility of the validated method. The implementation of a validated extraction and quantitation protocol adds consistency and reliability to field-collected data regarding transgenic products. PMID:25059137

Strain, Katherine E; Whiting, Sara A; Lydy, Michael J



Quantitative glycomics strategies.  


The correlations between protein glycosylation and many biological processes and diseases are increasing the demand for quantitative glycomics strategies enabling sensitive monitoring of changes in the abundance and structure of glycans. This is currently attained through multiple strategies employing several analytical techniques such as capillary electrophoresis, liquid chromatography, and mass spectrometry. The detection and quantification of glycans often involve labeling with ionic and/or hydrophobic reagents. This step is needed in order to enhance detection in spectroscopic and mass spectrometric measurements. Recently, labeling with stable isotopic reagents has also been presented as a very viable strategy enabling relative quantitation. The different strategies available for reliable and sensitive quantitative glycomics are herein described and discussed. PMID:23325767

Mechref, Yehia; Hu, Yunli; Desantos-Garcia, Janie L; Hussein, Ahmed; Tang, Haixu



Protein functional sites prediction using modified bio-basis function and quantitative indices.  


The prediction of functional sites in proteins is an important issue in protein function studies and drug design. To apply the kernel based pattern recognition algorithms such as support vector machines for protein functional sites prediction, a new string kernel function, termed as the modified bio-basis function, is proposed recently. The bio-basis strings for the new kernel function are selected by an efficient method that integrates the Fisher ratio and the concept of degree of resemblance. In this regard, this paper introduces some quantitative indices for evaluating the quality of selected bio-basis strings. Moreover, the effectiveness of the new string kernel function and bio-basis string selection method, along with a comparison with existing bio-basis function and related bio-basis string selection methods, is demonstrated on different protein data sets using the proposed quantitative indices and support vector machines. PMID:21266311

Maji, Pradipta; Das, Chandra



Concerted, Rapid, Quantitative, and Site-Specific Dual Labeling of Proteins  

PubMed Central

Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small molecules at physiological pH, temperature, and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-containing amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quantitative, site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, respectively. Protein double labeling in aqueous buffer at physiological pH, temperature, and pressure is quantitative in 30 min. PMID:24857040



Label-free quantitative proteomics reveals differentially regulated proteins in experimental gingivitis.  


We investigated the sequential protein expression in gingival crevicular fluid samples during the induction (I) and resolution (R) of experimental gingivitis. Periodontally and systemically healthy volunteers (n = 20) participated in a three-week experimental gingivitis protocol, followed by debridement and two weeks of regular plaque control. Gingival crevicular fluid (GCF) samples were collected at baseline, Day 7, 14, and 21 (induction; I-phase), and at Day 21, 25, 30, and 35 (resolution; R-phase). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for label-free quantitative proteomics was applied. A total of 287 proteins were identified including 254 human, 14 bacterial, 12 fungal, and 7 yeast proteins. Ontology analysis revealed proteins primarily involved in cytoskeletal rearrangements, immune response, antimicrobial function, protein degradation, and DNA binding. There was considerable variation in the number of proteins identified, both among subjects and within subjects across time points. After pooling of samples between subjects at each time point, the levels of 59 proteins in the I-phase and 73 proteins in the R-phase were quantified longitudinally. Our data demonstrate that LC-MS/MS label-free quantitative proteomics is valuable in the assessment of the protein content of the GCF and can facilitate a better understanding of the molecular mechanisms involved in the induction and resolution of plaque-induced gingival inflammation in humans. PMID:23244068

Bostanci, Nagihan; Ramberg, Per; Wahlander, Åsa; Grossman, Jonas; Jönsson, Daniel; Barnes, Virginia Monsul; Papapanou, Panos N



Identification of a Yellow gene-specific protein in Drosophila melanogaster by two-dimensional gel electrophoresis  

Microsoft Academic Search

Analysis of temperature-sensitive mutants suggests that the yellow (y) gene in Drosophila melanogaster is expressed at a different time in each cell type that gives rise to the various structures of the adult cuticle. An important step in analyzing the regulation of this gene requires identification of the y structural protein. A polypeptide has been identified which correlates with the

W. G. Nash; H. N. Kamerow; C. R. Merril



Using quantitative proteomics of Arabidopsis roots and leaves to predict metabolic activity  

Technology Transfer Automated Retrieval System (TEKTRAN)

Proteins isolated from developing roots and leaves of Arabidopsis thaliana were separated by high-resolution two-dimensional (2-D) electrophoresis. The resulting 2-D proteome maps are markedly different. Quantitative analysis of root and leaf protein spot pairs revealed that in most instances ther...


Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for quantitative parallel reaction monitoring of peptide abundance and single-shot proteomic analysis of a human cell line.  


We coupled capillary zone electrophoresis (CZE) with an ultrasensitive electrokinetically pumped nanospray ionization source for tandem mass spectrometry (MS/MS) analysis of complex proteomes. We first used the system for the parallel reaction monitoring (PRM) analysis of angiotensin II spiked in 0.45mg/mL of bovine serum albumin (BSA) digest. A calibration curve was generated between the loading amount of angiotensin II and intensity of angiotensin II fragment ions. CZE-PRM generated a linear calibration curve across over 4.5 orders of magnitude dynamic range corresponding to angiotensin II loading amount from 2amole to 150fmole. The relative standard deviations (RSDs) of migration time were <4% and the RSDs of fragment ion intensity were ?20% or less except 150fmole angiotensin II loading amount data (?36% RSD). We further applied the system for the first bottom up proteomic analysis of a human cell line using CZE-MS/MS. We generated 283 protein identifications from a 1h long, single-shot CZE MS/MS analysis of the MCF7 breast cancer cell line digest, corresponding to ?80ng loading amount. The MCF7 digest was fractionated using a C18 solid phase extraction column; single-shot analysis of a single fraction resulted in 468 protein identifications, which is by far the largest number of protein identifications reported for a mammalian proteomic sample using CZE. PMID:25082526

Sun, Liangliang; Zhu, Guijie; Mou, Si; Zhao, Yimeng; Champion, Matthew M; Dovichi, Norman J



Quantitative Determination of Expression of the Prostate Cancer Protein ?-Methylacyl-CoA Racemase Using Automated Quantitative Analysis (AQUA)  

PubMed Central

Despite years of discovery and attempts at validation, few molecular biomarkers achieve acceptance in the clinical setting. Tissue-based markers evaluated by immunohistochemistry suffer from a high degree of inter- and intraobserver variability. One recent advance in this field that promises to automate this process is the development of AQUA, a molecular-based method of quantitative assessment of protein expression. This system integrates a set of algorithms that allows for the rapid, automated, continuous, and quantitative analysis of tissue samples, including the separation of tumor from stromal elements and the subcellular localization of signals. This study uses the AQUA system to assess a recently described prostate cancer biomarker, ?-methylacyl-CoA-racemase (AMACR), and to determine the effectiveness of the quantitative measurement of this marker as a means for making the diagnosis of prostate cancer. Using a prostate cancer progression tissue microarray containing a wide range of prostate tissues, AQUA was directly compared to standard immunohistochemical evaluation for AMACR protein expression using the p504s monoclonal antibody. Both methods produced similar results showing AMACR protein expression to be strongest in the clinically localized prostate cancer, followed by the metastatic tumor samples. Benign prostate tissue was categorized as negative for most tissue samples by immunohistochemistry. However, AMACR was detectable using the AQUA system at low levels using the standard 1:25 dilution but also at 1:250 dilution, which is not detectable by light microscopy. The AQUA system was also able to discriminate foamy gland prostate cancers, which are known to have a lower AMACR expression than typical acinar prostate cancers, from benign prostate tissue samples. Finally, a receiver-operating-characteristic curve was plotted to determine the specificity of the AMACR AQUA Z-score (normalized AQUA score) to predict that a given tissue microarray sample contains cancer. The area under the curve was calculated at 0.90 (P < 0.00001; 95% CI, 0.84 to 0.95). At an AMACR AQUA Z-score score of ?0.3, 91% of the 70 samples classified as prostate cancer were correctly categorized without the intervention of a pathologist reviewing the tissue microarray slide. In conclusion, the AQUA system provides a continuous measurement of AMACR on a wide range of prostate tissue samples. In the future, the AMACR AQUA Z-score may be useful in the automated screening and evaluation of prostate tissue biomarkers. PMID:14982837

Rubin, Mark A.; Zerkowski, Maciej P.; Camp, Robert L.; Kuefer, Rainer; Hofer, Matthias D.; Chinnaiyan, Arul M.; Rimm, David L.



Profiling of experience-regulated proteins in the songbird auditory forebrain using quantitative proteomics  

E-print Network

substantial attention in this regard because it is involved in auditory processing of birdsong and is oneProfiling of experience-regulated proteins in the songbird auditory forebrain using quantitative: 2D-DIGE, auditory discrimination, auditory learning, NCM, plasticity, vocal learning Abstract

Jarvis, Erich D.


Coparative assessment of irradiated proteins in potato tuber with untreated control by High Performance Liquid Chromatography (HPLC) and gel electrophoresis  

NASA Astrophysics Data System (ADS)

About 2% of the weight of potato tuber is composed of proteins. In spite of their low quantity the proteins play a key role in the physiological activities leading to the break of the dormancy period and start of the cell division. This causes sprouting and also greening due to chlorophyll formation. This in turn is always accompanied by the production of the glycoalkaloid solanine in the flesh of tuber. For evaluation of radiation effect (dose range 50-250 Gy) and probable structural changes (amino acid release), analysis of selected proteins (molecular range 5 × 10^4 - 2 × 10^5 Dalton) of potato tuber in both irradiated and control type by HPLC showed no considerable changes in retention times, but qualitative assessment of amino acids by Pico-Tag^TM Pre-derivatizing method had some changes in quantity of amino acids like lysine which was increased 1 month after irradiation while Glutamic acid had considerable decreasment after the same time of irradiation.

Ghojaie, M.; Sayhoon, M.



Isolation of carboxyl-terminal peptides from proteins by diagonal electrophoresis: application to the entomocidal toxin from Bacillus thuringiensis.  


A procedure for the selective isolation of the C-terminal peptides from enzymatic digests of proteins is described. The methodology is based on the diagonal electrophoretic procedure described by R. G. Duggleby and H. Kaplan (1975) Anal. Biochem. 65, 346-354). The carboxyl groups in the protein are amidated with [14C]-methylamine followed by enzymatic digestion. Since only the C-terminal peptides lack a free carboxyl group, these peptides will lie on a diagonal line of a two-dimensional electrophoretogram run at pH 2.1 and 4.4. The diagonal line is delineated by autoradiography using [14C]taurine (net charge = 0 at pH 2.1 and 4.4) and [14C]choline (net charge = +1 at pH 2.1 and 4.4). Radioactive C-terminal peptides lie between these markers and can be directly excised for analysis. This procedure permits the detection and selective isolation of C-terminal peptides with minimal losses. The procedure was applied to the test proteins alpha-chymotrypsin and ribonuclease A. It was used to determine the C-terminus of the Bacillus thuringiensis toxin generated by tryptic cleavage of the protoxin. PMID:2817385

Bietlot, H P; Carey, P R; Pozsgay, M; Kaplan, H



Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*  

PubMed Central

High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms—including humans—are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)1 with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated ?-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius



Identification of DNA damage checkpoint-dependent protein interactions in Saccharomyces cerevisiae using quantitative mass spectrometry  

PubMed Central

Summary The DNA damage checkpoint (DDC) is an evolutionarily conserved signaling pathway that is crucial to maintain genomic integrity. In response to DNA damage, DDC kinases are rapidly activated and phosphorylate an elaborate network of substrates involved in multiple cellular processes. An important role of the DDC response is to assemble protein complexes. However, for most of the DDC substrates, how the DDC-dependent phosphorylation modulates their network of interactions remains to be established. Here, we present a protocol for the identification of DDC-dependent protein-protein interactions based on Stable Isotope Labeling of Amino acids in Cell culture (SILAC) followed by affinity-tagged protein purification and quantitative mass spectrometry analysis. Based on a model study using Saccharomyces cerevisiae, we provide a method that can be generally applied to study the role of kinases in mediating protein-protein interactions. PMID:24791994

Bastos de Oliveira, Francisco M.; Smolka, Marcus B.



Phylogenetic analyses and detection of viridans streptococci based on sequences and denaturing gradient gel electrophoresis of the rod shape-determining protein gene  

PubMed Central

Background Population analysis of viridans streptococci is important because these species are associated with dental caries, bacteremia, and subacute endocarditis, in addition to being important members of the human oral commensal microbiota. Design In this study, we phylogenetically analyzed the rod shape-determining protein gene (rodA), which is associated with cellular morphology, cell division, and sensitivity for antibiotics, and demonstrated that the diversity of the rodA gene is sufficient to identify viridans streptococci at the species level. Moreover, we developed a more convenient denaturing gradient gel electrophoresis (DGGE) method based on the diversity of the rodA gene (rodA-DGGE) for detecting nine dominant streptococcal species in human saliva, namely, Streptococcus sanguinis, Streptococcus oralis, Streptococcus mitis, Streptococcus parasanguinis, Streptococcus gordonii, Streptococcus vestibularis, Streptococcus salivarius, Streptococcus mutans, and Streptococcus sobrinus. Results This rodA-DGGE method proved useful in detecting viridans streptococci without cultivation, isolation, and phenotypic characterization. Conclusion Analysis of the oral microbiota by rodA-DGGE offers a higher resolution than the conventional DGGE using 16S rDNA and may be an alternative in the microbial diagnosis of streptococcal infection. PMID:21523207

Konishi, Ikuri; Hoshino, Tomonori; Kondo, Yoshio; Saito, Kan; Nishiguchi, Miyuki; Sato, Kyoko; Fujiwara, Taku



Comparison of Protein A Gene Sequencing with Pulsed-Field Gel Electrophoresis and Epidemiologic Data for Molecular Typing of Methicillin-Resistant Staphylococcus aureus  

PubMed Central

The epidemiologic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A (spa) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information. PMID:10747105

Tang, Yi-Wei; Waddington, Michael G.; Smith, Douglas H.; Manahan, Janice M.; Kohner, Peggy C.; Highsmith, Leanne M.; Li, Haijing; Cockerill, Franklin R.; Thompson, Rodney L.; Montgomery, Stacy O.; Persing, David H.



Quantitative and functional characterization of the hyper-conserved protein of Prochlorococcus and marine Synechococcus.  


A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly. PMID:25360678

Whidden, Caroline E; DeZeeuw, Katrina G; Zorz, Jackie K; Joy, Andrew P; Barnett, David A; Johnson, Milo S; Zhaxybayeva, Olga; Cockshutt, Amanda M



Quantitative and Functional Characterization of the Hyper-Conserved Protein of Prochlorococcus and Marine Synechococcus  

PubMed Central

A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly. PMID:25360678

Zorz, Jackie K.; Joy, Andrew P.; Barnett, David A.; Johnson, Milo S.; Zhaxybayeva, Olga; Cockshutt, Amanda M.



Starch-gel Electrophoresis of Anterior Pituitary Hormones  

Microsoft Academic Search

SEPARATION of pituitary hormones by zone electrophoresis on paper has not proved very successful, possibly due to complex formation between proteins1. Starch-gel zone electrophoresis introduced by Smithies2,3 separates proteins not only by their net charge but also by their molecular size. This procedure has superior resolving power to paper or agar-gel zone electrophoresis and has been successfully applied to the

K. A. Ferguson; A. L. C. Wallace



Gel-Free Electrophoresis of DNA and Proteins on Chips Featuring a 70 nm Capillary-Well Motif.  


We present an integrated glass capillary system on silicon for size-based sieving of distinct mixtures of proteins, short DNA, and long DNA fragments into sharp peaks. The minimum resolvable size difference achieved is noted as 3.45 kDa for 45-52.8 kDa proteins, 20 bp for 200-300 bp DNA strands, and 182 bp for 5.6-5.8 kbp DNA chains. This high-resolution sieving arises from vastly steep entropic barriers created at the onsets of extremely restrictive (resistive) capillary segments and their pivotal role in shifting the equilibrium entropic sieving to intense fields (>1000 V/cm). DNA fragments of various sizes are shown fully resolved in less than 7 min at a steady voltage of 2000 V being directly applied across the length of a 2 cm long sieve featuring thousands of entropic barriers. The utility of higher field strengths and longer sieves is also demonstrated without triggering dielectric breakdown by time-division multiplexing up to 2000 V across the 1 cm long sieve segments. The self-enclosed 70 nm diameter capillaries were fabricated using coarse (>1 ?m) photolithography and standard semiconductor manufacturing techniques. PMID:25535934

Cao, Zhen; Yobas, Levent



Introducing enzyme selectivity: a quantitative parameter to describe enzymatic protein hydrolysis.  


Enzyme selectivity is introduced as a quantitative parameter to describe the rate at which individual cleavage sites in a protein substrate are hydrolyzed relative to other cleavage sites. Whey protein isolate was hydrolyzed by Bacillus licheniformis protease, which is highly specific for Glu and Asp residues. The molar concentration of all peptides (58) from ?-lactoglobulin formed during hydrolysis was determined from the UV214 signal. The quality of identification and quantification of the peptides were described by newly defined parameters: the peptide sequence coverage (on average 94 %) and the molar sequence coverage (on average 75 %). The selectivity was calculated from the rate of hydrolysis of each cleavage site, and showed differences of up to a factor of 5,000. The ability to quantitatively discriminate the enzyme preference towards individual cleavage sites is considered essential to the understanding of enzymatic protein hydrolysis. PMID:25012360

Butré, Claire I; Sforza, Stefano; Gruppen, Harry; Wierenga, Peter A



Quantitative measurement of intracellular protein dynamics using photobleaching or photoactivation of fluorescent proteins.  


Unlike in vitro protein dynamics, intracellular protein dynamics are intricately regulated by protein-protein interactions or interactions between proteins and other cellular components, including nucleic acids, the plasma membrane and the cytoskeleton. Alteration of these dynamics plays a crucial role in physiological phenomena such as gene expression and cell division. Live-cell imaging via microscopy with the inherent properties of fluorescent proteins, i.e. photobleaching and photoconversion, or fluorescence correlation spectroscopy, provides insight into the movement of proteins and their interactions with cellular components. This article reviews techniques based on photo-induced changes in the physicochemical properties of fluorescent proteins to measure protein dynamics inside living cells, and it also discusses the strengths and weaknesses of these techniques. PMID:25268018

Matsuda, Tomoki; Nagai, Takeharu



Casein - whey protein interactions in heated milk  

Microsoft Academic Search

Heating of milk is an essential step in the processing of various dairy products, like for example yoghurt. A major consequence of the heat treatment is the denaturation of whey proteins, which either associate with the casein micelle or form soluble whey protein aggregates. By combination of enzymatic fractionation and capillary electrophoresis we were able to quantitatively determine the distribution

Astrid Jolanda Vasbinder



Kidney Cell Electrophoresis  

NASA Technical Reports Server (NTRS)

Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated, ground support in the form of analytical cell electrophoresis and flow cytometry was provided and cells returned from space flight were analyzed. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. The protocol established and utilized is given.

Todd, P.



Quantitative analysis of thymosin ?4 in whole saliva by capillary electrophoresis–mass spectrometry using multiple ions monitoring (CE-MIM-MS.).  


Thymosin ?4 (T?4) is a peptide present in almost any tissue and in extracellular media in mammals, having multiple amazing functions as wound healing, stimulation of angiogenesis, and suppression of inflammation. This study describes its determination in saliva through CE-MS using multiple ions monitoring scan mode by isolating the four most intense multicharged ions present in the MS spectra of the peptide. This scan modality, by reducing the baseline noise and interferences, increases the sensitivity and specificity in biological matrices. The CE-MS separation was optimized by studying different parameters influencing CE analysis, sample injection, and MS ionization, that is, the nebulizer gas flow, the sheath liquid, and BGE composition. The proposed technique can unambiguously identify in short time T?4 in saliva after a very fast and reduced sample pretreatment procedure. The method was validated for quantitation showing linearity of the response in the range 0.25 (lower limit of quantification) to 4 ?M (average R2 0.996 ± 0.005) and intra- and interassay precision and accuracy at three different concentrations with RSD values in the range of 7–16%. It was successfully applied to the analysis of T?4 in whole saliva showing a variable peptide content from individual to individual (in the range of 0.3–1.4 ?M) and in different days from the same individual. CE-MS in multiple ions monitoring scan mode provides a fast, selective, and economic method requiring only very few microliters of sample. PMID:23857244

Rossetti, Diana Valeria; Martelli, Claudia; Longhi, Renato; Iavarone, Federica; Castagnola, Massimo; Desiderio, Claudia



A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways.  


Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct ?-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement. PMID:25036637

Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan



Two-dimensional peptide separation improving sensitivity of selected reaction monitoring-based quantitative proteomics in mouse liver tissue: comparing off-gel electrophoresis and strong cation exchange chromatography.  


Protein expression analysis is one of the most powerful tools to further the understanding of biological systems. Progress in the field of mass spectrometry has shifted focus from gel-based approaches to the upcoming LC-selected reaction monitoring (SRM) technique which combines high technical accuracy with absolute quantification of proteins and the capability for high-throughput analyses. Due to these properties, LC-SRM has the potential to become the foundation for biomarker analysis, targeted hypothesis driven proteomic studies and contribute to the field of systems biology. While the performance of LC-SRM applied to samples from various bodily fluids, particularly plasma, and microorganisms has been extensively investigated, there is only little experience with its application to animal tissue samples. Here, we show that a conventional one-dimensional LC-SRM workflow applied to mouse liver tissue suffers from a shortcoming in terms of sensitivity for lower abundance proteins. This problem could be solved through the extension of the standard workflow by an additional dimension of separation at the peptide level prior to online LC-SRM. For this purpose, we used off-gel electrophoresis (OGE) which is also shown to outperform strong cation exchange (SCX) in terms of resolution, gain of signal intensity, and predictability of separation. The extension of the SRM workflow by a high resolving peptide separation technique is an ideal combination as it allows the addition of stable isotope standards directly after trytic digestion and will increase the dynamic range of protein abundances amenable by SRM in animal tissue. PMID:22994301

Schäfer, Alexander; von Toerne, Christine; Becker, Silke; Sarioglu, Hakan; Neschen, Susanne; Kahle, Melanie; Hauck, Stefanie M; Ueffing, Marius



Necrosis-inducing proteins of Rhynchosporium commune, effectors in quantitative disease resistance.  


The barley pathogen Rhynchosporium commune secretes necrosis-inducing proteins NIP1, NIP2, and NIP3. Expression analysis revealed that NIP1 transcripts appear to be present in fungal spores already, whereas NIP2 and NIP3 are synthesized after inoculation of host plants. To assess the contribution of the three effector proteins to disease development, deletion mutants were generated. The development of these fungal mutants on four barley cultivars was quantified in comparison with that of the parent wild-type strain and with two fungal strains failing to secrete an "active" NIP1 avirulence protein, using quantitative polymerase chain reaction as well as microscopic imaging after fungal green fluorescent protein tagging. The impact of the three deletions varied quantitatively depending on the host genotype, suggesting that the activities of the fungal effectors add up to produce stronger growth patterns and symptom development. Alternatively, recognition events of differing intensities may be converted into defense gene expression in a quantitative manner. PMID:22712509

Kirsten, S; Navarro-Quezada, A; Penselin, D; Wenzel, C; Matern, A; Leitner, A; Baum, T; Seiffert, U; Knogge, W



A Miniaturized Technique for Assessing Protein Thermodynamics and Function Using Fast Determination of Quantitative Cysteine Reactivity  

PubMed Central

Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity (fQCR), which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics. PMID:21387407

Isom, Daniel G.; Marguet, Philippe R.; Oas, Terrence G.; Hellinga, Homme W.



Label-free Quantitative Protein Profiling of vastus lateralis Muscle During Human Aging*  

PubMed Central

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers. PMID:24217021

Théron, Laëtitia; Gueugneau, Marine; Coudy, Cécile; Viala, Didier; Bijlsma, Astrid; Butler-Browne, Gillian; Maier, Andrea; Béchet, Daniel; Chambon, Christophe



Seasonal liver protein differences in a hibernator revealed by quantitative proteomics using whole animal isotopic labeling  

PubMed Central

Hibernation is an energy-saving strategy used by diverse species of mammals to survive winter. It is characterized by cycles between multi-day periods of torpor with low body temperature (Tb), and short periods of rapid, spontaneous rewarming. The ability to retain cellular integrity and function throughout torpor and rewarming is a key attribute of hibernation. Livers from winter hibernators are resistant to cellular damage induced by cold storage followed by warm reperfusion. Identifying proteins that differ between the summer-sensitive and winter-protected phenotypic states is one useful approach that may elucidate the molecular mechanisms that underlie this protection. Here we employ a novel quantitative proteomics screening strategy whereby a newly-weaned 13-lined ground squirrel was metabolically labeled by ingesting heavy-isotope substituted (15N) Spirulina. The liver protein extract from this animal provided a common reference for quantitative evaluation of protein differences by its addition to extracts from pooled samples of summer active (SA) or winter entrance (Ent) phase hibernating ground squirrels. We identified 61 significantly different proteins between the two groups and compared them to proteins identified previously in the same samples using 2D gels. Of the 20 proteins common to the two datasets, the direction and magnitude of their differences were perfectly concordant for 18, providing confidence that both sets of altered proteins reflect bona fide differences between the two physiological states. Furthermore, the 41 novel proteins recovered in this study included many new enzymes in pathways identified previously: specifically, additional enzymes belonging to the urea cycle, amino acid and carbohydrate degradation, and lipid biosynthetic pathways were decreased, whereas enzymes involved in ketone body synthesis, fatty acid utilization, protein synthesis and gluconeogenesis were increased in the samples from entrance hibernators compared to summer active animals, providing additional specific evidence for the importance of these pathways in the hibernating phenotype. PMID:21481655

Rose, J. Cameron; Epperson, L. Elaine; Carey, Hannah V.; Martin, Sandra L.



Acid- and base-induced proteins during aerobic and anaerobic growth of Escherichia coli revealed by two-dimensional gel electrophoresis.  


Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). The gad system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD. PMID:10094700

Blankenhorn, D; Phillips, J; Slonczewski, J L



Automatic multiple applicator electrophoresis  

NASA Technical Reports Server (NTRS)

Easy-to-use, economical device permits electrophoresis on all known supporting media. System includes automatic multiple-sample applicator, sample holder, and electrophoresis apparatus. System has potential applicability to fields of taxonomy, immunology, and genetics. Apparatus is also used for electrofocusing.

Grunbaum, B. W.



Electrophoresis of biological materials  

NASA Technical Reports Server (NTRS)

The selection of biological products was studied for electrophoresis in space. Free flow electrophoresis, isoelectric focusing, and isotachophoresis are described. The candidates discussed include: immunoglobulins and gamma globulins; isolated islet of langerhans from pancreas; bone marrow; tumor cells; kidney cells, cryoprecipitate; and column separated cultures.



Ubiquitin Ligase Substrate Identification through Quantitative Proteomics at Both the Protein and Peptide Levels  

PubMed Central

Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His6-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology. PMID:21987572

Lee, Kimberly A.; Hammerle, Lisa P.; Andrews, Paul S.; Stokes, Matthew P.; Mustelin, Tomas; Silva, Jeffrey C.; Black, Roy A.; Doedens, John R.



Quantification of heat-induced casein–whey protein interactions in milk and its relation to gelation kinetics  

Microsoft Academic Search

This paper describes a quantitative study on the distribution of denatured whey proteins over whey protein aggregates and whey proteins associated with the casein micelles in heated milk. This was done by combination of enzymatic fractionation and capillary electrophoresis. More severe heat treatment at the natural pH of milk caused more denaturation, but the ratio of denatured whey proteins associated

Astrid J Vasbinder; Arno C Alting; Kees G de Kruif



Quantitative variability of 342 plasma proteins in a human twin population.  


The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2-7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis-SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood-based biomarker studies. PMID:25652787

Liu, Yansheng; Buil, Alfonso; Collins, Ben C; Gillet, Ludovic Cj; Blum, Lorenz C; Cheng, Lin-Yang; Vitek, Olga; Mouritsen, Jeppe; Lachance, Genevieve; Spector, Tim D; Dermitzakis, Emmanouil T; Aebersold, Ruedi



The Influence of Histatin 5 on Candida albicans Mitochondrial Protein Expression Assessed by Quantitative Mass Spectrometry  

PubMed Central

Individual aspects of the mode of action of histatin 5, a human salivary antifungal protein, have been partially elucidated, but the mechanism likely involves a complex set of events which have not been characterized. Previous evidence points toward histatin-induced alterations in mitochondrial function. The purpose of the present study was to verify and quantify changes in the mitochondrial proteome of C. albicans treated with histatin 5. Cell killing was determined by plating and differential protein expression levels in the mitochondrial samples were determined by quantitative proteomics approaches employing mTRAQ and ICAT labeling and Western blotting. Relative quantitation ratios were established for 144 different proteins. Up-regulated mitochondrial proteins were predominantly involved in genome maintenance and gene expression, whereas proteins that constitute the respiratory enzyme complexes were mostly down-regulated. The differential expression of ATP synthase gamma chain and elongation factor 1-alpha were confirmed by Western blotting by comparison to levels of cytochrome c which were unchanged upon histatin treatment. The mTRAQ and ICAT proteomics results suggest that key steps in the histatin 5 antifungal mechanism involve a bioenergetic collapse of C. albicans, caused essentially by a decrease in mitochondrial ATP synthesis. PMID:21080726

Komatsu, Tomoko; Salih, Erdjan; Helmerhorst, Eva J.; Offner, Gwynneth D.; Oppenheim, Frank G.



Qualitative and Quantitative Detection of Protein and Genetic Traits in Genetically Modified Food  

Microsoft Academic Search

Due to the market introduction of genetically modified organisms (GMOs) in crops, foods, and ingredients, legislation worldwide came face to face with the question of the use and labeling requirements on GMO crops and their derivatives. In this review, protein- and DNA-based methods, such as enzyme-linked immunosorbent assay, western blots, and qualitative and quantitative polymerase chain reaction PCR (Q-PCR) are

P. Markoulatos; N. Siafakas; A. Papathoma; E. Nerantzis; B. Betzios; V. Dourtoglou; M. Moncany



Quantitative evaluation of the lengths of homobifunctional protein cross-linking reagents used as molecular rulers  

PubMed Central

Homobifunctional chemical cross-linking reagents are important tools for functional and structural characterization of proteins. Accurate measures of the lengths of these molecules currently are not available, despite their widespread use. Stochastic dynamics calculations now provide quantitative measures of the lengths, and length dispersions, of 32 widely used molecular rulers. Significant differences from published data have been found. Supplemental material: See PMID:11420431

Green, Nora S.; Reisler, Emil; Houk, K.N.



Quantitative phosphoproteomics of cytotoxic T cells to reveal protein kinase d 2 regulated networks.  


The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labeling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T-cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription, and translation. In other cell types, PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages. PMID:25266776

Navarro, María N; Goebel, Juergen; Hukelmann, Jens L; Cantrell, Doreen A



Quantitative Phosphoproteomics of Cytotoxic T Cells to Reveal Protein Kinase D 2 Regulated Networks*  

PubMed Central

The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labeling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T-cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription, and translation. In other cell types, PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages. PMID:25266776

Navarro, María N.; Goebel, Juergen; Hukelmann, Jens L.; Cantrell, Doreen A.



Identification, quantitation, and characterization of biomolecules by capillary electrophoretic analysis of binding interactions.  


The high resolving power of capillary electrophoresis combined with the specificity of binding interactions may be used with advantage to characterize the structure-function relationship of biomolecules, to quantitate specific analytes in complex sample matrices, and to determine the purity of pharmaceutical and other molecules. We here review recent and innovative methodologies and applications of high resolution affinity electrophoresis within the fields of binding constant determination, structure-activity studies, quantitative microassays, analysis of drug purity and protein conformation, and immobilized affinity ligands. Despite the virtues of these approaches with respect to applicability, resolving power, speed, and low sample consumption, problems remain with respect to analyte identification and low concentration limits of detection. The ongoing development of new detector technologies for capillary electrophoresis such as mass spectrometry, and possibly nuclear magnetic resonance and other spectroscopic methods, is therefore very promising for the continued increased use of affinity capillary electrophoresis. PMID:10596820

Heegaard, N H; Kennedy, R T



Quantitative Modeling of Membrane Deformations by Multihelical Membrane Proteins: Application to G-Protein Coupled Receptors  

PubMed Central

The interpretation of experimental observations of the dependence of membrane protein function on the properties of the lipid membrane environment calls for a consideration of the energy cost of protein-bilayer interactions, including the protein-bilayer hydrophobic mismatch. We present a novel (to our knowledge) multiscale computational approach for quantifying the hydrophobic mismatch-driven remodeling of membrane bilayers by multihelical membrane proteins. The method accounts for both the membrane remodeling energy and the energy contribution from any partial (incomplete) alleviation of the hydrophobic mismatch by membrane remodeling. Overcoming previous limitations, it allows for radially asymmetric bilayer deformations produced by multihelical proteins, and takes into account the irregular membrane-protein boundaries. The approach is illustrated by application to two G-protein coupled receptors: rhodopsin in bilayers of different thickness, and the serotonin 5-HT2A receptor bound to pharmacologically different ligands. Analysis of the results identifies the residual exposure that is not alleviated by bilayer adaptation, and its quantification at specific transmembrane segments is shown to predict favorable contact interfaces in oligomeric arrays. In addition, our results suggest how distinct ligand-induced conformations of G-protein coupled receptors may elicit different functional responses through differential effects on the membrane environment. PMID:22067146

Mondal, Sayan; Khelashvili, George; Shan, Jufang; Andersen, Olaf S.; Weinstein, Harel



Label-free quantitation of protein modifications by pseudo selected reaction monitoring with internal reference peptides.  


Liquid chromatography tandem mass spectrometry (LC-MS/MS) based methods provide powerful tools for the quantitative analysis of modified proteins. We have developed a label-free approach using internal reference peptides (IRP) from the target protein for signal normalization without the need for isotope labeling. Ion-trap mass spectrometry and pseudo-selected reaction monitoring (pSRM) were used to acquire full MS/MS and MS(3) spectra from target peptides. Skyline, a widely used software for SRM experiments, was used for chromatographic ion extraction. Phosphopeptides spiked into a BSA background yielded concentration response curves with high correlation coefficients (typically >0.9) and low coefficients of variation (?15%) over a 200-fold concentration range. Stable isotope dilution (SID) and IRP methods were compared for quantitation of six site-specific phosphorylations in the epidermal growth factor receptor (EGFR) in epidermal growth factor-stimulated A431 cells with or without the addition of EGFR inhibitors cetuximab and gefitinib. Equivalent responses were observed with both IRP and SID methods, although analyses using the IRP method typically had higher median CVs (22-31%) than SID (10-20%). Analyses using both methods were consistent with immunoblot using site-selective antibodies. The ease of implementation and the suitability for targeted quantitative comparisons make this method suitable for broad application in protein biochemistry. PMID:22559222

Sherrod, Stacy D; Myers, Matthew V; Li, Ming; Myers, Jeremy S; Carpenter, Kristin L; Maclean, Brendan; Maccoss, Michael J; Liebler, Daniel C; Ham, Amy-Joan L



Protein Expression Profiling of Coccidioides posadasii by Two-Dimensional Differential In-Gel Electrophoresis and Evaluation of a Newly Recognized Peroxisomal Matrix Protein as a Recombinant Vaccine Candidate  

PubMed Central

Coccidioides posadasii and Coccidioides immitis are dimorphic, soil-dwelling pathogenic ascomycetes endemic to the southwestern United States. Infection can result from inhalation of a very few arthroconidia, but following natural infection, long-lived immunity is the norm. Previous work in the field has shown that spherule-derived vaccines afford more protection than those from mycelia. We have used two-dimensional differential in-gel electrophoresis coupled with nano-high-performance liquid chromatography-tandem mass spectrometry to directly assess both absolute abundance and differential expression of proteins in the spherule and the mycelial phases of C. posadasii with the intent to identify potential vaccine candidates. Peptides derived from 40 protein spots were analyzed and a probable identity was assigned to each. One spherule-abundant protein, identified as Pmp1, showed homology to allergens from Aspergillus fumigatus and other fungi, all of which exhibit similarity to yeast thiol peroxidases. Recombinant Pmp1 was reactive with serum from individuals with both acute and protracted disease, and evoked protection in two murine models of infection with C. posadasii. These results demonstrate the utility of proteomic analysis as a point of discovery for protective antigens for possible inclusion in a vaccine candidate to prevent coccidioidomycosis. PMID:16495561

Orsborn, Kris I.; Shubitz, Lisa F.; Peng, Tao; Kellner, Ellen M.; Orbach, Marc J.; Haynes, Paul A.; Galgiani, John N.



Quantitative Proteomic Analysis of Serum Proteins from Oral Cancer Patients: Comparison of Two Analytical Methods  

PubMed Central

Serum proteomic analysis can be a valuable approach for the discovery of protein biomarkers for early detection or monitoring of a disease. In this study, two analytical methods were compared for quantification of serum proteins in patients with oral cancer. In the first approach, we quantified serum proteins between oral squamous cell carcinoma (OSCC) and healthy control subjects by performing in-solution digestion of serum proteins, isobaric tags for relative and absolute quantification (iTRAQ) labeling of the resulting peptides, strong cation exchange (SCX) fractionation of labeled peptides and finally capillary liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of the peptides. In the second approach, we first separated serum proteins with SDS-PAGE. The gel-separated proteins were then digested with trypsin and the resulting peptides were labeled with iTRAQ and analyzed with LC-MS/MS for protein quantification. A total of 319 serum proteins were quantified with the first proteomic approach whereas a total of 281 proteins were quantified by the second proteomic approach. Most of the proteins were identified and quantified by both approaches, suggesting that these methods are similarly effective for serum proteome analysis. This study provides compelling evidence that quantitative serum proteomic analysis of OSCC is a valuable approach for identifying differentially expressed proteins in cancer patients’ circulation systems that may be used as potential biomarkers for disease detection. Further validation in large oral cancer patient populations may lead to a simple and low invasive clinical tool for OSCC diagnosis or monitoring. PMID:25196439

Yang, Yan; Huang, Junwei; Rabii, Bahareh; Rabii, Ramin; Hu, Shen



Improved Protein Arrays for Quantitative Systems Analysis of the Dynamics of Signaling Pathway Interactions  

SciTech Connect

Astronauts and workers in nuclear plants who repeatedly exposed to low doses of ionizing radiation (IR, <10 cGy) are likely to incur specific changes in signal transduction and gene expression in various tissues of their body. Remarkable advances in high throughput genomics and proteomics technologies enable researchers to broaden their focus from examining single gene/protein kinetics to better understanding global gene/protein expression profiling and biological pathway analyses, namely Systems Biology. An ultimate goal of systems biology is to develop dynamic mathematical models of interacting biological systems capable of simulating living systems in a computer. This Glue Grant is to complement Dr. Boothman’s existing DOE grant (No. DE-FG02-06ER64186) entitled “The IGF1/IGF-1R-MAPK-Secretory Clusterin (sCLU) Pathway: Mediator of a Low Dose IR-Inducible Bystander Effect” to develop sensitive and quantitative proteomic technology that suitable for low dose radiobiology researches. An improved version of quantitative protein array platform utilizing linear Quantum dot signaling for systematically measuring protein levels and phosphorylation states for systems biology modeling is presented. The signals are amplified by a confocal laser Quantum dot scanner resulting in ~1000-fold more sensitivity than traditional Western blots and show the good linearity that is impossible for the signals of HRP-amplification. Therefore this improved protein array technology is suitable to detect weak responses of low dose radiation. Software is developed to facilitate the quantitative readout of signaling network activities. Kinetics of EGFRvIII mutant signaling was analyzed to quantify cross-talks between EGFR and other signaling pathways.




Polyacrylamide gel electrophoretic methods in the separation of structural muscle proteins.  

SciTech Connect

Polyacrylamide gel electrophoresis plays a major role in analyzing the function of muscle structural proteins. This review describes one- and two-dimensional gel electrophoretic methods for qualitative and quantitative investigation of the muscle proteins, with special emphasis on determination of protein phosphorylation. The electrophoretic studies established the subunit structures of the muscle proteins, characterized their multiple forms, revealed changes in subunit composition or shifts in isoform distribution of specific proteins during development, upon stimulation or denervation of the muscle. Protein phosphorylation during muscle contraction is preferentially studied by two-dimensional gel electrophoresis. The same method demonstrated protein alterations in human neuromuscular diseases.

Barany, K.; Barany, M.; Giometti, C. S.; Center for Mechanistic Biology and Biotechnology; Univ. of Illinois at Chicago



Western Blotting using Capillary Electrophoresis  

PubMed Central

A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. Although discrete protein zones could be detected, bands were broadened by ~1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Western blotting using capillary electrophoresis shows promise to analyze low volume samples with reduced reagents and time, while retaining the information content of a typical Western blot. PMID:21265514

Anderson, Gwendolyn J.; Cipolla, Cynthia; Kennedy, Robert T.



Genome-scale quantitative characterization of bacterial protein localization dynamics throughout the cell cycle  

PubMed Central

Bacterial cells display both spatial and temporal organization, and this complex structure is known to play a central role in cellular function. Although nearly one-fifth of all proteins in Escherichia coli localize to specific subcellular locations, fundamental questions remain about how cellular-scale structure is encoded at the level of molecular-scale interactions. One significant limitation to our understanding is that the localization behavior of only a small subset of proteins has been characterized in detail. As an essential step toward a global model of protein localization in bacteria, we capture and quantitatively analyze spatial and temporal protein localization patterns throughout the cell cycle for nearly every protein in E. coli that exhibits nondiffuse localization. This genome-scale analysis reveals significant complexity in patterning, notably in the behavior of DNA-binding proteins. Complete cell-cycle imaging also facilitates analysis of protein partitioning to daughter cells at division, revealing a broad and robust assortment of asymmetric partitioning behaviors. PMID:25353361

Kuwada, Nathan J; Traxler, Beth; Wiggins, Paul A



Genome-scale quantitative characterization of bacterial protein localization dynamics throughout the cell cycle.  


Bacterial cells display both spatial and temporal organization, and this complex structure is known to play a central role in cellular function. Although nearly one-fifth of all proteins in Escherichia coli localize to specific subcellular locations, fundamental questions remain about how cellular-scale structure is encoded at the level of molecular-scale interactions. One significant limitation to our understanding is that the localization behavior of only a small subset of proteins has been characterized in detail. As an essential step toward a global model of protein localization in bacteria, we capture and quantitatively analyze spatial and temporal protein localization patterns throughout the cell cycle for nearly every protein in E.?coli that exhibits nondiffuse localization. This genome-scale analysis reveals significant complexity in patterning, notably in the behavior of DNA-binding proteins. Complete cell-cycle imaging also facilitates analysis of protein partitioning to daughter cells at division, revealing a broad and robust assortment of asymmetric partitioning behaviors. PMID:25353361

Kuwada, Nathan J; Traxler, Beth; Wiggins, Paul A



Proteomic analysis of cow, yak, buffalo, goat and camel milk whey proteins: quantitative differential expression patterns.  


To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species. PMID:23464874

Yang, Yongxin; Bu, Dengpan; Zhao, Xiaowei; Sun, Peng; Wang, Jiaqi; Zhou, Lingyun



Interferences of suspended clay fraction in protein quantitation by several determination methods.  


Seven current methods of protein quantitation, Bradford (standard, micro, and 590/450 nm ratio), Lowry, bicinchoninic acid (BCA), UV spectrophotometry at 280 nm, and Quant-iT fluorescence-based determination, were compared with regard to their susceptibility to interferences due to the presence of suspended and not easily detectable clay particles. Bovine serum albumin (BSA) and Na-Wyoming montmorillonite were selected as model protein and reference clay, respectively. Protein-clay suspension mixtures were freshly prepared for each assay to simulate supernatants not completely centrifuged in batch sorption/kinetic experiments. Seven fixed increasing levels of clay (0.0, 0.00725, 0.0145, 0.029, 0.058, 0.145, 0.435 mg ml(-1)) were mixed with different levels of BSA in an appropriate range for each assay. To ascertain the interfering effect of different levels of clay, the theoretical concentrations of BSA were plotted against the estimated BSA concentrations of the samples, as obtained from the calibration curve of each method. A correct quantitation of the BSA concentration not influenced by clay would be described by a regression line with slope (b) not significantly different from 1 and an intercept (a) not significantly different from zero. At the lowest clay levels (0.00725 mg ml(-1)) a significant interference was evident for Bradford micro, Bradford 590/450, UV, and fluorescence. The three methods (Bradford standard, Lowry, and BCA) that seemed to show the better performances in the presence of clay after this first screening step also underwent an ANCOVA analysis, with the measured BSA concentrations as dependent variable and the clay concentrations as covariate. The Bradford standard and BCA methods were affected by a clay-dependent interference on BSA quantitation. The Lowry assay was the only method that gave correct estimates of BSA concentrations in the presence of any of the clay levels tested. PMID:18314004

Lozzi, I; Pucci, A; Pantani, O L; D'Acqui, L P; Calamai, L



Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation.  


The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and alpha-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome. PMID:20533907

Chouchani, Edward T; Hurd, Thomas R; Nadtochiy, Sergiy M; Brookes, Paul S; Fearnley, Ian M; Lilley, Kathryn S; Smith, Robin A J; Murphy, Michael P



Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation  

PubMed Central

The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and ?-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome. PMID:20533907

Chouchani, Edward T.; Hurd, Thomas R.; Nadtochiy, Sergiy M.; Brookes, Paul S.; Fearnley, Ian M.; Lilley, Kathryn S.; Smith, Robin A. J.; Murphy, Michael P.



Quantitative Analysis of Multivalent Ligand Presentation on Gold Glyconanoparticles and Their Effects on Protein Binding  

NASA Astrophysics Data System (ADS)

Bio-functionalized nanomaterials, which combine functions of biological ligands and unique properties of nano-sized building blocks, have exhibited increased potential applications in biosensing, therapeutics, and diagnostics. Glyconanoparitcles carrying a monolayer of carbohydrate ligands on nanoparticles provide an excellent platform for sensitive protein recognitions. Using Au nanoparticles as the scaffold, multivalent interactions between glycan ligands and proteins have been demonstrated. However, quantitative analysis especially the binding affinity of the resulting glyconanoparticles is challenging to determine. Here we present a new characterization technique, based on fluorescent competition binding assays, for measuring dissociation constants for glyconanoparticles-protein interactions. Au nanoparticles coupled with a series of un-derivatized carbohydrates were prepared by a photocoupling chemistry. Dramatic binding affinity enhancement was observed due to the high ligand density on nanoparticles, which was highly relevant to ligand display, controlled by the linker type, chain length, ligand size and density.

Wang, Xin; Ramström, Olof; Yan, Mingdi



Complex mixture analysis using protein expression as a qualitative and quantitative tool  

SciTech Connect

Some proteins in organisms exposed to chemicals in stressful amounts or toxic concentrations show increased expression; others show decreased expression. These inducible and repressible proteins together potentially provide qualitative and quantitative diagnoses of components in complex mixtures of chemicals. The authors examined sets of proteins synthesized by Daphnia magna after exposure to mixtures of a cationic polyamide epichlorhydrin adduct (Kymene) and a combined assortment of water-extractable substances from chemi-thermal-mechanical pulp (CTMP) in lab water. Proteins were identified, after extracting from Daphnia magna, by gel filtration and silver staining, or by radiolabeling and then gel separation. Patterns of proteins induced by Kymene[reg sign] and by CTMP extracts were distinguishable in lab water, but there was interaction between them. The method of identifying and quantifying Kymene, however, was successful using lab simulations of mixtures. The method was tested using wastewater samples from a paper manufacturing plant. Kymene could be detected against variable levels and types of additional substances. But, again, there was interference, perhaps due to Kymene binding to other anionic polymers sometimes present in the samples. Interpretation from analyses of protein expression were consistent with results from sublethal Ceriodaphnia dubia assays.

Bradley, B.P.; Gonzalez, C.M.; Bond, J.A. (Univ. of Maryland, Baltimore, MD (United States). Dept. of Biological Sciences); Tepper, B.E. (Procter and Gamble Co., Cincinnati, OH (United States). Paper Products Division)



Quantitative electrical detection of immobilized protein using gold nanoparticles and gold enhancement on a biochip  

NASA Astrophysics Data System (ADS)

Electrical detection of the concentration of protein immobilized on a biochip is demonstrated. The concentration of the direct immobilized protein can be determined by the resistance values measured by an ohm-meter directly. Indium tin oxide interdigitated electrodes were utilized as the detection sites on the biochip. Protein, i.e. antibody, of certain concentration was first immobilized on the detection site. Gold nanoparticles were then applied to indicate the immobilized protein. Since the gold nanoparticles were tiny, a detectable electrical signal could not be generated. Hence, a gold enhancement process was performed for signal amplification. Gold nanoparticles were enlarged physically, such that a conductive metal layer was formed on the detection site. The presence and concentration of protein can be determined by the resistance value across the electrode measured by an ohm-meter. An immobilized protein concentration ranging from 50 to 1000 ng ml-1 can be detected quantitatively by the resistance values from 4300 to 1700 ?. The proposed technique is potentially extended for the detection of immunoassay on the biochip. Since the protocol of the electrical detection does not involve sophisticated equipment, it can therefore be used for the development of a portable immunoassay device.

Fong Lei, Kin



Quantitative proteomics at different depths in human articular cartilage reveals unique patterns of protein distribution.  


The articular cartilage of synovial joints ensures friction-free mobility and attenuates mechanical impact on the joint during movement. These functions are mediated by the complex network of extracellular molecules characteristic for articular cartilage. Zonal differences in the extracellular matrix (ECM) are well recognized. However, knowledge about the precise molecular composition in the different zones remains limited. In the present study, we investigated the distribution of ECM molecules along the surface-to-bone axis, using quantitative non-targeted as well as targeted proteomics.\\ In a discovery approach, iTRAQ mass spectrometry was used to identify all extractable ECM proteins in the different layers of a human lateral tibial plateau full thickness cartilage sample. A targeted MRM mass spectrometry approach was then applied to verify these findings and to extend the analysis to four medial tibial plateau samples. In the lateral tibial plateau sample, the unique distribution patterns of 70 ECM proteins were identified, revealing groups of proteins with a preferential distribution to the superficial, intermediate or deep regions of articular cartilage. The detailed analysis of selected 29 proteins confirmed these findings and revealed similar distribution patterns in the four medial tibial plateau samples. The results of this study allow, for the first time, an overview of the zonal distribution of a broad range of cartilage ECM proteins and open up further investigations of the functional roles of matrix proteins in the different zones of articular cartilage in health and disease. PMID:25193283

Müller, Catharina; Khabut, Areej; Dudhia, Jayesh; Reinholt, Finn P; Aspberg, Anders; Heinegård, Dick; Önnerfjord, Patrik



Towards Quantitative Classification of Folded Proteins in Terms of Elementary Functions  

E-print Network

A comparative classification scheme provides a good basis for several approaches to understand proteins, including prediction of relations between their structure and biological function. But it remains a challenge to combine a classification scheme that describes a protein starting from its well organized secondary structures and often involves direct human involvement, with an atomary level Physics based approach where a protein is fundamentally nothing more than an ensemble of mutually interacting carbon, hydrogen, oxygen and nitrogen atoms. In order to bridge these two complementary approaches to proteins, conceptually novel tools need to be introduced. Here we explain how the geometrical shape of entire folded proteins can be described analytically in terms of a single explicit elementary function that is familiar from nonlinear physical systems where it is known as the kink-soliton. Our approach enables the conversion of hierarchical structural information into a quantitative form that allows for a folded protein to be characterized in terms of a small number of global parameters that are in principle computable from atomary level considerations. As an example we describe in detail how the native fold of the myoglobin 1M6C emerges from a combination of kink-solitons with a very high atomary level accuracy. We also verify that our approach describes longer loops and loops connecting $\\alpha$-helices with $\\beta$-strands, with same overall accuracy.

Shuangwei Hu; Andrei Krokhotin; Antti J. Niemi; Xubiao Peng



Towards quantitative classification of folded proteins in terms of elementary functions  

NASA Astrophysics Data System (ADS)

A comparative classification scheme provides a good basis for several approaches to understand proteins, including prediction of relations between their structure and biological function. But it remains a challenge to combine a classification scheme that describes a protein starting from its well-organized secondary structures and often involves direct human involvement, with an atomary-level physics-based approach where a protein is fundamentally nothing more than an ensemble of mutually interacting carbon, hydrogen, oxygen, and nitrogen atoms. In order to bridge these two complementary approaches to proteins, conceptually novel tools need to be introduced. Here we explain how an approach toward geometric characterization of entire folded proteins can be based on a single explicit elementary function that is familiar from nonlinear physical systems where it is known as the kink soliton. Our approach enables the conversion of hierarchical structural information into a quantitative form that allows for a folded protein to be characterized in terms of a small number of global parameters that are in principle computable from atomary-level considerations. As an example we describe in detail how the native fold of the myoglobin 1M6C emerges from a combination of kink solitons with a very high atomary-level accuracy. We also verify that our approach describes longer loops and loops connecting ? helices with ? strands, with the same overall accuracy.

Hu, Shuangwei; Krokhotin, Andrei; Niemi, Antti J.; Peng, Xubiao



Detection and quantitation of succinimide in intact protein via hydrazine trapping and chemical derivatization.  


The formation of aspartyl succinimide is a common post-translational modification of protein pharmaceuticals under acidic conditions. We present a method to detect and quantitate succinimide in intact protein via hydrazine trapping and chemical derivatization. Succinimide, which is labile under typical analytical conditions, is first trapped with hydrazine to form stable hydrazide and can be directly analyzed by mass spectrometry. The resulting aspartyl hydrazide can be selectively derivatized by various tags, such as fluorescent rhodamine sulfonyl chloride that absorbs strongly in the visible region (570 nm). Our tagging strategy allows the labeled protein to be analyzed by orthogonal methods, including HPLC-UV-Vis, liquid chromatography mass spectrometry (LC-MS), and SDS-PAGE coupled with fluorescence imaging. A unique advantage of our method is that variants containing succinimide, after derivatization, can be readily resolved via either affinity enrichment or chromatographic separation. This allows further investigation of individual factors in a complex protein mixture that affect succinimide formation. Some additional advantages are imparted by fluorescence labeling including the facile detection of the intact protein without proteolytic digestion to peptides; and high sensitivity, for example, without optimization, 0.41% succinimide was readily detected. As such, our method should be useful for rapid screening, optimization of formulation conditions, and related processes relevant to protein pharmaceuticals. PMID:25043726

Klaene, Joshua J; Ni, Wenqin; Alfaro, Joshua F; Zhou, Zhaohui Sunny



Preparation of heteroelement-incorporated and stable isotope-labeled protein standards for quantitative proteomics.  


A major obstacle for further development of quantitative proteomics is the lack of accurately quantified protein standards. The following protocol describes innovative methods for the production of stable isotope-labeled protein standards. Their production is achieved by cell-free protein synthesis, which enables simultaneous incorporation of selenomethionine and stable isotope-labeled amino acids. The selenium tag allows sensitive and accurate quantification by inductively coupled plasma mass spectrometry (ICP-MS). The stable isotope label allows internal standardization in mass spectrometry-based proteomics by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Both label types can be placed within a single protein RISQ standard (recombinant isotope-labeled and selenium quantified) or can be distributed over two types of related RSQ and RIQ standards for the same target protein (recombinant selenium quantified and recombinant isotope-labeled and quantified). The combination of cell-free synthesis as production method with ICP-MS and ESI-MS/MS as detection methods results in protein standards, which are quantified at an outstanding level of accuracy. PMID:24792000

Konopka, Anna; Zinn, Nico; Wild, Christina; Lehmann, Wolf D



Protein profiling of human lung telocytes and microvascular endothelial cells using iTRAQ quantitative proteomics.  


Telocytes (TCs) are described as a particular type of cells of the interstitial space ( Their main characteristics are the very long telopodes with alternating podoms and podomers. Recently, we performed a comparative proteomic analysis of human lung TCs with fibroblasts, demonstrating that TCs are clearly a distinct cell type. Therefore, the present study aims to reinforce this idea by comparing lung TCs with endothelial cells (ECs), since TCs and ECs share immunopositivity for CD34. We applied isobaric tag for relative and absolute quantification (iTRAQ) combined with automated 2-D nano-ESI LC-MS/MS to analyse proteins extracted from TCs and ECs in primary cell cultures. In total, 1609 proteins were identified in cell cultures. 98 proteins (the 5th day), and 82 proteins (10th day) were confidently quantified (screened by two-sample t-test, P < 0.05) as up- or down-regulated (fold change >2). We found that in TCs there are 38 up-regulated proteins at the 5th day and 26 up-regulated proteins at the 10th day. Bioinformatics analysis using Panther revealed that the 38 proteins associated with TCs represented cellular functions such as intercellular communication (via vesicle mediated transport) and structure morphogenesis, being mainly cytoskeletal proteins and oxidoreductases. In addition, we found 60 up-regulated proteins in ECs e.g.: cell surface glycoprotein MUC18 (15.54-fold) and von Willebrand factor (5.74-fold). The 26 up-regulated proteins in TCs at 10th day, were also analysed and confirmed the same major cellular functions, while the 56 down-regulated proteins confirmed again their specificity for ECs. In conclusion, we report here the first extensive comparison of proteins from TCs and ECs using a quantitative proteomics approach. Our data show that TCs are completely different from ECs. Protein expression profile showed that TCs play specific roles in intercellular communication and intercellular signalling. Moreover, they might inhibit the oxidative stress and cellular ageing and may have pro-proliferative effects through the inhibition of apoptosis. The group of proteins identified in this study needs to be explored further for the role in pathogenesis of lung disease. PMID:25059386

Zheng, Yonghua; Cretoiu, Dragos; Yan, Guoquan; Cretoiu, Sanda Maria; Popescu, Laurentiu M; Fang, Hao; Wang, Xiangdong



Electrophoresis and Gel Analysis  

NSDL National Science Digital Library

In this animation produced by WGBH and Digizyme, Inc., see how molecules of DNA are separated using gel electrophoresis, and how this process enables scientists to compare the molecular variations of two or more DNA samples.



Quantitative proteomic analysis reveals concurrent RNA–protein interactions and identifies new RNA-binding proteins in Saccharomyces cerevisiae  

PubMed Central

A growing body of evidence supports the existence of an extensive network of RNA-binding proteins (RBPs) whose combinatorial binding affects the post-transcriptional fate of every mRNA in the cell—yet we still do not have a complete understanding of which proteins bind to mRNA, which of these bind concurrently, and when and where in the cell they bind. We describe here a method to identify the proteins that bind to RNA concurrently with an RBP of interest, using quantitative mass spectrometry combined with RNase treatment of affinity-purified RNA–protein complexes. We applied this method to the known RBPs Pab1, Nab2, and Puf3. Our method significantly enriched for known RBPs and is a clear improvement upon previous approaches in yeast. Our data reveal that some reported protein–protein interactions may instead reflect simultaneous binding to shared RNA targets. We also discovered more than 100 candidate RBPs, and we independently confirmed that 77% (23/30) bind directly to RNA. The previously recognized functions of the confirmed novel RBPs were remarkably diverse, and we mapped the RNA-binding region of one of these proteins, the transcriptional coactivator Mbf1, to a region distinct from its DNA-binding domain. Our results also provided new insights into the roles of Nab2 and Puf3 in post-transcriptional regulation by identifying other RBPs that bind simultaneously to the same mRNAs. While existing methods can identify sets of RBPs that interact with common RNA targets, our approach can determine which of those interactions are concurrent—a crucial distinction for understanding post-transcriptional regulation. PMID:23636942

Klass, Daniel M.; Scheibe, Marion; Butter, Falk; Hogan, Gregory J.; Mann, Matthias; Brown, Patrick O.



Quantitative and rapid analysis of transglutaminase activity using protein arrays in mammalian cells.  


We developed a novel on-chip activity assay using protein arrays for quantitative and rapid analysis of transglutami-nase activity in mammalian cells. Transglutaminases are a family of Ca2+-dependent enzymes involved in cell regulation as well as human diseases such as neurodegenerative disorders, inflammatory diseases and tumor progression. We fabricated the protein arrays by immobilizing N,N'-dimethylcasein (a substrate) on the amine surface of the arrays. We initiated transamidating reaction on the protein arrays and determined the transglutaminase activity by analyzing the fluorescence intensity of biotinylated casein. The on-chip transglutaminase activity assay was proved to be much more sensitive than the [3H]putrescine-incorporation assay. We successfully applied the on-chip assay to a rapid and quantitative analysis of the transgluta-minase activity in all-trans retinoic acid-treated NIH 3T3 and SH-SY5Y cells. In addition, the on-chip transglutaminase activity assay was sufficiently sensitive to determine the transglutaminase activity in eleven mammalian cell lines. Thus, this novel on-chip transglutaminase activity assay was confirmed to be a sensitive and high-throughput approach to investigating the roles of transglutaminase in cellular signaling, and, moreover, it is likely to have a strong potential for monitoring human diseases. PMID:19326081

Kwon, Mi-Hye; Jung, Jae-Wan; Jung, Se-Hui; Park, Jin-Young; Kim, Young-Myeong; Ha, Kwon-Soo



Conformational analysis by quantitative NOE measurements of the ?-proton pairs across individual disulfide bonds in proteins.  


NOEs between the ?-protons of cysteine residues across disulfide bonds in proteins provide direct information on the connectivities and conformations of these important cross-links, which are otherwise difficult to investigate. With conventional [U-(13)C, (15)N]-proteins, however, fast spin diffusion processes mediated by strong dipolar interactions between geminal ?-protons prohibit the quantitative measurements and thus the analyses of long-range NOEs across disulfide bonds. We describe a robust approach for alleviating such difficulties, by using proteins selectively labeled with an equimolar mixture of (2R, 3S)-[?-(13)C; ?,?-(2)H(2)] Cys and (2R, 3R)-[?-(13)C; ?,?-(2)H(2)] Cys, but otherwise fully deuterated. Since either one of the prochiral methylene protons, namely ?2 (proS) or ?3 (proR), is always replaced with a deuteron and no other protons remain in proteins prepared by this labeling scheme, all four of the expected NOEs for the ?-protons across disulfide bonds could be measured without any spin diffusion interference, even with long mixing times. Therefore, the NOEs for the ?2 and ?3 pairs across each of the disulfide bonds could be observed at high sensitivity, even though they are 25% of the theoretical maximum for each pair. With the NOE information, the disulfide bond connectivities can be unambiguously established for proteins with multiple disulfide bonds. In addition, the conformations around disulfide bonds, namely ?(2) and ?(3), can be determined based on the precise proton distances of the four ?-proton pairs, by quantitative measurements of the NOEs across the disulfide bonds. The feasibility of this method is demonstrated for bovine pancreatic trypsin inhibitor, which has three disulfide bonds. PMID:22131165

Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune



Quantitative Liver-Specific Protein Fingerprint in Blood: A Signature for Hepatotoxicity  

PubMed Central

We discuss here a new approach to detecting hepatotoxicity by employing concentration changes of liver-specific blood proteins during disease progression. These proteins are capable of assessing the behaviors of their cognate liver biological networks for toxicity or disease perturbations. Blood biomarkers are highly desirable diagnostics as blood is easily accessible and baths virtually all organs. Fifteen liver-specific blood proteins were identified as markers of acetaminophen (APAP)-induced hepatotoxicity using three proteomic technologies: label-free antibody microarrays, quantitative immunoblotting, and targeted iTRAQ mass spectrometry. Liver-specific blood proteins produced a toxicity signature of eleven elevated and four attenuated blood protein levels. These blood protein perturbations begin to provide a systems view of key mechanistic features of APAP-induced liver injury relating to glutathione and S-adenosyl-L-methionine (SAMe) depletion, mitochondrial dysfunction, and liver responses to the stress. Two markers, elevated membrane-bound catechol-O-methyltransferase (MB-COMT) and attenuated retinol binding protein 4 (RBP4), report hepatic injury significantly earlier than the current gold standard liver biomarker, alanine transaminase (ALT). These biomarkers were perturbed prior to onset of irreversible liver injury. Ideal markers should be applicable for both rodent model studies and human clinical trials. Five of these mouse liver-specific blood markers had human orthologs that were also found to be responsive to human hepatotoxicity. This panel of liver-specific proteins has the potential to effectively identify the early toxicity onset, the nature and extent of liver injury and report on some of the APAP-perturbed liver networks. PMID:24465277

Hu, Zhiyuan; Lausted, Christopher; Yoo, Hyuntae; Yan, Xiaowei; Brightman, Amy; Chen, Jiankui; Wang, Weizhi; Bu, Xiangli; Hood, Leroy



Quantitative imaging of protein adsorption on patterned organic thin-film arrays using secondary electron emission.  


Secondary electron emission is developed as a means to quantify and image protein binding to Au surfaces modified with patterned organic thin-film arrays. Alkane thiols were patterned via microcontact printing on gold, and their effects on the secondary electron (SE) yield of the surface, systematically quantified. We show that a self-assembled monolayer (SAM) of hexadecane thiol significantly increases the SE yield over the native gold surface, a yield that increases as a function of alkane chain length (C8-C16). This effect is linearly correlated with the surface potentials and wetting properties of these SAMs. Surface layers comprised of poly(ethylene glycol) (PEG) grafted polyacrylamide polymers behave differently, affecting the SE yield by attenuation according to the polymer thickness. These results demonstrate the relative contributions of factors related to the adsorbate molecular structures that serve to strongly mediate the SE yield, providing a foundation for exploiting them as a quantitative electron imaging probe. The latter capability is demonstrated using a model microfluidic assay in which a series of proteins was spatially addressed to a SAM-based pixel array. The gray scale contrasts seen with protein adsorption are directly correlated with both protein molecular weight and mass coverage. These methods are used in two model protein assay experiments: (1) the measurement of the concentration dependent adsorption isotherm for a model protein (fibrinogen); and (2) the selective recognition of a biotinylated protein layer by avidin. These results demonstrate a unique approach to imaging protein binding processes on surfaces with both high analytical and spatial sensitivity. PMID:16771500

Mack, Nathan H; Dong, Rui; Nuzzo, Ralph G



Quantitative analysis of highly homologous proteins: the challenge of assaying the "CYP-ome" by mass spectrometry.  


Cytochrome P450 enzymes comprise families of highly homologous proteins. These proteins play a pivotal role in oxidative drug metabolism and are important targets in drug discovery research. Proteomics today is a valuable tool for the analysis of proteins. In the past, qualitative analysis of the proteome was the main focus of research, but in the last few years interest in the mathematical modelling of protein networks has been growing and so has the demand on quantitative proteome analysis. As a thorough understanding of cytochrome P450 dependent metabolism is crucial for drug discovery, it is thus not astounding that cytochrome P450 enzymes are a target for quantitative proteomics research. In this article, we review the techniques available for quantitative proteome analysis and to what extent these techniques have been used for the quantification of cytochrome P450 enzymes and give a brief outlook of the techniques that have promising potential for the analysis of these proteins in the future. PMID:18846368

Langenfeld, Elmar; Meyer, Helmut E; Marcus, Katrin



Gel Electrophoresis on a Budget to Dye for  

ERIC Educational Resources Information Center

Gel electrophoresis is one of the most important tools used in molecular biology and has facilitated the entire field of genetic engineering by enabling the separation of nucleic acids and proteins. However, commercial electrophoresis kits can cost up to $800 for each setup, which is cost prohibitive for most classroom budgets. This article…

Yu, Julie H.



Quantitative Correlation Between the Protein Primary Sequences and Secondary Structures in Spider Dragline Silks  

PubMed Central

Synthetic spider silk holds great potential for use in various applications spanning medical uses to ultra lightweight armor, however producing synthetic fibers with mechanical properties comparable to natural spider silk has eluded the scientific community. Natural dragline spider silks are commonly made from proteins that contain highly repetitive amino acid motifs, adopting an array of secondary structures. Before further advances can be made in the production of synthetic fibers based on spider silk proteins, it is imperative to know the percentage of each amino acid in the protein that forms a specific secondary structure. Linking these percentages to the primary amino acid sequence of the protein will establish a structural foundation for synthetic silk. In this study, Nuclear Magnetic Resonance (NMR) techniques are used to quantify the percentage of Ala, Gly, and Ser that form both ?-sheet and helical secondary structures. The fraction of these three amino acids and their secondary structure are quantitatively correlated to the primary amino acid sequence for the proteins that comprise major and minor ampullate silk from the Nephila clavipes spider providing a blueprint for synthetic spider silks. PMID:20000730

Jenkins, Janelle E.; Creager, Melinda S.; Lewis, Randolph V.; Holland, Gregory P.; Yarger, Jeffery L.



A large-scale quantitative proteomic approach to identifying sulfur mustard-induced protein phosphorylation cascades.  


Sulfur mustard [SM, bis-(2-chloroethyl) sulfide] is a potent alkylating agent and chemical weapon. While there are no effective treatments for SM-induced injury, current research focuses on understanding the molecular changes upon SM exposure. Indeed, efforts that seek a more comprehensive analysis of proteins and post-translational modifications are critical for understanding SM-induced toxicity on a more global scale. Furthermore, these studies can uncover proteins previously uncharacterized in SM-exposed cells, which in turn leads to potential targets for therapeutic intervention. Here, we apply a quantitative proteomic approach termed stable isotope-labeling with amino acids in cell culture combined with immobilized metal affinity chromatography to study the large-scale protein phosphorylation changes resulting from SM exposure in a human keratinocyte cell culture model. This resulted in the characterization of over 2300 nonredundant phosphorylation sites, many of which exhibit altered levels in response to SM. Our results point toward several proteins previously implicated in SM-induced toxicity as well as many additional proteins previously uncharacterized. Further de novo analysis of these phosphoproteins using interaction mapping software revealed both known and novel pathways that may serve as future therapeutic targets of SM exposure. PMID:19845377

Everley, Patrick A; Dillman, James F



A Chip-Capillary Hybrid Device for Automated Transfer of Sample Pre-Separated by Capillary Isoelectric Focusing to Parallel Capillary Gel Electrophoresis for Two-Dimensional Protein Separation  

PubMed Central

In this report, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to re-route the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed. PMID:22830584

Lu, Joann J.; Wang, Shili; Li, Guanbin; Wang, Wei; Pu, Qiaosheng; Liu, Shaorong



Quantitative Time-course Profiling of Parasite and Host Cell Proteins in the Human Malaria Parasite Plasmodium falciparum*  

PubMed Central

Studies of the Plasmodium falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intra-erythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade in which most expressed genes exhibit a single transcriptional peak. Because the biochemical and cellular functions of most genes are mediated by the encoded proteins, understanding the relationship between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. Although studies on other organisms show that <50% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on oligonucleotide microarrays and two-dimensional differential gel electrophoresis protein gels. We find that most proteins are represented by more than one isoform, presumably because of post-translational modifications. Like transcripts, most proteins exhibit cyclic abundance profiles with one peak during the IDC, whereas the presence of functionally related proteins is highly correlated. In contrast, the abundance of most parasite proteins peaks significantly later (median 11 h) than the corresponding transcripts and often decreases slowly in the second half of the IDC. Computational modeling indicates that the considerable and varied incongruence between transcript and protein abundance may largely be caused by the dynamics of translation and protein degradation. Furthermore, we present cyclic abundance profiles also for parasite-associated human proteins and confirm the presence of five human proteins with a potential role in antioxidant defense within the parasites. Together, our data provide fundamental insights into transcript-protein relationships in P. falciparum that are important for the correct interpretation of transcriptional data and that may facilitate the improvement and development of malaria diagnostics and drug therapy. PMID:21558492

Foth, Bernardo Javier; Zhang, Neng; Chaal, Balbir Kaur; Sze, Siu Kwan; Preiser, Peter Rainer; Bozdech, Zbynek



Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation  

PubMed Central

Background Fluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs) for further analysis which is subjective and does not allow for comparing different cells and experimental settings. Results We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp) function is fitted to fluorescence loss (FL) inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP), we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ) disease proteins like mutant huntingtin (mtHtt) can form large aggregates called inclusion bodies (IB’s). The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt) between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and experiments. Conclusions We present two complementary methods for quantitative analysis of FLIP experiments in living cells. They provide spatial maps of exchange dynamics and absolute binding parameters of fluorescent molecules to moving intracellular entities, respectively. Our methods should be of great value for quantitative studies of intracellular transport. PMID:23148417



Lost in centrifugation: accounting for transporter protein losses in quantitative targeted absolute proteomics.  


In drug development, considerable efforts are made to extrapolate from in vitro and preclinical findings to predict human drug disposition by using in vitro-in vivo extrapolation (IVIVE) approaches. Use of IVIVE strategies linked with physiologically based pharmacokinetic (PBPK) modeling is widespread, and regulatory agencies are accepting and occasionally requesting model analysis to support licensing submissions. Recently, there has been a drive to improve PBPK models by characterizing the absolute abundance of enzymes, transporters, and receptors within mammalian tissues and in vitro experimental systems using quantitative targeted absolute proteomics (QTAP). The absolute abundance of proteins relevant to processes governing drug disposition provided by QTAP will enable IVIVE-PBPK to incorporate terms for the abundance of enzymes and transporters in target populations. However, most studies that report absolute abundances of enzymes and transporter proteins do so in enriched membrane fractions so as to increase the abundance per sample, and thus the assay's sensitivity, rather than measuring the expected lower abundance in the more biologically meaningful whole cells or tissues. This communication discusses the balance between protein enrichment and potential loss during the preparation of membrane fractions from whole cells or tissues. Accounting for losses with recovery factors throughout the fractionation procedure provides a means to correct for procedural losses, thereby enabling the scaling of protein abundance from subcellular fractions to whole-cell or organ abundances. PBPK models based on corrected abundances will more closely resemble biological systems and facilitate development of more meaningful IVIVE scaling factors, producing more accurate quantitative predictions of drug disposition. PMID:25061159

Harwood, Matthew D; Russell, Matthew R; Neuhoff, Sibylle; Warhurst, Geoffrey; Rostami-Hodjegan, Amin




E-print Network

with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism and metalloproteins in open tubular capillary electrochromatography with etched chemically modified columns J. J

Xuan, Xiangchun "Schwann"


Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein.  


The mechanism of G protein-coupled receptor (GPCR) signaling in yeasts is similar to that in mammalian cells. Therefore, yeasts can be used in GPCR assays, and several ligand detection systems using a pheromone signaling pathway in yeasts have been developed by employing yeasts with disrupted chromosomal genes that code for proteins producing specific effects. In this study, the construction of yeast strains that can detect ligand binding mediated by interactions between the G protein and GPCR using either fluorescence or auxotrophic selectivity is demonstrated. The strain was constructed by integrating the fusion gene of pheromone-responsive protein (FUS1), enhanced green fluorescence protein (EGFP), and auxotrophic marker protein (HIS3) into the FUS1 locus. Moreover, the influence of gene disruptions on the yeast signal transduction cascade is closely investigated with respect to both quantitative and dynamic aspects to further develop a high-throughput screening system for the GPCR assay using yeasts. Yeast strains with a disrupted SST2 gene, which is a member of the RGS (regulator of G protein signaling) family, and a disrupted FAR1 gene, which mediates cell cycle arrest in response to a pheromone, were monitored by measuring their fluorescence and growth rate. This method will be applicable to other comprehensive GPCR ligand screening methods. PMID:16889369

Ishii, Jun; Matsumura, Shizuka; Kimura, Sakurako; Tatematsu, Kenji; Kuroda, Shun'ichi; Fukuda, Hideki; Kondo, Akihiko



Application of Microchip Electrophoresis for Clinical Tests  

NASA Astrophysics Data System (ADS)

Microchip electrophoresis has recently attracted much attention in the field of nuclear acid analysis due to its high efficiency, ease of operation, low consumption of samples and reagents, and relatively low costs. In addition, the analysis has expanded to an analytical field like not only the analysis of DNA but also the analysis of RNA, the protein, the sugar chain, and the cellular function, etc. In this report, we showed that high-performance monitoring systems for human blood glucose levels and ?-amylase activity in human plasma using microchip electrophoresis.

Yatsushiro, Shouki; Kataoka, Masatoshi


Quantitative analysis in the characterization and optimization of protein crystal growth.  


Protein crystal growth often depends on the combination of many different factors. Some affect protein solubility directly; others may act indirectly by causing conformational changes. Systematic characterization of these factors can be important for generating good crystals. It can also provide useful insight into the biochemical behavior of the protein being crystallized. Here we focus on statistical methods to achieve these two objectives. (1) Characterization of a protein system by analyzing patterns of crystal polymorphism under different levels of biochemical parameters, such as ligands and pH. Tests of the reproducibility of crystal growth experiments indicate that quantitative scales of crystal quality can be statistically significant. Analysis of variance for a replicated, full-factorial design in which four factors were tested at two levels has been used to demonstrate highly significant, biochemically relevant, two-factor interactions strongly implicating pH and ligand-dependent conformational changes. (2) Optimization of crystal growth via response-surface methods. 'Minimum predicted variance' designs provide for efficient response-surface experiments aimed at constructing quadratic models in several dimensions. We have used such models to improve crystal size and quality significantly for three forms of Bacillus stearothermophilus tryptophanyl-tRNA synthetase. In one case we can now avoid having to increase the size by repeated seeding, a difficult procedure that also produces unwanted growth of satellite crystals. Graphs of two-dimensional level surfaces reveal a number of ridges, where the same result is obtained for many combinations of the factors usually varied when trying to improve crystals. An important inference is that it may be better to sample simultaneously for the effects of protein concentration and supersaturation. For a system involving only one crystallizing agent, supersaturation can be approximated as the product of protein and precipitant concentrations. Use of this search direction significantly improves the performance of response-surface experiments. Advantages of growing crystals at stationary points of their response surfaces include better crystals and higher reproducibility, since crystal growth at stationary points is insulated from the deleterious effects of experimental fluctuations. This arises because the derivatives of the response are by definition zero with respect to the experimental variables. Quantitative analysis of appropriately designed crystal growth experiments can thus be a powerful way to characterize complex and interacting biochemical dependencies in macromolecular systems and optimize parameters important to the crystallography. PMID:15299421

Carter, C W; Yin, Y



Gel Electrophoresis of Dyes  

NSDL National Science Digital Library

In this experiment related to plant biotechnology, learners discover how to prepare and load an electrophoresis gel. They will then run the gels in an electrophoresis system to separate several dyes that are of different molecular sizes and carry different charges. This technique is fundamental to many of the procedures used in biotechnology. This lesson guide includes background information for the educator, safety precautions, and questions with answers for learners. For safety reasons, adult supervision is recommended. Modifications for use with younger learners are described in a related PDF (see related resource).

Stephens, Janice; Leach, Jan



Determination and quantification of Escherichia coli by capillary electrophoresis.  


Capillary electrophoresis (CE) is widely employed for the separation of nucleic acids or protein, but it is rarely applied in the quantification of Escherichia coli (E. coli). Here, we have analysed E. coli by CE with mercury lamp induced fluorescence, and demonstrated the relationship between its fluorescence intensity with the concentration of E. coli for the first time. The gradient concentration of E. coli was obtained by polymerase chain reaction (PCR) with different amplification cycles and dilution certain PCR products of E. coli, respectively. Results show that the peak area was linearly related to the logarithm of the concentration of E. coli and the logarithm of PCR replication numbers. The correlation coefficients R(2) are 0.957 and 0.966, respectively. The limit of detection (LOD) was found to be about 8.913 × 10(-15) mol ?l(-1). The reproducibility of capillary electrophoresis may make this technique possible for quantitative measurement of bacteria in bio-analytical science. PMID:25307062

Li, Zhenqing; Li, De; Zhang, Dawei; Yamaguchi, Yoshinori



Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling  

PubMed Central

We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope 18O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level 16O and 18O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in ?gspD mutant cells of many outer membrane proteins including the outer membrane c-cype cytochromes OmcA and MtrC, in agreement with previously investigation demonstrating that these proteins are substrates of the type II secretion system. PMID:20380418

Zhang, Haizhen; Brown, Roslyn N.; Qian, Wei-Jun; Monroe, Matthew E.; Purvine, Samuel O.; Moore, Ronald J.; Gritsenko, Marina A.; Shi, Liang; Romine, Margaret F; Fredrickson, James K.; Paša-Toli?, Ljiljana; Smith, Richard D.; Lipton, Mary S.



Ultrarapid quantitation of maize proteins by perfusion and monolithic reversed-phase high-performance liquid chromatography.  


The main objective of this study was to develop a new methodology alternative to the classical Kjeldahl analysis for determining maize proteins in maize products and seeds. For that purpose, two different chromatographic methodologies using perfusion and monolithic stationary phases, both enabling rapid separations of maize proteins, were investigated. Due to the difficulty to find suitable standards for this type of analysis, three different maize products were initially tested as proteins standards: zein F4000, corn gluten meal, and maize flour. Different figures of merit (i.e., linearity, correlation coefficient, precision, limits of detection and quantitation), as well as the presence of matrix inferences, were investigated. The results obtained for the different chromatographic stationary phases and protein standards were compared in order to select the most suitable analytical conditions. Despite both perfusion and monolithic methodologies resulting, in general, as appropriate for the quantitation of maize proteins, the highest reduction of analysis time and lowest detection and determination limits provided by perfusion methodology enabled to select this one as the method of choice for the quantitation of maize proteins. Regarding the different protein standards studied in this work, in general the best results were obtained using the zein standard. Compared to Kjeldahl methodology, perfusion chromatography yields total protein contents in shorter analysis time while enabling the separation of the different kinds of proteins. Due to the high diversity and complexity of industrial maize products, the proposed chromatographic method could be a very useful tool for their routine analysis. PMID:19323473

Rodríguez-Nogales, J M; del Alamo, M; García, M C; Cifuentes, A; Marina, M L



Quantitation of femtomolar protein levels via direct readout with the electrochemical proximity assay.  


We have developed a separation-free, electrochemical assay format with direct readout that is amenable to highly sensitive and selective quantitation of a wide variety of target proteins. Our first generation of the electrochemical proximity assay (ECPA) is composed of two thrombin aptamers which form a cooperative complex only in the presence of target molecules, moving a methylene blue (MB)-conjugated oligonucleotide close to a gold electrode. Without washing steps, electrical current is increased in proportion to the concentration of a specific target protein. By employing a DNA-based experimental model with the aptamer system, we show that addition of a short DNA competitor can reduce background current of the MB peak to baseline levels. As such, the detection limit of aptamer-based ECPA for human thrombin was 50 pM via direct readout. The dual-probe nature of ECPA gave high selectivity and 93% recovery of signal from 2.5 nM thrombin in 2% bovine serum albumin (BSA). To greatly improve the flexibility of ECPA, we then proved the system functional with antibody-oligonucleotide conjugates as probes; the insulin detection limit was 128 fM with a dynamic range of over 4 orders of magnitude in concentration, again with high assay selectivity. ECPA thus allows separation-free, highly sensitive, and highly selective protein detection with a direct electrochemical readout. This method is extremely flexible, capable of detecting a wide variety of protein targets, and is amenable to point-of-care protein measurement, since any target with two aptamers or antibodies could be assayed via direct electrochemical readout. PMID:22452720

Hu, Jiaming; Wang, Tanyu; Kim, Joonyul; Shannon, Curtis; Easley, Christopher J



Dynamics of natural killer cell receptor revealed by quantitative analysis of photoswitchable protein.  


Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies that rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report an apparently novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, after a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 ± 0.06 ?m(2) s(-1)) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse, and remains surprisingly stable there. Unexpectedly, however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected intersynaptic exchange of protein. PMID:24209843

Pageon, Sophie V; Aquino, Gerardo; Lagrue, Kathryn; Köhler, Karsten; Endres, Robert G; Davis, Daniel M



Quantitation of a cationic antimicrobial granule protein of human polymorphonuclear leukocytes by ELISA.  


The quantitation of CAP57, a highly hydrophobic, native cationic antigen of human polymorphonuclear leukocytes has been achieved using ELISA. An important feature determining the sensitivity and precision of the ELISA was the reduction of non-specific protein-protein binding, particularly in the inhibition assays, thus eliminating high backgrounds obtained with presently available methodology. Washing of the solid phase-bound antigen and blocking of the non-specific binding sites using a potassium phosphate buffer containing heparin largely contributed to this increased sensitivity. The inhibition assays were conducted using antigen concentrations over the range of 0.9-120 ng. The assay is highly specific and can be performed using monoclonal antibodies and polyclonal antibodies. Non-specific reactions were observed only when high concentrations of antigen (greater than 100 ng) were present in the inhibition mixture. The technique as described is extremely simple, highly reproducible and could be of value in the detection of cationic antimicrobial proteins in the clinical setting in the future. PMID:2913156

Pereira, H A; Martin, L E; Spitznagel, J K



Fraction collector for electrophoresis  

NASA Technical Reports Server (NTRS)

Rotating-tube electrophoresis apparatus employs rotating jet of eluting buffer to reduce effects of convection during separation. Designed for separation of microorganisms and biological species, system combines gravity/gradient compensating of lumen with buffer flush at fraction outlet to increase separation efficiency.

Bier, M.



Methods for quantitative evaluation of dynamics of repair proteins within irradiated cells  

NASA Astrophysics Data System (ADS)

Living HeLa cells are irradiated well directed with single 100 MeV oxygen ions by the superconducting ion microprobe SNAKE, the Superconducting Nanoscope for Applied Nuclear (= Kern-) Physics Experiments, at the Munich 14 MV tandem accelerator. Various proteins, which are involved directly or indirectly in repair processes, accumulate as clusters (so called foci) at DNA-double strand breaks (DSBs) induced by the ions. The spatiotemporal dynamics of these foci built by the phosphorylated histone ?-H2AX are studied. For this purpose cells are irradiated in line patterns. The ?-H2AX is made visible under the fluorescence microscope using immunofluorescence techniques. Quantitative analysis methods are developed to evaluate the data of the microscopic images in order to analyze movement of the foci and their changing size.

Hable, V.; Dollinger, G.; Greubel, C.; Hauptner, A.; Krücken, R.; Dietzel, S.; Cremer, T.; Drexler, G. A.; Friedl, A. A.; Löwe, R.



Strigolactone-Regulated Proteins Revealed by iTRAQ-Based Quantitative Proteomics in Arabidopsis  

SciTech Connect

Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. Here, a quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found SLs regulate the expression of about three dozens of proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

Li, Zhou [ORNL] [ORNL; Czarnecki, Olaf [ORNL] [ORNL; Chourey, Karuna [ORNL] [ORNL; Yang, Jun [ORNL] [ORNL; Tuskan, Gerald A [ORNL] [ORNL; Hurst, Gregory {Greg} B [ORNL; Pan, Chongle [ORNL] [ORNL; Chen, Jay [ORNL] [ORNL



Quantitative Fitness Analysis Shows That NMD Proteins and Many Other Protein Complexes Suppress or Enhance Distinct Telomere Cap Defects  

PubMed Central

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70?). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70?, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70? mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70?. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology. PMID:21490951

Yu, Min; Chapman, Kaye; Banks, A. Peter; Ngo, Hien-Ping; Maringele, Laura; Taschuk, Morgan; Young, Alexander; Ciesiolka, Adam; Lister, Allyson Lurena; Wipat, Anil; Wilkinson, Darren James; Lydall, David



Quantitative single-molecule detection of protein based on DNA tetrahedron fluorescent nanolabels.  


A highly sensitive method for single-molecule quantitative detection of human IgG is presented by the employment of a new fluorescent nanolabel. In this method, fluorescent nanolabels were assembled by inserting SYBR Green I into DNA tetrahedron nanostructure. The bio-nanolabels were attached to the streptavidin-antihuman antibody by a specific reaction between biotin and streptavidin. The antibody was combined with the target antigen, human IgG, which was immobilized on the silanized glass subtrate surface. Finally, epi-fluorescence microscopy (EFM) coupled with an electron multiplying charge-coupled device was employed for fluorescence imaging. The fluorescent spots corresponding to single protein molecule on images were counted and further used for the quantitative detection. It was found that the new nanolabel shows good photostability, biocompatiblity and exhibits no blinking compared to traditional labels like fluorescence dyes and quantum dot (QDs). In addition, the number of fluorescence spots on the images has a linear relationship with the concentration of human IgG in the range of 3.0×10(-14) to 1.0×10(-12)mol L(-1). What is more, this method showed an excellent specificity and a low matrix effect. PMID:24840462

Ding, Yongshun; Liu, Xingti; Zhu, Jing; Wang, Lei; Jiang, Wei



Absolute quantitation of proteins in human blood by multiplexed multiple reaction monitoring mass spectrometry.  


Multiple reaction monitoring (MRM)-mass spectrometry (MS) with stable isotope-labeled standards (SIS) has proven adept in rapidly, precisely, and accurately quantifying proteins in complex biological samples. The impetus behind the early use of multiplexed MRM in proteomics was to expedite the verification and validation stages of the protein biomarker pipeline for clinical utility, which involves the analysis of hundreds or even thousands of samples. Moreover, once a multiplexed assay has been developed, however, it can be turned around and used for biomarker discovery, as has been demonstrated for cancer biomarkers by our laboratory and by others. Overall, these MRM-based methods compare favorably with antibody-based techniques, such as ELISAs or protein arrays, in that MRM-based methods are less expensive and can be developed more rapidly. There are two MRM-based platforms that are currently being developed: a standard-flow and a nano-flow LC/ESI-MRM-MS (liquid chromatography-electrospray ionization) platform. In this book chapter, we describe a recent study in which we evaluated these two platforms, both interfaced to the same mass spectrometer. This study demonstrated the enhanced performance metrics (in terms of sensitivity, dynamic range, and robustness) of the standard-flow ultra-high performance liquid chromatography (UHPLC) system compared to the nano-flow HPLC-Chip for the absolute quantitation of 48 plasma proteins. Using the standard-flow platform, we also developed two high-throughput assays for the analysis of a panel of 67 cardiovascular disease (CVD) biomarkers in non-depleted and non-enriched human plasma and a panel of 25 putative biomarkers in dried human blood spots (DBS). Since the nanoLC/MRM-MS platform has advantages under sample-limited conditions and for the analysis of certain specific peptides, the protocols for both systems are described here. PMID:23585092

Percy, Andrew J; Chambers, Andrew G; Parker, Carol E; Borchers, Christoph H



A Slot Blot Immunoassay for Quantitative Detection of Plasmodium falciparum Circumsporozoite Protein in Mosquito Midgut Oocyst  

PubMed Central

There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB) for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP) and native PfCSP from Oocysts (Pf Oocyst) developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5–20 pg; R2?=?0.9505). We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1–4, R2?=?0.9795) and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5–3 pg) of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV) for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes and transmission-blocking interventions in endemic areas. PMID:25531543

Kumar, Sanjai; Zheng, Hong; Deng, Bingbing; Mahajan, Babita; Grabias, Bryan; Kozakai, Yukiko; Morin, Merribeth J.; Locke, Emily; Birkett, Ashley; Miura, Kazutoyo; Long, Carole



Analysis of protein composition of red wine in comparison with rose? and white wines by electrophoresis and high-pressure liquid chromatography-mass spectrometry (HPLC-MS).  


Wine proteins not only influence wine stability but are also being discussed as potential allergens. Proteins from red, rose?, and white wines were enriched by dialysis and lyophilization followed by separation by SDS-PAGE. Significant differences were detected in the protein compositions of the analyzed wine varieties, and the major protein bands were identified by mass spectrometry after in-gel digestion with trypsin. In German Portugieser red wine, a total of 121 tryptic peptides were identified, which were attributed to 12 grape proteins and 6 proteins derived from yeast. Among the identified constituents are several proteins considered to influence wine stability and previously described potential grape allergens. The pathogenesis-related proteins represent the main proteins in all of the wines, but only some red wines show a band with a molecular mass of 12 kDa, identified as a lipid transfer protein (LTP). The occurrence and distribution of LTP depend on the wine variety. PMID:19385597

Wigand, Petra; Tenzer, Stefan; Schild, Hansjoerg; Decker, Heinz



Bayesian analysis of high-throughput quantitative measurement of protein-DNA interactions.  


Transcriptional regulation depends upon the binding of transcription factor (TF) proteins to DNA in a sequence-dependent manner. Although many experimental methods address the interaction between DNA and proteins, they generally do not comprehensively and accurately assess the full binding repertoire (the complete set of sequences that might be bound with at least moderate strength). Here, we develop and evaluate through simulation an experimental approach that allows simultaneous high-throughput quantitative analysis of TF binding affinity to thousands of potential DNA ligands. Tens of thousands of putative binding targets can be mixed with a TF, and both the pre-bound and bound target pools sequenced. A hierarchical Bayesian Markov chain Monte Carlo approach determines posterior estimates for the dissociation constants, sequence-specific binding energies, and free TF concentrations. A unique feature of our approach is that dissociation constants are jointly estimated from their inferred degree of binding and from a model of binding energetics, depending on how many sequence reads are available and the explanatory power of the energy model. Careful experimental design is necessary to obtain accurate results over a wide range of dissociation constants. This approach, which we call Simultaneous Ultra high-throughput Ligand Dissociation EXperiment (SULDEX), is theoretically capable of rapid and accurate elucidation of an entire TF-binding repertoire. PMID:22069446

Pollock, David D; de Koning, A P Jason; Kim, Hyunmin; Castoe, Todd A; Churchill, Mair E A; Kechris, Katerina J



Quantitative Imaging of Protein Secretions from Single Cells in Real Time  

PubMed Central

Protein secretions from individual cells create spatially and temporally varying concentration profiles in the extracellular environment, which guide a wide range of biological processes such as wound healing and angiogenesis. Fluorescent and colorimetric probes for the detection of single cell secretions have time resolutions that range from hours to days, and as a result, little is known about how individual cells may alter their protein secretion rates on the timescale of minutes or seconds. Here, we present a label-free technique based upon nanoplasmonic imaging, which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from single hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell and from the bursts we estimated the diffusion constant of the secreted antibodies through the extracellular media. The design also incorporates transmitted light and fluorescence microscopy capabilities for monitoring cellular morphological changes and intracellular fluorescent labels. We anticipate that this technique can be adapted as a general tool for the quantitative study of paracrine signaling in both adherent and nonadherent cell lines. PMID:23931308

Raphael, Marc P.; Christodoulides, Joseph A.; Delehanty, James B.; Long, James P.; Byers, Jeff M.



Quantitative imaging of protein secretions from single cells in real time.  


Protein secretions from individual cells create spatially and temporally varying concentration profiles in the extracellular environment, which guide a wide range of biological processes such as wound healing and angiogenesis. Fluorescent and colorimetric probes for the detection of single cell secretions have time resolutions that range from hours to days, and as a result, little is known about how individual cells may alter their protein secretion rates on the timescale of minutes or seconds. Here, we present a label-free technique based upon nanoplasmonic imaging, which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from single hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell and from the bursts we estimated the diffusion constant of the secreted antibodies through the extracellular media. The design also incorporates transmitted light and fluorescence microscopy capabilities for monitoring cellular morphological changes and intracellular fluorescent labels. We anticipate that this technique can be adapted as a general tool for the quantitative study of paracrine signaling in both adherent and nonadherent cell lines. PMID:23931308

Raphael, Marc P; Christodoulides, Joseph A; Delehanty, James B; Long, James P; Byers, Jeff M



Relative, label-free protein quantitation: spectral counting error statistics from nine replicate MudPIT samples.  


Nine replicate samples of peptides from soybean leaves, each spiked with a different concentration of bovine apotransferrin peptides, were analyzed on a mass spectrometer using multidimensional protein identification technology (MudPIT). Proteins were detected from the peptide tandem mass spectra, and the numbers of spectra were statistically evaluated for variation between samples. The results corroborate prior knowledge that combining spectra from replicate samples increases the number of identifiable proteins and that a summed spectral count for a protein increases linearly with increasing molar amounts of protein. Furthermore, statistical analysis of spectral counts for proteins in two- and three-way comparisons between replicates and combined replicates revealed little significant variation arising from run-to-run differences or data-dependent instrument ion sampling that might falsely suggest differential protein accumulation. In these experiments, spectral counting was enabled by PANORAMICS, probability-based software that predicts proteins detected by sets of observed peptides. Three alternative approaches to counting spectra were also evaluated by comparison. As the counting thresholds were changed from weaker to more stringent, the accuracy of ratio determination also changed. These results suggest that thresholds for counting can be empirically set to improve relative quantitation. All together, the data confirm the accuracy and reliability of label-free spectral counting in the relative, quantitative analysis of proteins between samples. PMID:20541435

Cooper, Bret; Feng, Jian; Garrett, Wesley M



Identification of Protein Network Alterations upon Retinal Ischemia-Reperfusion Injury by Quantitative Proteomics Using a Rattus norvegicus Model  

PubMed Central

Retinal ischemia is a common feature associated with several ocular diseases, including diabetic retinopathy. In this study, we investigated the effect of a retinal ischemia and reperfusion (I/R) injury on protein levels via a quantitative shotgun strategy using stable isotope dimethyl labeling combined with LC-MS/MS analysis. Based on the relative quantitation data of 1088 proteins, 234 proteins showed a greater than 1.5-fold change following I/R injury, 194 of which were up-regulated and 40 were down-regulated. Gene ontology analysis revealed that after I/R injury, there was an increase in the metabolic-process related proteins but a decline in cell communication, system process and transport-related proteins. A ribosome protein network and a secreted protein network consisting of many protease inhibitors were identified among the up-regulated proteins, despite a suppression of the mammalian target of rapamycin (mTOR) pathway following the I/R injury. A synaptic-related protein network was found to be significantly down-regulated, implicating a functional reduction of neurons following a retinal I/R injury. Our results provide new systems-biology clues for the study of retinal ischemia. PMID:25549249

Tian, Han; Wang, Leilei; Cai, Ruiqi; Zheng, Ling; Guo, Lin



Analysis of electrophoresis performance  

NASA Technical Reports Server (NTRS)

The SAMPLE computer code models electrophoresis separation in a wide range of conditions. Results are included for steady three dimensional continuous flow electrophoresis (CFE), time dependent gel and acetate film experiments in one or two dimensions and isoelectric focusing in one dimension. The code evolves N two dimensional radical concentration distributions in time, or distance down a CFE chamber. For each time or distance increment, there are six stages, successively obtaining the pH distribution, the corresponding degrees of ionization for each radical, the conductivity, the electric field and current distribution, and the flux components in each direction for each separate radical. The final stage is to update the radical concentrations. The model formulation for ion motion in an electric field ignores activity effects, and is valid only for low concentrations; for larger concentrations the conductivity is, therefore, also invalid.

Roberts, G. O.



Happy bicentennial, electrophoresis!  


A short survey of electrophoresis and a celebration of its bicentennial, with some remarkable mementos and a list of books that shaped the field. Where one also learns of a secret production plant with a huge-scale electrophoretic apparatus for skimming of latex from Hevea brasiliensis and keeping the wheels of the Ally Army running during World War II. And of cyber (mammoth) 2D gels of 1.5 x 1 m in size accommodating >12,000 spots. PMID:19938305

Righetti, Pier Giorgio



Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR  

PubMed Central

Background Accurate interpretation of quantitative PCR (qPCR) data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. coli. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in E. coli. Results In this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various E. coli strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, rrsA encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in E. coli cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes cysG, hcaT, and idnT but not rrsA and ihfB as highly invariant in two E. coli strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression. Conclusion This study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli. PMID:21513543



Protein alterations associated with pancreatic cancer and chronic pancreatitis found in human plasma using global quantitative proteomics profiling  

PubMed Central

Pancreatic cancer is a lethal disease that is difficult to diagnose at early stages when curable treatments are effective. Biomarkers that can improve current pancreatic cancer detection would have great value in improving patient management and survival rate. A large scale quantitative proteomics study was performed to search for the plasma protein alterations associated with pancreatic cancer. The enormous complexity of the plasma proteome and the vast dynamic range of protein concentration therein present major challenges for quantitative global profiling of plasma. To address these challenges, multi-dimensional fractionation at both protein and peptide levels was applied to enhance the depth of proteomics analysis. Employing stringent criteria, more than thirteen hundred proteins total were identified in plasma across 8-orders of magnitude in protein concentration. Differential proteins associated with pancreatic cancer were identified, and their relationship with the proteome of pancreatic tissue and pancreatic juice from our previous studies was discussed. A subgroup of differentially expressed proteins was selected for biomarker testing using an independent cohort of plasma and serum samples from well-diagnosed patients with pancreatic cancer, chronic pancreatitis and non-pancreatic disease controls. Using ELISA methodology, the performance of each of these protein candidates was benchmarked against CA19-9, the current gold standard for a pancreatic cancer blood test. A composite marker of TIMP1 and ICAM1 demonstrate significantly better performance than CA19-9 in distinguishing pancreatic cancer from the non-pancreatic disease controls and chronic pancreatitis controls. In addition, protein AZGP1 was identified as a biomarker candidate for chronic pancreatitis. The discovery and technical challenges associated with plasma-based quantitative proteomics are discussed and may benefit the development of plasma proteomics technology in general. The protein candidates identified in this study provide a biomarker candidate pool for future investigations. PMID:21443201

Pan, Sheng; Chen, Ru; Crispin, David A.; May, Damon; Stevens, Tyler; McIntosh, Martin; Bronner, Mary P.; Ziogas, Argyrios; Anton-Culver, Hoda; Brentnall, Teresa A.



Quantitative proteomic analysis reveals proteins involved in the neurotoxicity of marine medaka Oryzias melastigma chronically exposed to inorganic mercury.  


Mercury is a ubiquitous environmental contaminant which exerts neurotoxicity upon animals. Nevertheless, the molecular mechanisms involved in inorganic mercury neurotoxicity are unknown. We investigated protein profiles of marine medaka, chronically exposed to mercuric chloride using two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS) analysis. The mercury accumulation and ultrastructure were also examined in the brain. The results showed that mercury was significantly accumulated in the treated brain, and subsequently caused a noticeable damage. The comparison of 2D-DIGE protein profiles between the control and treatment revealed that 16 protein spots were remarkably altered in abundance, which were further submitted for MALDI-TOF-TOF MS analysis. The identified proteins indicated that inorganic mercury may cause neurotoxicity through the induction of oxidative stress, cytoskeletal assembly dysfunction and metabolic disorders. Thus, this study provided a basis for a better understanding of the molecular mechanisms involved in mercury neurotoxicity. PMID:25460752

Wang, Yuyu; Wang, Dazhi; Lin, Lin; Wang, Minghua



Qualitative and quantitative characterization of plasma proteins when incorporating traveling wave ion mobility into a liquid chromatography-mass spectrometry workflow for biomarker discovery: use of product ion quantitation as an alternative data analysis tool for label free quantitation.  


Discovery of protein biomarkers in clinical samples necessitates significant prefractionation prior to liquid chromatography-mass spectrometry (LC-MS) analysis. Integrating traveling wave ion mobility spectrometry (TWIMS) enables in-line gas phase separation which when coupled with nanoflow liquid chromatography and data independent acquisition tandem mass spectrometry, confers significant advantages to the discovery of protein biomarkers by improving separation and inherent sensitivity. Incorporation of TWIMS leads to a packet of concentrated ions which ultimately provides a significant improvement in sensitivity. As a consequence of ion packeting, when present at high concentrations, accurate quantitation of proteins can be affected due to detector saturation effects. Human plasma was analyzed in triplicate using liquid-chromatography data independent acquisition mass spectrometry (LC-DIA-MS) and using liquid-chromatography ion-mobility data independent acquisition mass spectrometry (LC-IM-DIA-MS). The inclusion of TWIMS was assessed for the effect on sample throughput, data integrity, confidence of protein and peptide identification, and dynamic range. The number of identified proteins is significantly increased by an average of 84% while both the precursor and product mass accuracies are maintained between the modalities. Sample dynamic range is also maintained while quantitation is achieved for all but the most abundant proteins by incorporating a novel data interpretation method that allows accurate quantitation to occur. This additional separation is all achieved within a workflow with no discernible deleterious effect on throughput. Consequently, TWIMS greatly enhances proteome coverage and can be reliably used for quantification when using an alternative product ion quantification strategy. Using TWIMS in biomarker discovery in human plasma is thus recommended. PMID:24397486

Daly, Charlotte E; Ng, Leong L; Hakimi, Amirmansoor; Willingale, Richard; Jones, Donald J L



Qualitative and Quantitative Characterization of Plasma Proteins When Incorporating Traveling Wave Ion Mobility into a Liquid Chromatography–Mass Spectrometry Workflow for Biomarker Discovery: Use of Product Ion Quantitation As an Alternative Data Analysis Tool for Label Free Quantitation  

PubMed Central

Discovery of protein biomarkers in clinical samples necessitates significant prefractionation prior to liquid chromatography–mass spectrometry (LC–MS) analysis. Integrating traveling wave ion mobility spectrometry (TWIMS) enables in-line gas phase separation which when coupled with nanoflow liquid chromatography and data independent acquisition tandem mass spectrometry, confers significant advantages to the discovery of protein biomarkers by improving separation and inherent sensitivity. Incorporation of TWIMS leads to a packet of concentrated ions which ultimately provides a significant improvement in sensitivity. As a consequence of ion packeting, when present at high concentrations, accurate quantitation of proteins can be affected due to detector saturation effects. Human plasma was analyzed in triplicate using liquid-chromatography data independent acquisition mass spectrometry (LC-DIA-MS) and using liquid-chromatography ion-mobility data independent acquisition mass spectrometry (LC-IM-DIA-MS). The inclusion of TWIMS was assessed for the effect on sample throughput, data integrity, confidence of protein and peptide identification, and dynamic range. The number of identified proteins is significantly increased by an average of 84% while both the precursor and product mass accuracies are maintained between the modalities. Sample dynamic range is also maintained while quantitation is achieved for all but the most abundant proteins by incorporating a novel data interpretation method that allows accurate quantitation to occur. This additional separation is all achieved within a workflow with no discernible deleterious effect on throughput. Consequently, TWIMS greatly enhances proteome coverage and can be reliably used for quantification when using an alternative product ion quantification strategy. Using TWIMS in biomarker discovery in human plasma is thus recommended. PMID:24397486



Protocols of two-dimensional difference gel electrophoresis to investigate mechanisms of toxicity.  


In recent years, several global omics technologies have been increasingly used to better understand the molecular mechanisms of drug toxicity. Two-dimensional difference gel electrophoresis (2D-DIGE) is a large-scale proteomics high-resolution gel-based quantitative method widely used to detect protein expression alterations after drug treatment. The 2D-DIGE technology is based on the labeling of proteins with different fluorescent dyes, allowing the separation of different samples on the same gel with the use of an internal standard, thus reducing the complexity of spot pattern comparison and providing a reliable method applied to toxicology studies for the detection of modulated proteins in targeted organs. PMID:20972754

Com, Emmanuelle; Gruhler, Albrecht; Courcol, Martine; Gautier, Jean-Charles



Applications of space-electrophoresis in medicine. [for cellular separations in molecular biology  

NASA Technical Reports Server (NTRS)

The nature of electrophoresis is reviewed and potential advances realizable in the field of biology and medicine from a space electrophoresis facility are examined. The ground-based applications of electrophoresis: (1) characterization of an ionized species; (2) determination of the quantitative composition of a complex mixture; and (3) isolation of the components of a mixture, separation achieved on the basis of the difference in transport rates is reviewed. The electrophoresis of living cells is considered, touching upon the following areas: the separation of T and B lymphocytes; the genetic influence on mouse lymphocyte mobilities; the abnormal production of specific and monoclonal immunoproteins; and the study of cancer. Schematic diagrams are presented of three types of electrophoresis apparatus: the column assembly for the static electrophoresis experiment on the Apollo-Soyuz mission, the continuous flow apparatus used in the same mission and a miniaturized electrophoresis apparatus.

Bier, M.



Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition.  


The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, we previously described proteome remodeling during Drosophila egg activation. Here, we describe our quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey, to our knowledge, of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. We show that Muskelin, found to be up-regulated during oocyte maturation, is required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. We find down-regulation of this class of proteins to be mediated partially by APC/C(CORT), a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C). PMID:25349405

Kronja, Iva; Whitfield, Zachary J; Yuan, Bingbing; Dzeyk, Kristina; Kirkpatrick, Joanna; Krijgsveld, Jeroen; Orr-Weaver, Terry L



Quantitation of Serum Prostate-specific Membrane Antigen by a Novel Protein Biochip Immunoassay Discriminates Benign from Malignant Prostate Disease1  

Microsoft Academic Search

The lack of a sensitive immunoassay for quantitating serum prostate- specific membrane antigen (PSMA) hinders its clinical utility as a diagnostic\\/ prognostic biomarker. An innovative protein biochip immunoassay was used to quantitate and compare serum PSMA levels in healthy men and patients with either benign or malignant prostate disease. PSMA was captured from serum by anti-PSMA antibody bound to ProteinChip

Zhen Xiao; Bao-Ling Adam; Lisa H. Cazares; John W. Davis; Paul F. Schellhammer; Enrique A. Dalmasso; George L. Wright


The use of passive haemolysis in the quantitative estimation of anti-protein antibodies  

PubMed Central

The conditions for the establishment of a method for the quantitative assay of antiprotein antibodies by the use of passive haemolysis were studied by using an anti-BSA—BSA system as a model. When haemolytic assaying of antibody was carried out under standard conditions with a fixed concentration of optimally sensitized red cells, in the presence of excess complement (10 C?H50) reproducible titrations could be performed to detect 0.12 ?g. N Ab with an error of ±10 per cent. Factors affecting the specific lysis of optimally sensitized cells were studied and some parameters fixed. The amounts of coated erythrocytes and complement and the length of time of reaction were seen to influence the final degree of lysis given by a fixed amount of antibody. The conditions of optimal sensitization of erythrocytes with protein antigen by the use of bis-diazotized benzidine (BDB) are indicated. It was found that, when conjugation was carried out at 0° with proper amounts of a standardized cell suspension, BDB and antigen, the resulting coated cells were highly sensitive to immune haemolysis and highly resistant to `spontaneous' lysis. PMID:5294634

Rangel, H.; Repka, D.



Shape-Shifting 3D Protein Microstructures with Programmable Directionality via Quantitative Nanoscale Stiffness Modulation.  


The ability to shape-shift in response to a stimulus increases an organism's survivability in nature. Similarly, man-made dynamic and responsive "smart" microtechnology is crucial for the advancement of human technology. Here, 10-30 ?m shape-changing 3D BSA protein hydrogel microstructures are fabricated with dynamic, quantitative, directional, and angle-resolved bending via two-photon photolithography. The controlled directional responsiveness is achieved by spatially controlling the cross-linking density of BSA at a nanometer lengthscale. Atomic force microscopy measurements of Young's moduli of structures indicate that increasing the laser writing distance at the z-axis from 100-500 nm decreases the modulus of the structure. Hence, through nanoscale modulation of the laser writing z-layer distance at the nanoscale, control over the cross-linking density is possible, allowing for the swelling extent of the microstructures to be quantified and controlled with high precision. This method of segmented moduli is applied within a single microstructure for the design of shape-shifting microstructures that exhibit stimulus-induced chirality, as well as for the fabrication of a free-standing 3D microtrap which is able to open and close in response to a pH change. PMID:25264141

Lee, Mian Rong; Phang, In Yee; Cui, Yan; Lee, Yih Hong; Ling, Xing Yi



A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays  

PubMed Central

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays. PMID:24022739

Helka, Blake-Joseph; Brennan, John D.



Non Linear Programming (NLP) formulation for quantitative modeling of protein signal transduction pathways.  


Modeling of signal transduction pathways plays a major role in understanding cells' function and predicting cellular response. Mathematical formalisms based on a logic formalism are relatively simple but can describe how signals propagate from one protein to the next and have led to the construction of models that simulate the cells response to environmental or other perturbations. Constrained fuzzy logic was recently introduced to train models to cell specific data to result in quantitative pathway models of the specific cellular behavior. There are two major issues in this pathway optimization: i) excessive CPU time requirements and ii) loosely constrained optimization problem due to lack of data with respect to large signaling pathways. Herein, we address both issues: the former by reformulating the pathway optimization as a regular nonlinear optimization problem; and the latter by enhanced algorithms to pre/post-process the signaling network to remove parts that cannot be identified given the experimental conditions. As a case study, we tackle the construction of cell type specific pathways in normal and transformed hepatocytes using medium and large-scale functional phosphoproteomic datasets. The proposed Non Linear Programming (NLP) formulation allows for fast optimization of signaling topologies by combining the versatile nature of logic modeling with state of the art optimization algorithms. PMID:23226239

Mitsos, Alexander; Melas, Ioannis N; Morris, Melody K; Saez-Rodriguez, Julio; Lauffenburger, Douglas A; Alexopoulos, Leonidas G



Development of an isoform-specific tandem mass spectrometry assay for absolute quantitation of maize lipid transfer proteins.  


Precise and accurate quantitation of maize grain allergens is important for seed and food industries. The major allergen in maize grain is Zea m 14, a lipid transfer protein (LTP). The B73 maize genome encodes for at least six LTPs sharing 15%-87% sequence identity to Zea m 14. Phylogenetic analysis of the maize LTP family revealed one gene that corresponds to Zea m 14 (denoted as LTPa) and two other genes sharing 43% (LTPc) and 74% (LTPb) identity with Zea m 14 that are putative homologues. Using stable isotope peptide mimics as internal standards for LTPs, we present a multiple reaction monitoring mass spectrometry approach for multiplexed, absolute quantitation of all three LTP proteins and alternative transcript models therein. To validate quantitative accuracy, a redundant peptide, simultaneously representing the two most abundant LTPs, was included. Analysis of 21 maize varieties revealed LTPa was most prominently expressed in maize grain, ranging from 9 to 32 ?g LTP/mg protein. Proteins belonging to the LTPb and LTPc gene models were also expressed but at approximately 10- and 100-fold lower levels than LTPa, respectively. The quantitative results provided by the redundant peptide show around 95% agreement with the sum of the two unique peptides, thus providing support for the LTP gene models and validating the accuracy of this method. Though not all Zea m 14-related LTPs are abundant in grain, their high sequence homology and detectable expression in maize grain signify that LTPb and LTPc are putative allergens and should be accounted for in any quantitation strategy for maize LTP allergens. PMID:25540820

Stevenson, Severin E; McClain, Scott; Thelen, Jay J



Determination of opium alkaloids in crude opium using non-aqueous capillary electrophoresis  

Microsoft Academic Search

A method for the quantitative determination of the opium alkaloids morphine, codeine, thebaine, noscapine and papaverine in crude opium and in drug preparations based on non-aqueous capillary electrophoresis has been developed. The non-aqueous mode provides high separation selectivity and new possibilities for regulating the selectivity in capillary electrophoresis. The nature of the organic solvent, the acidity of the electrolytes as

Inga Bjornsdottir; Steen Honoré Hansen



Glycation Isotopic Labeling with 13C-Reducing Sugars for Quantitative Analysis of Glycated Proteins in Human Plasma*  

PubMed Central

Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [13C6]glucose incubation to evaluate the native glycated proteins labeled with [12C6]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive approach was also tested to detect potential glycation sites under high glucose concentration. PMID:19955080

Priego-Capote, Feliciano; Scherl, Alexander; Müller, Markus; Waridel, Patrice; Lisacek, Frédérique; Sanchez, Jean-Charles



High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli.  


Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (, our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays. PMID:25146501

Saez, Natalie J; Nozach, Hervé; Blemont, Marilyne; Vincentelli, Renaud



Flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction.  


Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction. PMID:23979513

Wang, Li; Carnegie, Graeme K



Kinetic methods in capillary electrophoresis and their applications  

NASA Astrophysics Data System (ADS)

In recent years, capillary electrophoresis (CE) has been one of rapidly growing analytical techniques to study affinity interactions. Quick analysis, high efficiency, high resolving power, low sample consumption, and wide range of possible analytes make CE an indispensable tool for studies of biomolecules and, in particular, studies of their interactions. In the article, we discuss kinetic methods in CE. The spectrum of proven applications of kinetic CE methods includes: (i) measuring equilibrium and rate constants of protein-ligand interaction from a single experiment, (ii) quantitative affinity analyses of proteins, (iii) measuring temperature in CE, (iv) studying thermochemistry of affinity interactions, and (v) kinetic selection of ligands from combinatorial libraries. We demonstrate that new kinetic CE method can serve as a "Swiss army knife" in the development and utilization of oligonucleotide aptamers. Uniquely, they can facilitate selection of smart aptamers - aptamers with pre-defined binding parameters. We believe that further development of kinetic CE methods will provide a variety of methodological schemes for high-throughput screening of combinatorial libraries for affinity probes and drug candidates using CE as a universal instrumental platform.

Berezovski, Maxim V.; Okhonin, Victor; Petrov, Alex; Krylov, Sergey N.



Improved Methodical Approach for Quantitative BRET Analysis of G Protein Coupled Receptor Dimerization  

PubMed Central

G Protein Coupled Receptors (GPCR) can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V2 vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, ?2 adrenergic and CB1 cannabinoid receptors. PMID:25329164

Szalai, Bence; Hoffmann, Péter; Prokop, Susanne; Erdélyi, László; Várnai, Péter; Hunyady, László



Development and plasticity-related changes in protein expression patterns in cat visual cortex: a fluorescent two-dimensional difference gel electrophoresis approach.  


During early postnatal brain development, changes in visual input can lead to specific alteration of function and connectivity in mammalian visual cortex. In cat, this so-called critical period exhibits maximal sensory-driven adaptations around postnatal day 30 (P30), and ceases toward adulthood. We examined the molecular framework that directs age- and experience-dependent plasticity in cat visual cortex, by comparing protein expression profiles at eye opening (postnatal day 10 (P10), when experience-dependent plasticity starts), the peak of the critical period (P30), and in adulthood. Using 2-D DIGE, we performed comparisons of P10-P30 and P30-adult brain protein samples. Sixty protein spots showed statistically significant intensity changes in at least one comparison. Fifty-one spots were identified using quadrupole-TOF MS/MS or LC-MS/MS, containing 37 different proteins. The progressive increase or decrease in protein expression levels could be correlated to age-dependent postnatal brain development. Four spots containing transferrin, 14-3-3 alpha/beta and cypin, showed maximal protein expression levels at P30, thereby showing a positive correlation to critical period plasticity. Western analysis indeed revealed a clear effect of visual deprivation on cypin expression in cat visual cortex. Our results therefore demonstrate the power of 2-D DIGE as a tool toward understanding the molecular basis of nervous system development and plasticity. PMID:16739136

Van den Bergh, Gert; Clerens, Stefan; Firestein, Bonnie L; Burnat, Kalina; Arckens, Lutgarde



Static continuous electrophoresis device  

NASA Technical Reports Server (NTRS)

An apparatus is disclosed for carrying out a moving wall type electrophoresis process for separation of cellular particles. The apparatus includes a water-tight housing containing an electrolytic buffer solution. A separation chamber in the housing is defined by spaced opposed moving walls and spaced opposed side walls. Substrate assemblies, which support the moving wall include vacuum ports for positively sealing the moving walls against the substrate walls. Several suction conduits communicate with the suction ports and are arranged in the form of valleys in a grid plate. The raised land portion of the grid plat supports the substrate walls against deformation inwardly under suction. A cooling chamber is carried on the back side of plate. The apparatus also has tensioner means including roller and adjustment screws for maintaining the belts in position and a drive arrangement including an electric motor with a gear affixed to its output shaft. Electrode assemblies are disposed to provide the required electric field.

Rhodes, P. H. (inventor)



Electrophoresis experiment for space  

NASA Technical Reports Server (NTRS)

The Apollo 16 electrophoresis experiment was analyzed, demonstrating that the separation of the two different-size monodisperse latexes did indeed take place, but that the separation was obscured by the pronounced electroosmotic flow of the liquid medium. The results of this experiment, however, were dramatic since it is impossible to carry out a similar separation on earth. It can be stated unequivocally from this experiment that any electrophoretic separation will be enhanced under microgravity conditions. The only question is the degree of this enhancement, which can be expected to vary from one experimental technique to another. The low-electroosmotic-mobility coating (Z6040-MC) developed under this program was found to be suitable for a free-fluid electrophoretic separation such as the experiment designed for the ASTP flight. The problem with this coating, however, is that its permanency is limited because of the slow desorption of the methylcellulose from the coated surface.

Vanderhoff, J. W.; Micale, F. J.



Methyl jasmonate responsive proteins in Brassica napus guard cells revealed by iTRAQ-based quantitative proteomics.  


Stomata on leaf epidermis formed by pairs of guard cells control CO(2) intake and water transpiration, and respond to different environmental conditions. Stress-induced stomatal closure is mediated via an intricate hormone network in guard cells. Although methyl jasmonate (MeJA) has been intensively studied for its function in plant defense, the molecular mechanisms underlying its function in stomatal movement are not fully understood. Here we report the effects of MeJA on Brassica napus stomatal movement and H(2)O(2) production. Using the isobaric tags for relative and absolute quantitation (iTRAQ) approach, we have identified 84 MeJA-responsive proteins in B. napus guard cells. Most of the genes encoding these proteins contain jasmonate-responsive elements in the promoters, indicating that they are potentially regulated at the transcriptional level. Among the identified proteins, five protein changes after MeJA treatment were validated using Western blot analysis. The identification of the MeJA-responsive proteins has revealed interesting molecular mechanisms underlying MeJA function in guard cells, which include homeostasis of H(2)O(2) production and scavenging, signaling through calcium oscillation and protein (de)phosphorylation, gene transcription, protein modification, energy balance, osmoregulation, and cell shape modulation. The knowledge of the MeJA-responsive proteins has improved our understanding of MeJA signaling in stomatal movement, and it may be applied to crop engineering for enhanced yield and stress tolerance. PMID:22639841

Zhu, Mengmeng; Dai, Shaojun; Zhu, Ning; Booy, Aaron; Simons, Brigitte; Yi, Sarah; Chen, Sixue



Comparison of Colorimetric Assays with Quantitative Amino Acid Analysis for Protein Quantification of Generalized Modules for Membrane Antigens (GMMA).  


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. PMID:25223624

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



Electrophoresis demonstration on Apollo 16  

NASA Technical Reports Server (NTRS)

Free fluid electrophoresis, a process used to separate particulate species according to surface charge, size, or shape was suggested as a promising technique to utilize the near zero gravity condition of space. Fluid electrophoresis on earth is disturbed by gravity-induced thermal convection and sedimentation. An apparatus was developed to demonstrate the principle and possible problems of electrophoresis on Apollo 14 and the separation boundary between red and blue dye was photographed in space. The basic operating elements of the Apollo 14 unit were used for a second flight demonstration on Apollo 16. Polystyrene latex particles of two different sizes were used to simulate the electrophoresis of large biological particles. The particle bands in space were extremely stable compared to ground operation because convection in the fluid was negligible. Electrophoresis of the polystyrene latex particle groups according to size was accomplished although electro-osmosis in the flight apparatus prevented the clear separation of two particle bands.

Snyder, R. S.



Kidney cell electrophoresis, continuing task  

NASA Technical Reports Server (NTRS)

Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated to provide ground support in the form of analytical cell electrophoresis and flow cytometry. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. Cells were prepared in suspension prior to flight in electrophoresis buffer and 10% calf serum. Electrophoretic separation proceeded in electrophoresis buffer without serum in the Continuous Flow Electrophoretic Separator, and fractions were collected into sample bags containing culture medium and concentrated serum. Fractions that yielded enough progeny cells were analyzed for morphology and electrophoretic mobility distributions. It is noted that the lowest mobility fraction studied produced higher mobility progeny while the other fractions produced progeny cells with mobilities related to the fractions from which they were collected.

Todd, P. W.



Expanding the capabilities of microfluidic gradient elution moving boundary electrophoresis for complex samples.  


Gradient elution moving boundary electrophoresis (GEMBE) is a robust, continuous injection separation technique that uses electrophoresis to drive electrically charged analytes into a capillary or microfluidic channel for detection, while opposing electroosmosis and controlled variable pressure-driven flow prevent other sample components-for example, cells, proteins, or particulates in complex samples that can interfere with analysis-from entering the channel. This work expands the sample-in/answer-out analytical capabilities of GEMBE for complex samples by demonstrating the quantitative analysis of anions, implementing aqueous background electrolyte (BGE) solutions at neutral pH, and introducing the use of additives to the sample solution to optimize performance. Dirt was analyzed quantitatively, with the sole preparatory step of suspension in an aqueous BGE solution at neutral pH, for dissolved chloride, nitrite, nitrate, sulfate, and oxalate using GEMBE with capacitively-coupled contactless conductivity detection. In addition to altering the pH of the BGE solution, optimization of the analysis of dirt and whole blood was achieved using various commercially available additives. These results, taken together with previous demonstrations of GEMBE for the analysis of complex samples, underscore the uncomplicated versatility of GEMBE, facilitate effective analysis of biological complex samples using BGE solutions at physiological pH, and offer a sufficient set of techniques and tools to build a foundation for the analysis of a broad range of complex samples. PMID:21766783

Strychalski, Elizabeth A; Henry, Alyssa C; Ross, David



A simple quantitative model of macromolecular crowding effects on protein folding: Application to the murine prion protein(121-231)  

NASA Astrophysics Data System (ADS)

A model of protein folding kinetics is applied to study the effects of macromolecular crowding on protein folding rate and stability. Macromolecular crowding is found to promote a decrease of the entropic cost of folding of proteins that produces an increase of both the stability and the folding rate. The acceleration of the folding rate due to macromolecular crowding is shown to be a topology-dependent effect. The model is applied to the folding dynamics of the murine prion protein (121-231). The differential effect of macromolecular crowding as a function of protein topology suffices to make non-native configurations relatively more accessible.

Bergasa-Caceres, Fernando; Rabitz, Herschel A.



WebProAnalyst: an interactive tool for analysis of quantitative structure–activity relationships in protein families  

PubMed Central

WebProAnalyst is a web-accessible analysis tool () designed for scanning quantitative structure–activity relationships in protein families. The tool allows users to search correlations between protein activity and physicochemical characteristics (i.e. hydrophobicity or alpha-helical amphipathicity) in queried sequences. WebProAnalyst uses aligned amino acid sequences and data on protein activity (pK, Km, ED50, among others). WebProAnalyst implements methods of the known ProAnalyst package, including the multiple linear regression analysis and the sequence–activity correlation coefficient. In addition, WebProAnalyst incorporates a method based on neural networks. The WebProAnalyst reports a list of sites in protein family, the regression analysis parameters (including correlation values) for the relationships between the amino acid physicochemical characteristics in the site and the protein activity values. WebProAnalyst is useful in search of the amino acid residues that are important for protein function/activity. Furthermore, WebProAnalyst may be helpful in designing the protein-engineering experiments. PMID:15980590

Ivanisenko, Vladimir A.; Eroshkin, Alexey M.; Kolchanov, Nickolay A.



Colostrum protein uptake in neonatal lambs examined by descriptive and quantitative liquid chromatography-tandem mass spectrometry.  


Colostrum intake is a key factor for newborn ruminant survival because the placenta does not allow the transfer of immune components. Therefore, newborn ruminants depend entirely on passive immunity transfer from the mother to the neonate, through the suckling of colostrum. Understanding the importance of specific colostrum proteins has gained significant attention in recent years. However, proteomics studies of sheep colostrum and their uptake in neonate lambs has not yet been presented. The aim of this study was to describe the proteomes of sheep colostrum and lamb blood plasma, using sodium dodecyl sulfate-PAGE for protein separation and in-gel digestion, followed by liquid chromatography-tandem mass spectrometry of resulting tryptic peptides for protein identification. An isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics approach was subsequently used to provide relative quantification of how neonatal plasma protein concentrations change as an effect of colostrum intake. The results of this study describe the presence of 70 proteins in the ovine colostrum proteome. Furthermore, colostrum intake resulted in an increase of 8 proteins with important immune functions in the blood plasma of lambs. Further proteomic studies will be necessary, particularly using the selected reaction monitoring approach, to describe in detail the role of specific colostrum proteins for immune transfer to the neonate. PMID:25465637

Hernández-Castellano, Lorenzo E; Argüello, Anastasio; Almeida, André M; Castro, Noemí; Bendixen, Emøke



Quantitative model of calcium/calmodulin-dependent protein kinase II activation  

NASA Astrophysics Data System (ADS)

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key element in the calcium second messenger cascades that lead to long term potentiation (LTP) of synaptic strength. In this thesis, I have constructed kinetic models of activation of CaMKII and measured some of the unknown parameters of the model. I used the models to elucidate mechanisms of activation of CaMKII and to study the kinetics of its activation under conditions similar to those in dendritic spines.In chapter 2, I developed a new experimental method to rapidly stop the autophosphorylation reaction. I used this method to measure the catalytic turnover number of CaMKII. To quantitatively characterize CaMKII atophosphorylation in nonsaturating calcium, I also measured the autophosphorylation turnover number when CaMKII is activated by calmodulin mutants that can bind calcium ions only in either the amino or the carboxyl lobes.Previous models of CaMKII activation assumed that binding of calmodulins to individual CaMKII subunits is independent and that autophosphorylation occurs within a ring of 6 subunits. However, a recent structure of CaMKII suggests that pairs of subunits cooperate in binding calmodulin and raises the possibility that the autophosphorylation occurs within pairs of subunits. In chapter 3, I constructed a model in which CaMKII subunits cooperate in binding calmodulin. This model reconciled previous experimental results from the literature that appeared contradictory. In chapter 4, I constructed two models for CaMKII autophosphorylation, in which autophosphorylation can occur either in rings or pairs, and used them to design experiments aimed at differentiating between these possibilities. Previously published measurements and the measurements that I performed are more consistent with autophosphorylation occurring within pairs.In chapter 5, I constructed a model for simultaneous interactions among calcium, calmodulin, and CaMKII, and I used an automatic parameter search algorithm to fit the parameters for this model. I used it to characterize which of the parameters of calcium transients are critical for CaMKII activation.This modeling work is part of a continuing effort to realistically model the spatial and temporal aspects of calcium second messenger signaling in dendritic spines.

Mihalas, Stefan


Biochemical Identification of the Two Races of Radopholus similis by Polyacrylamide Gel Electrophoresis.  


Analysis of proteins of the banana and citrus race of Radopholus similis was carried out by several different types of polyacrylamide gel electrophoresis. These included standard slab gel, SDS slab gel, gradient slab gel, and two-ditnensional slab gel electrophoresis. A major band difference was detected between the two races by slab gel electrophoresis. However, several other poorly resolved but consistent hands of high molecular weight proteins near the gel origin also were considered as diagnostic. Resolution of protein bands was greatly improved by SDS and gradient slab gel electrophoresis, but no differences could be detected among the proteins resolved between the two rares with these techniques. Two-dimensional gels revealed a large number of proteins, but background staining obscured them hindering interpretation. When nematode races were reared on three different host plants, no differences in protein patterns were detected between them, indicating host preferences does not play a role in determining the types proteins occurring in these nematodes. PMID:19295815

Huettel, R N; Dickson, D W; Kaplan, D T



The development and application of a quantitative Peptide microarray based approach to protein interaction domain specificity space.  


Protein interaction domain (PID) linear peptide motif interactions direct diverse cellular processes in a specific and coordinated fashion. PID specificity, or the interaction selectivity derived from affinity preferences between possible PID-peptide pairs is the basis of this ability. Here, we develop an integrated experimental and computational cellulose peptide conjugate microarray (CPCMA) based approach for the high throughput analysis of PID specificity that provides unprecedented quantitative resolution and reproducibility. As a test system, we quantify the specificity preferences of four Src Homology 2 domains and 124 physiological phosphopeptides to produce a novel quantitative interactome. The quantitative data set covers a broad affinity range, is highly precise, and agrees well with orthogonal biophysical validation, in vivo interactions, and peptide library trained algorithm predictions. In contrast to preceding approaches, the CPCMAs proved capable of confidently assigning interactions into affinity categories, resolving the subtle affinity contributions of residue correlations, and yielded predictive peptide motif affinity matrices. Unique CPCMA enabled modes of systems level analysis reveal a physiological interactome with expected node degree value decreasing as a function of affinity, resulting in minimal high affinity binding overlap between domains; uncover that Src Homology 2 domains bind ligands with a similar average affinity yet strikingly different levels of promiscuity and binding dynamic range; and parse with unprecedented quantitative resolution contextual factors directing specificity. The CPCMA platform promises broad application within the fields of PID specificity, synthetic biology, specificity focused drug design, and network biology. PMID:25135669

Engelmann, Brett W; Kim, Yohan; Wang, Miaoyan; Peters, Bjoern; Rock, Ronald S; Nash, Piers D



Copolymers For Capillary Gel Electrophoresis  


This invention relates to an electrophoresis separation medium having a gel matrix of at least one random, linear copolymer comprising a primary comonomer and at least one secondary comonomer, wherein the comonomers are randomly distributed along the copolymer chain. The primary comonomer is an acrylamide or an acrylamide derivative that provides the primary physical, chemical, and sieving properties of the gel matrix. The at least one secondary comonomer imparts an inherent physical, chemical, or sieving property to the copolymer chain. The primary and secondary comonomers are present in a ratio sufficient to induce desired properties that optimize electrophoresis performance. The invention also relates to a method of separating a mixture of biological molecules using this gel matrix, a method of preparing the novel electrophoresis separation medium, and a capillary tube filled with the electrophoresis separation medium.

Liu, Changsheng (State College, PA); Li, Qingbo (State College, PA)



A capillary electrophoresis-tandem mass spectrometry methodology for the determination of non-protein amino acids in vegetable oils as novel markers for the detection of adulterations in olive oils.  


A new analytical methodology based on capillary electrophoresis-mass spectrometry (CE-MS(2)) is presented in this work, enabling the identification and determination of six non-protein amino acids (ornithine, ?-alanine, GABA, alloisoleucine, citrulline and pyroglutamic acid) in vegetable oils. This methodology is based on a previous derivatization with butanol and subsequent separation using acidic conditions followed by on-line coupling to an ion trap analyzer for MS(2) detection established through an electrospray-coaxial sheath flow interface. The electrophoretic and interface parameters were optimized obtaining the separation of all compounds in less than 15 min and with resolutions higher than 5. The proposed method was validated by assessing its accuracy, precision (RSD<7% for corrected peak areas), LODs and LOQs (between 0.04-0.19 ng/g and 0.06-0.31 ng/g, respectively) and linearity range (R(2)>0.99), and it was used in order to identify the selected non-protein amino acids in soybean oils, sunflower oils, corn oils and extra virgin olive oils. MS(2) experiments performed the fingerprint fragmentation of these compounds allowing to corroborate ornithine and alloisoleucine in seed oils but not in olive oils. The method was applied to identify and quantify olive oil adulterations with soybean oil detecting in a single run the amino acids in mixtures up to 2% (w/w). The results showed a high potential in using these compounds as novel markers for the detection of adulterations of extra virgin olive oils with seed oils. Thus, the developed method could be considered a simple, rapid and reliable method for the quality evaluation of extra virgin olive oil permitting its authentication. PMID:21306720

Sánchez-Hernández, Laura; Marina, Maria Luisa; Crego, Antonio L



Assessment of ERCC1 and XPF Protein Expression Using Quantitative Immunohistochemistry in Nasopharyngeal Carcinoma Patients Undergoing Curative Intent Treatment  

SciTech Connect

Purpose: We sought to evaluate the prognostic/predictive value of ERCC1 and XPF in patients with nonmetastatic nasopharyngeal carcinoma (NPC) treated with curative intent. Methods and Materials: ERCC1 and XPF protein expression was evaluated by immunofluorescence combined with automated quantitative analysis (AQUA) using the FL297 and 3F2 antibodies, respectively. ERCC1 and XPF protein expression levels were correlated with clinical outcomes. Results: Patient characteristics were as follows: mean age 52 years (range, 18-85 years), 67% male, 72% Karnofsky performance status (KPS) ?90%, World Health Organization (WHO) type 1/2/3 = 12%/28%/60%, stage III/IV 65%. With a median follow-up time of 50 months (range, 2.9 to 120 months), the 5-year overall survival (OS) was 70.8%. Median standardized nuclear AQUA scores were used as cutpoints for ERCC1 (n=138) and XPF (n=130) protein expression. Agreement between dichotomized ERCC1 and XPF scores was high at 79.4% (kappa = 0.587, P<.001). Neither biomarker predicted locoregional recurrence, DFS, or OS after adjustment for age and KPS, irrespective of stratification by stage, WHO type, or treatment. Conclusions: Neither ERCC1 nor XPF, analyzed by quantitative immunohistochemistry using the FL297 and 3F2 antibodies, was prognostic or predictive in this cohort of NPC patients.

Jagdis, Amanda [Department of Internal Medicine, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia (Canada)] [Department of Internal Medicine, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia (Canada); Phan, Tien [Department of Radiation Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada) [Department of Radiation Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Faculty of Medicine, University of Calgary, Calgary, Alberta (Canada); Klimowicz, Alexander C. [Department of Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada) [Department of Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Faculty of Medicine, University of Calgary, Calgary, Alberta (Canada); Laskin, Janessa J. [Department of Medical Oncology, British Columbia Cancer Agency–Vancouver, Vancouver, British Columbia (Canada) [Department of Medical Oncology, British Columbia Cancer Agency–Vancouver, Vancouver, British Columbia (Canada); Faculty of Medicine, University of British Columbia, Vancouver, British Columbia (Canada); Lau, Harold Y. [Department of Radiation Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada) [Department of Radiation Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Faculty of Medicine, University of Calgary, Calgary, Alberta (Canada); Petrillo, Stephanie K. [Functional Tissue Imaging Unit, Translational Research Laboratory, Tom Baker Cancer Centre, Calgary, Alberta (Canada)] [Functional Tissue Imaging Unit, Translational Research Laboratory, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Siever, Jodi E. [Department of Biostatistics, Public Health Innovation and Decision Support Population and Public Health, Alberta Health Services, Calgary, Alberta (Canada)] [Department of Biostatistics, Public Health Innovation and Decision Support Population and Public Health, Alberta Health Services, Calgary, Alberta (Canada); Thomson, Thomas A. [Department of Pathology, British Columbia Cancer Agency–Vancouver, Vancouver, British Columbia (Canada) [Department of Pathology, British Columbia Cancer Agency–Vancouver, Vancouver, British Columbia (Canada); Faculty of Medicine, University of British Columbia, Vancouver, British Columbia (Canada); Magliocco, Anthony M. [Department of Pathology, Tom Baker Cancer Centre, Calgary, Alberta (Canada) [Department of Pathology, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Faculty of Medicine, University of Calgary, Calgary, Alberta (Canada); Hao, Desirée, E-mail: [Department of Medical Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada) [Department of Medical Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Faculty of Medicine, University of Calgary, Calgary, Alberta (Canada)



Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens*  

PubMed Central

Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection. PMID:22311637

Dengjel, Jörn; Høyer-Hansen, Maria; Nielsen, Maria O.; Eisenberg, Tobias; Harder, Lea M.; Schandorff, Søren; Farkas, Thomas; Kirkegaard, Thomas; Becker, Andrea C.; Schroeder, Sabrina; Vanselow, Katja; Lundberg, Emma; Nielsen, Mogens M.; Kristensen, Anders R.; Akimov, Vyacheslav; Bunkenborg, Jakob; Madeo, Frank; Jäättelä, Marja; Andersen, Jens S.



Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action  

PubMed Central

Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments. PMID:23776212

Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J.; Zhang, Huiming; Tao, W. Andy; Zhu, Jian-Kang



Label-Free Quantitative Mass Spectrometry Reveals a Panel of Differentially Expressed Proteins in Colorectal Cancer  

PubMed Central

To identify potential biomarkers involved in CRC, a shotgun proteomic method was applied to identify soluble proteins in three CRCs and matched normal mucosal tissues using high-performance liquid chromatography and mass spectrometry. Label-free protein profiling of three CRCs and matched normal mucosal tissues were then conducted to quantify and compare proteins. Results showed that 67 of the 784 identified proteins were linked to CRC (28 upregulated and 39 downregulated). Gene Ontology and DAVID databases were searched to identify the location and function of differential proteins that were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, and so on. Among the differentially expressed proteins, tropomyosin-3 (TPM3), endoplasmic reticulum resident protein 29 (ERp29), 18?kDa cationic antimicrobial protein (CAMP), and heat shock 70?kDa protein 8 (HSPA8) were verified to be upregulated in CRC tissue and seven cell lines through western blot analysis. Furthermore, the upregulation of TPM3, ERp29, CAMP, and HSPA8 was validated in 69 CRCs byimmunohistochemistry (IHC) analysis. Combination of TPM3, ERp29, CAMP, and HSPA8 can identify CRC from matched normal mucosal achieving an accuracy of 73.2% using IHC score. These results suggest that TPM3, ERp29, CAMP, and HSPA8 are great potential IHC diagnostic biomarkers for CRC.

Fan, Nai-Jun; Gao, Jiang-Ling; Liu, Yan; Song, Wei; Zhang, Zhan-Yang; Gao, Chun-Fang



Biomacromolecular quantitative structure-activity relationship (BioQSAR): a proof-of-concept study on the modeling, prediction and interpretation of protein-protein binding affinity.  


Quantitative structure-activity relationship (QSAR), a regression modeling methodology that establishes statistical correlation between structure feature and apparent behavior for a series of congeneric molecules quantitatively, has been widely used to evaluate the activity, toxicity and property of various small-molecule compounds such as drugs, toxicants and surfactants. However, it is surprising to see that such useful technique has only very limited applications to biomacromolecules, albeit the solved 3D atom-resolution structures of proteins, nucleic acids and their complexes have accumulated rapidly in past decades. Here, we present a proof-of-concept paradigm for the modeling, prediction and interpretation of the binding affinity of 144 sequence-nonredundant, structure-available and affinity-known protein complexes (Kastritis et al. Protein Sci 20:482-491, 2011) using a biomacromolecular QSAR (BioQSAR) scheme. We demonstrate that the modeling performance and predictive power of BioQSAR are comparable to or even better than that of traditional knowledge-based strategies, mechanism-type methods and empirical scoring algorithms, while BioQSAR possesses certain additional features compared to the traditional methods, such as adaptability, interpretability, deep-validation and high-efficiency. The BioQSAR scheme could be readily modified to infer the biological behavior and functions of other biomacromolecules, if their X-ray crystal structures, NMR conformation assemblies or computationally modeled structures are available. PMID:23306464

Zhou, Peng; Wang, Congcong; Tian, Feifei; Ren, Yanrong; Yang, Chao; Huang, Jian



Quantitative statistical analysis of standard and human blood proteins from liquid chromatography, electrospray ionization, and tandem mass spectrometry.  


It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion m/z and intensity data in the mascot generic files from LC-ESI-MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC-ESI-MS/MS data from human blood. PMID:22316523

Bowden, Peter; Thavarajah, Thanusi; Zhu, Peihong; McDonell, Mike; Thiele, Herbert; Marshall, John G



08-136.doc; 3/10/09 Electrophoresis, a technique which uses electrical  

E-print Network

08-136.doc; 3/10/09 Electrophoresis, a technique which uses electrical energy to separate molecules such as proteins or nucleic acids by their size, structure and electrical charge, is frequently used in laboratories. Electrophoresis work poses potential electrical, chemical and physical safety hazards. ELECTRICAL

Ford, James


Mass spectrometry-based quantitative proteomic analysis of Salmonella enterica serovar Enteritidis protein expression upon exposure to hydrogen peroxide  

PubMed Central

Background Salmonella enterica, a common food-borne bacterial pathogen, is believed to change its protein expression profile in the presence of different environmental stress such as that caused by the exposure to hydrogen peroxide (H2O2), which can be generated by phagocytes during infection and represents an important antibacterial mechanism of host cells. Among Salmonella proteins, the effectors of Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) are of particular interest since they are expressed during host infection in vivo and are important for invasion of epithelial cells and for replication in organs during systemic infection, respectively. However, the expression profiles of these proteins upon exposure to H2O2 or to host cells in vivo during the established phase of systemic infection have not been extensively studied. Results Using stable isotope labeling coupled with mass spectrometry, we performed quantitative proteomic analysis of Salmonella enterica serovar Enteritidis and identified 76 proteins whose expression is modulated upon exposure to H2O2. SPI-1 effector SipC was expressed about 3-fold higher and SopB was expressed approximately 2-fold lower in the presence of H2O2, while no significant change in the expression of another SPI-1 protein SipA was observed. The relative abundance of SipA, SipC, and SopB was confirmed by Western analyses, validating the accuracy and reproducibility of our approach for quantitative analysis of protein expression. Furthermore, immuno-detection showed substantial expression of SipA and SipC but not SopB in the late phase of infection in macrophages and in the spleen of infected mice. Conclusions We have identified Salmonella proteins whose expression is modulated in the presence of H2O2. Our results also provide the first direct evidence that SipC is highly expressed in the spleen at late stage of salmonellosis in vivo. These results suggest a possible role of SipC and other regulated proteins in supporting survival and replication of Salmonella under oxidative stress and during its systemic infection in vivo. PMID:20529336



Quantitative analysis of complex protein mixtures using isotope-coded affinity tags  

Microsoft Academic Search

We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source.

Steven P. Gygi; Beate Rist; Scott A. Gerber; Frantisek Turecek; Michael H. Gelb; Ruedi Aebersold



Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system  

Microsoft Academic Search

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that

Marino DiFranco; Patricia Ñeco; Joana Capote; Pratap Meera; Julio L. Vergara



Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry.  


To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers for metastatic progression, we have developed a highly metastatic variant of human MDA-MB-435 breast cancer cell line through in vivo stepwise selection of pulmonary metastatic cells caused by parental MDA-MB-435 cells in the athymic mice. Comparative proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein spots were reproducibly altered more than three-fold between the selected variant and its parental counterpart. Eleven differentially expressed protein spots were identified with high confidence using SEQUEST with uninterpreted tandem mass raw data. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (TPM1), TPM2, TPM3, TPM4, 14-3-3 protein epsilon, and tumor protein D54 were up-regulated in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parental counterpart. Differential expression was confirmed for four proteins including PDX6, CRAB, TPM4, and HSP60 by real-time quantitative PCR and Western blotting analysis in our model. Immunohistochemical analysis in 80 breast cancer donors demonstrated a significant association of TPM4 (p = 0.002), HSP60 (p = 0.001), PDX6 (p = 0.002) but not CRAB (p = 0.113) staining with the presence of lymph node metastasis. In addition, TPM4 staining was also associated with clinical stage (p = 0.000), but no significant association was found between TPM4, PDX6, CRAB, and HSP60 expression and tumor size, hormone receptor, and HER-2 status (p > 0.05). The functional implication of these identified proteins was also discussed. These proteomic data are valuable and informative for understanding breast cancer metastasis and searching for potential markers for metastatic progression. PMID:16637015

Li, Da-Qiang; Wang, Lei; Fei, Fei; Hou, Yi-Feng; Luo, Jian-Min; Zeng, Rong; Wu, Jiong; Lu, Jin-Song; Di, Gen-Hong; Ou, Zhou-Luo; Xia, Qi-Chang; Shen, Zhen-Zhou; Shao, Zhi-Min



A quantitative model of odor deactivation based on the redox shift of the pheromone-binding protein im moth antennae.  


Recent in vitro experiments with homogenates of isolated olfactory hairs of Antheraea polyphemus suggest that the pheromone-binding protein (PBP) is involved not only in pheromone solubilization and transport but also in pheromone deactivation. PBP occurs in a reduced form with one or two disulfide bridges (PBP(red)) and in the oxidized form with three bridges (PBP(ox)). From kinetic experiments it was concluded that the pheromone is first bound to PBP(red). This complex activates the receptor molecules and then turns into the oxidized form which--according to our working hypothesis--is unable to activate further receptor molecules. Apparently, the pheromone bound to the PBP (both forms) is protected from enzymatic degradation into nonexcitatory metabolites. A quantitative kinetic model of pheromone deactivation was developed (in collaboration with J. Thorson, Oxford) in which the receptor molecules are considered to act as enzymes catalyzing the redox shift of the binding protein. PMID:10049225

Kaissling, K E



Quantitative description of the parameters affecting the adsorption behaviour of globular proteins.  


The adsorption behaviour of proteins depends significantly on their molecular properties and system conditions. To study this relation, the effect of relative exposed hydrophobicity, protein concentration and ionic strength on the adsorption rate and adsorbed amount is studied using ?-lactoglobulin, ovalbumin and lysozyme. The curves of surface elastic modulus versus surface pressure of all three proteins, under different conditions (i.e. concentration and ionic strength) superimposed. This showed that the interactions between the adsorbed proteins are similar and that the adsorbed proteins retain their native state. In addition, the adsorption rate (kadsorb) was shown to scale with the relative hydrophobicity and ionic strength. Moreover, the adsorbed amount was shown to be dependent on the protein charge and the ionic strength. Based on these results, a model is proposed to predict the maximum adsorbed amount (?max). The model approximates the adsorbed amount as a close-packed monolayer using a hard-sphere approximation with an effective protein radius which depends on the electrostatic repulsion. The theoretical adsorbed amount was in agreement with experimental ?max (±10%). PMID:25280607

Delahaije, Roy J B M; Gruppen, Harry; Giuseppin, Marco L F; Wierenga, Peter A



Silver Staining of 2D Electrophoresis Gels Thierry Rabilloud  

E-print Network

and 10 times higher than colloidal Coomassie Blue. However, the first silver staining protocols wereSilver Staining of 2D Electrophoresis Gels Thierry Rabilloud CEA-DSV-iRTSV/LCBM and UMR CNRS Head: Silver staining #12;i. Abstract Silver staining is used to detect proteins after electrophoretic

Paris-Sud XI, Université de


Metabolic labeling of plant cell cultures with K15NO3 as a tool for quantitative analysis of proteins and metabolites  

Microsoft Academic Search

Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that address biological questions beyond the establishment of protein lists. Here, we propose the use of 15N-KNO3 as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population. Proteins from such metabolically labeled culture are distinguishable from

Wolfgang R Engelsberger; Alexander Erban; Joachim Kopka; Waltraud X Schulze



A Porous SiO2 Interferometric Biosensor for Quantitative Determination of Protein Interactions: Binding of Protein A to Immunoglobulins Derived from Different Species  

PubMed Central

Determination of kinetic and thermodynamic protein binding constants using interferometry from a porous Si Fabry-Perot layer is presented. A protein A capture probe is adsorbed within the pores of an oxidized porous Si matrix, and binding of immunoglobulin G (IgG) antibodies derived from different species is investigated. The relative protein A/IgG binding affinity is human > rabbit > goat, in agreement with literature values. The equilibrium binding constant (Ka) for human IgG binding to surface-immobilized protein A is determined to be 3.0 ± 0.5 × 107 M-1 using an equilibrium Langmuir model. Kinetic rate constants are calculated to be kd = 2.1 ± 0.2 × 10-4 s-1 and ka = 1.2 ± 0.4 × 104 M-1s-1 using non-linear least squares analysis, yielding an equilibrium binding constant of Ka = 5.5 ± 1.5 × 107 M-1. Both steady state and time-dependent measurements yield equilibrium binding constants that are consistent with literature values. Kinetic rate constants determined through non-linear least squares analysis are also in agreement with protein A/IgG binding on a surface. Dosing with a high concentration of analyte leads to deviations from ideal binding behavior, interpreted in terms of restricted analyte diffusion within the porous SiO2 matrix. It is shown that the diffusion limitations can be minimized if the kinetic measurements are performed at low analyte concentrations or under conditions in which the protein A capture probe is not saturated with analyte. Potential limitations of the use of porous SiO2 interferometers for quantitative determination of protein binding constants are discussed. PMID:17194157

Schwartz, Michael P.; Alvarez, Sara D.; Sailor, Michael J.



Quantitation of Total Protein using OPA Total protein content is a measurement common to many applications in basic science and  

E-print Network

that eliminate many of the problems associated with the traditional absorbance-based colorimetric methods protocol to a 96-well microplate based format. Over the years, many different absorbance-based colorimetric-binding protein assays were developed; the most commonly used being the method described by Bradford (3

Raizada, Manish N.


Quantitative Proteomic Profiling Reveals Differentially Regulated Proteins in Cystic Fibrosis Cells  

PubMed Central

The most prevalent cause of cystic fibrosis (CF) is the deletion of a phenylalanine residue at position 508 in CFTR (?F508-CFTR) protein. The mutated protein fails to fold properly, is retained in the endoplasmic reticulum via the action of molecular chaperones, and is tagged for degradation. In this study, the differences in protein expression levels in CF cell models were assessed using a systems biology approach aided by the sensitivity of MudPIT proteomics. Analysis of the differential proteome modulation without a priori hypotheses has the potential to identify markers that have not yet been documented. These may also serve as the basis for developing new diagnostic and treatment modalities for CF. Several novel differentially expressed proteins observed in our study are likely to play important roles in the pathogenesis of CF and may serve as a useful resource for the CF scientific community. PMID:24818864

Rauniyar, Navin; Gupta, Vijay; Balch, William E.; Yates, John R.



Inhibition of Bacterial Conjugation by Phage M13 and Its Protein g3p: Quantitative Analysis and Model  

PubMed Central

Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes. PMID:21637841

Lin, Abraham; Jimenez, Jose; Derr, Julien; Vera, Pedro; Manapat, Michael L.; Esvelt, Kevin M.; Villanueva, Laura; Liu, David R.; Chen, Irene A.



Quantitative Atlas of Membrane Transporter Proteins: Development and Application of a Highly Sensitive Simultaneous LC\\/MS\\/MS Method Combined with Novel In-silico Peptide Selection Criteria  

Microsoft Academic Search

Purpose  To develop an absolute quantification method for membrane proteins, and to construct a quantitative atlas of membrane transporter\\u000a proteins in the blood–brain barrier, liver and kidney of mouse.\\u000a \\u000a \\u000a \\u000a Methods  Mouse tissues were digested with trypsin, and mixed with stable isotope labeled-peptide as a quantitative standard. The amounts\\u000a of transporter proteins were simultaneously determined by liquid chromatography–tandem mass spectrometer (LC\\/MS\\/MS).\\u000a \\u000a \\u000a \\u000a Results  The target

Junichi Kamiie; Sumio Ohtsuki; Ryo Iwase; Ken Ohmine; Yuki Katsukura; Kazunari Yanai; Yumi Sekine; Yasuo Uchida; Shingo Ito; Tetsuya Terasaki



A quantitative proteomic approach to identify significantly altered protein networks in the serum of patients with lymphangioleiomyomatosis (LAM).  


Lymphangioleiomyomatosis (LAM) is a rare and progressive cystic lung condition affecting approximately 3.4-7.5/million women, with an average lag time between symptom onset and diagnosis of upwards of 4 years. The aim of this work was to identify altered proteins in LAM serum which may be potential biomarkers of disease. Serum from LAM patient volunteers and healthy control volunteers were pooled and analysis carried out using quantitative 4-plex iTRAQ technology. Differentially expressed proteins were validated using ELISAs and pathway analysis was carried out using Ingenuity Pathway Analysis. Fourteen proteins were differentially expressed in LAM serum compared to control serum (p<0.05). Further screening validated the observed differences in extracellular matrix remodelling proteins including fibronectin (30% decrease in LAM, p?=?0.03), von Willebrand Factor (40% reduction in LAM, p?=?0.03) and Kallikrein III (25% increase in LAM, p?=?0.03). Pathway networks elucidated the relationships between the ECM and cell trafficking in LAM. This study was the first to highlight an imbalance in networks important for remodelling in LAM, providing a set of novel potential biomarkers. These understandings may lead to a new effective treatment for LAM in the future. PMID:25133674

Banville, Nessa; Burgess, Janette K; Jaffar, Jade; Tjin, Gavin; Richeldi, Luca; Cerri, Stefania; Persiani, Elisa; Black, Judith L; Oliver, Brian G



Micro-size polyacrylamide gel electrophoresis system  

NASA Astrophysics Data System (ADS)

The development and characterization of a micro-size two-dimensional polyacrylamide gel electrophoresis system is described. Some of the techniques which have evolved with use of the system are also discussed. This apparatus has unique features which provide advantages over other small scale units. Up to ten first- and second-dimension gels can be processed simultaneously with excellent resolution of protein regions. Consistent reproducibility is possible from protein samples as small as 400 ng and individual protein regions as small as 1 pg can be visualized by silver staining of the two-dimensional gels. Similar sensitivities are achieved in autoradiographs of 3H-labeled proteins extracted from the nuclei of cultured cells. The application of this system in conjunction with flow cytometric examination of nuclear DNA and electrostatic cell sorting of specific cell nuclei to provide homogeneous sample populations, allows subtle variations in isotope incorporation in proteins to be detected; whereas many times in generalized tissue samples these changes are masked. Also, these techniques elucidate the effects of external stimuli (chemicals, drugs, or environment) on protein synthesis and phosphorylation for analyses and comparison. Fabrication drawings are available upon request.

Hinson, W. G.; Pipkin, J. L.; Anson, J. F.; Casciano, D. A.; Burns, E. R.



Fluid flow electrophoresis in space  

NASA Technical Reports Server (NTRS)

Four areas relating to free-flow electrophoresis in space were investigated. The first was the degree of improvement over earthbound operations that might be expected. The second area of investigation covered the problems in developing a flowing buffer electrophoresis apparatus. The third area of investigation was the problem of testing on the ground equipment designed for use in space. The fourth area of investigation was the improvement to be expected in space for purification of biologicals. The results of some ground-based experiments are described. Other studies included cooling requirements in space, fluid sealing techniques, and measurement of voltage drop across membranes.

Griffin, R. N.



Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis  

PubMed Central

Background The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids. Results After initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide) and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine), showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed. Conclusion This study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis. PMID:15941475

Churchward, Matthew A; Butt, R Hussain; Lang, John C; Hsu, Kimberly K; Coorssen, Jens R



Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities  

PubMed Central

We established a novel technique for differential activity-based gel electrophoresis (DABGE) of lipolytic enzymes from two different biological samples. For this purpose, a set of three fluorescent suicide inhibitors was developed. These probes possess the same substrate analogous structures but carry different cyanine dyes (Cy2b, Cy3, and Cy5) as reporter fluorophores. For comparison of enzyme profiles, two samples are individually labeled with a different probe followed by mixing, gel electrophoresis, fluorescence imaging, and identification of the tagged proteins by MS/MS. Protocols for quantitative determination of active enzymes were developed on the basis of lipolytic proteomes that had been admixed with defined amounts of known lipases and esterases. A detailed analysis of the fluorescence intensities showed that the found enzyme ratios very closely reflected the relative amounts of the labeled enzymes that were used for spiking. The DABGE method was used to compare the lipolytic proteomes of brown and white adipose tissue showing specific enzyme patterns of both samples. This study represents the first application of this technology for comparative analysis of lipases and esterases. Further applications of this technique can be expected to provide entirely new information on lipid enzymology in health and disease with high precision. PMID:19282273

Morak, Maria; Schmidinger, Hannes; Krempl, Peter; Rechberger, Gerald; Kollroser, Manfred; Birner-Gruenberger, Ruth; Hermetter, Albin



High efficiency and quantitatively reproducible protein digestion by trypsin-immobilized magnetic microspheres.  


Aldehyde- and NHS-activated magnetic microspheres were used to immobilize trypsin (CHO-trypsin and NHS-trypsin), and their performance for protein digestion was evaluated by reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry using an LTQ Orbitrap Velos instrument. NHS-trypsin provided greater sequence coverage and identified more peptides for the digestion of bovine serum albumin. A 1-min digestion at room temperature using the immobilized trypsin also identified more peptides (96±6 vs. 48±1) and produced higher sequence coverage (90±2% vs. 75±2%) than traditional free trypsin digestion for 12h at 37 °C. Analysis of 15 nM (0.001 mg/mL) BSA digested by NHS-trypsin in 1 min at room temperature consistently yielded one detected peptide; 150 nM BSA generated 22 peptides. Peptide intensity and protein spectral count were used to evaluate the run-to-run digestion reproducibility of NHS-trypsin with a three-protein-mixture. Three high intensity peptides for each protein generated intensity ratios from 0.70 to 1.09 and spectral count ratios from 0.78 to 1.18. Finally, RAW 264.7 cell lysates were digested by NHS-trypsin for 10 min and 30 min at room temperature, 604 and 697 protein groups, respectively, were identified by RPLC-ESI-MS/MS, with a peptide false discovery rate of less than 1%. Digestion by solution phase trypsin for 12h at 37 °C resulted in identification of 878 protein groups. PMID:22176736

Sun, Liangliang; Li, Yihan; Yang, Ping; Zhu, Guijie; Dovichi, Norman J



Quantitative analysis of backbone motion in proteins using MAS solid-state NMR spectroscopy  

Microsoft Academic Search

We present a comprehensive analysis of protein dynamics for a micro-crystallin protein in the solid-state. Experimental data\\u000a include 15N T\\u000a 1 relaxation times measured at two different magnetic fields as well as 1H–15N dipole, 15N CSA cross correlated relaxation rates which are sensitive to the spectral density function J(0) and are thus a measure of T\\u000a 2 in the solid-state.

Veniamin Chevelkov; Uwe Fink; Bernd Reif



Peptide production and decay rates affect the quantitative accuracy of protein cleavage isotope dilution mass spectrometry (PC-IDMS).  


No consensus has been reached on the proper time to add stable-isotope labeled (SIL) peptides in protein cleavage isotope dilution mass spectrometry workflows. While quantifying 24 monolignol pathway enzymes in the xylem tissue of Populus trichocarpa, we compared the protein concentrations obtained when adding the SIL standard peptides concurrently with the enzyme or after quenching of the digestion (i.e. postdigestion) and observed discrepancies for nearly all tryptic peptides investigated. In some cases, greater than 30-fold differences were observed. To explain these differences and potentially correct for them, we developed a mathematical model based on pseudo-first-order kinetics to account for the dynamic production and decay (e.g. degradation and precipitation) of the native peptide targets in conjunction with the decay of the SIL peptide standards. A time course study of the digests confirmed the results predicted by the proposed model and revealed that the discrepancy between concurrent