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1

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

2010-04-01

2

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

PubMed Central

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 2 D 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 hr 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

2009-01-01

3

Protein electrophoresis - serum  

MedlinePLUS

This lab test measures the types of protein in the fluid (serum) part of a blood sample. Other electrophoresis tests that measure proteins in the serum include: Immunoelectrophoresis Immunofixation Globulin electrophoresis

4

Capillary Electrophoresis of Proteins  

Microsoft Academic Search

1.1. Capillary Electrophoresis of Proteins Capillary electrophoresis (CE) is a separation technique that combines aspects of both gel electrophoresis and high performance liquid chromatography (HPLC). As is the case for gel electrophoresis, the separation in CE is based upon differential migration in an electrical field. Like HPLC, the detection of the migrating sample analytes may be monitored on-line or postcolumn\\/capillary

Mark Strege

5

Quantitative analysis of microRNA in blood serum with protein-facilitated affinity capillary electrophoresis.  

PubMed

MicroRNAs play an important role in gene regulation and disease etiology and are blood-based biomarkers of diseases. Here, we describe a protein-facilitated affinity capillary electrophoresis (ProFACE) method for ultra-sensitive direct miRNA detection as low as 300,000 molecules in 1 mL of blood serum, using single-stranded DNA binding protein (SSB) and double-stranded RNA binding protein (p19) as separation enhancers. This method utilizes either the selective binding of SSB to a fluorescent single-stranded DNA/RNA probe or the binding of p19 to miRNA-RNA probe duplex. PMID:24026701

Berezovski, Maxim V; Khan, Nasrin

2013-01-01

6

Optimization of coomassie staining for quantitative densitometry of soybean storage proteins in gradient gel electrophoresis  

Microsoft Academic Search

Soybean (Glycine maxL. Merr., cv. Dare) protein subunits were separated by gradient gel electrophoresis and analyzed by two-dimensional densitometry\\u000a with computer-aided volume integration. Significant differences in the time required to achieve equilibrium staining with\\u000a Coomassie Blue were revealed among the various polypeptides. Bands corresponding to lipoxygenase reached staining equilibrium\\u000a in 2.7 h, whereas longer periods were required for polypeptides of

Prachuab Kwanyuen; Richard F. Wilson

2000-01-01

7

Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis.  

PubMed

We investigated the ability of gradient elution moving boundary electrophoresis (GEMBE) with capacitively coupled contactless conductivity detection (C(4) D) to assay total protein concentration using the bicinchoninic acid (BCA) reaction. We chose this format because GEMBE-C(4) D behaves as a concentration dependent detection system, unlike optical methods that also rely on pathlength (due to Beer's law). This system tolerates proteins well compared with other capillary electrophoretic methods, allowing the capillary to be reused without coatings or additional hydroxide wash steps. The typical reaction protocol was modified by reducing the pH slightly from 11.25 to 9.4, which enabled elimination of tartrate from the reagents. We estimated that copper (I) could be detected at approximately 3.0 ?mol/L, which agrees with similar GEMBE and CZE systems utilizing C(4) D. Under conditions similar to the BCA "micro method" assay, we determined the LOD for three common proteins (insulin, BSA, and bovine gamma globulin) and found that they agree well with the existing spectroscopic detection methods. Further, we investigated how long reaction times impact the LOD and found that the conversion was proportional to log(time). This indicated that little sensitivity is gained by extending the reaction past 1 h. Hence, GEMBE provides an alternative platform for total protein assays while maintaining the excellent sensitivity of the optical-based methods. PMID:24648165

Kralj, Jason G; Munson, Matthew S; Ross, David

2014-07-01

8

Quantitative analysis of microRNA in blood serum with protein-facilitated affinity capillary electrophoresis.  

PubMed

MicroRNAs (miRNAs) are small (?22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding protein (SSB) and double-stranded RNA binding protein (p19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of p19 to miRNA-RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by p19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30?000 miRNA molecules in 1 mL of serum as a potential source of nai?ve miRNAs. PMID:21714529

Khan, Nasrin; Cheng, Jenny; Pezacki, John Paul; Berezovski, Maxim V

2011-08-15

9

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.

2014-01-01

10

Quantitative Analysis of Human Milk Oligosaccharides by Capillary Electrophoresis  

Microsoft Academic Search

Human milk oligosaccharides may have important biological activities. 1 We developed a sensitive, convenient, quantitative method for the routine study of sialylated (acidic, negatively charged) oligosaccharides in large numbers of milk samples. Capillary electrophoresis (CE) with detection at 205 nm was sensitive to the femtomole level and could resolve and quantify nine acidic oligosaccharides in milk, ranging from tri- to

David S. Newburg; Zuojun Shen; Christopher D. Warren

11

Using Gel Electrophoresis To Illustrate Protein Diversity and Isoelectric Point.  

ERIC Educational Resources Information Center

Demonstrates the differences in protein structures by focusing on isoelectric point with an experiment that is observable under certain pH levels in gel electrophoresis. Explains the electrophoresis procedure and reports results of the experiments. (YDS)

Browning, Mark; Vanable, Joseph

2002-01-01

12

Quantitative Analysis of Corn Zeins by Capillary Electrophoresis  

Microsoft Academic Search

Cereal Chem. 74(6):766-770 Kjeldahl analysis is commonly used to measure zein proteins in corn maize (N ? 5.7) with no attempt to eliminate contribution from other nitrogen sources. In this study, dry milled corn was extracted with 70% ethanol or 0.1N NaOH and the zein content of the extract measured using capillary electrophoresis in the presence of sodium dodecyl sulfate.

Nicholas Parris; Leland Dickey; James Craig

1997-01-01

13

INTERVAL CARRIER FREE ELECTROPHORESIS FOR HIGH RESOLUTION PROTEIN PURIFICATION  

Microsoft Academic Search

Interval free flow zone electrophoresis is a new mode of free flow zone electrophoresis (FFZE), which facilitates purification of proteins and other molecular substances at very high resolution. It can be performed in the commercially available free flow electrophoresis (FFE) apparatus Octopus. The specimens are loaded and unloaded as usual with the help of a thin buffer film flowing between

Johann Bauer; Gerhard Weber

1998-01-01

14

Analysis of proteins in solution using affinity capillary electrophoresis.  

PubMed

Analysis of protein interactions by means of capillary electrophoresis (CE) has unique challenges and rewards. The choice of analysis conditions, especially involving electrophoresis buffers, are crucial and not universal for protein analysis. If conditions for analysis can be worked out, it is possible to utilize CE quantitatively and qualitatively to characterize protein-ligand binding involving unmodified molecules in solution and taking place under physiological conditions. This chapter deals with the most important practical considerations in capillary electrophoretic affinity approaches, affinity CE (ACE). The text emphasizes the most critical factors for successful analyses and has application examples illustrating various types of information offered by ACE-based studies. Also included are step-by-step accounts of the two main classes of experimental design: the pre-equilibration ACE (in the form of CE-frontal analysis (CE-FA)) and mobility shift ACE together with examples of their use. The ACE approaches for binding assays of proteins should be considered when the biological material is scarce, when any kind of labeling is not possible or desired, when the interacting molecules are the same size and when rapid and simple method development is a priority. PMID:18826064

Heegaard, Niels H H; Schou, Christian; Ostergaard, Jesper

2008-01-01

15

Studies on the Serum Proteins IV. The Dye-Binding of Purified Serum Proteins Separated by Continuous-Flow Electrophoresis  

Microsoft Academic Search

HE FRACTIONATION OF SERUM PROTEINS by paper electrophoresis, first described in 1949 (1), has had ever-widening clinical application dur- ing the past decade. Discrepancies between quantitative analyses by paper electrophoresis and by the older, moving-boundary technics stimulated the development of conversion factors to render compara- ble the results obtained by the 2 methods (2-8). These conversion fac- tors have been

F. William Sunderman

16

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

PubMed

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

2013-07-01

17

Protein concentration by precipitation with pyrogallol red prior to electrophoresis.  

PubMed

The pyrogallol red protein assay (Clinical Chemistry 1986, 32, 1551-1554) is based upon formation of a blue protein-dye complex in the presence of molybdate under acidic conditions. However, centrifugation of the assay mixture results in loss of color yield and precipitation of the protein-dye complex which can be recovered and resolubilized to achieve protein concentration prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The method has been evaluated relative to trichloroacetic acid (TCA) precipitation for recovery and electrophoresis of commercial protein and peptide molecular weight markers. Precipitation with pyrogallol red-molybdate (PRM) gives better and more uniform recovery of both proteins and peptides as compared to TCA. The lower limit of PRM precipitation is similar to TCA and corresponds to 1 microgram protein per mL assay mixture. This is equivalent to 100 microL of 10 micrograms/mL protein using the standard protein assay or 1 microgram/mL protein using a modified assay incorporating a fivefold concentrate of the dye reagent. Application of the method is demonstrated by concentration of urinary proteins. The method is simple and economic and useful for conserving trace amounts of precious sample as it allows recovery of protein for electrophoresis following protein assay. PMID:7737088

Marshall, T; Abbott, N J; Fox, P; Williams, K M

1995-01-01

18

Isolation of membrane protein complexes by blue native electrophoresis.  

PubMed

Blue native PAGE is a discontinuous electrophoretic system that allows the separation of membrane protein complexes in a native, enzymatically active state with high resolution. Membrane protein complexes are solubilized by neutral, nondenaturing detergents like n-dodecyl-beta-D-maltoside. After addition of Coomassie G250 that binds to the surface of the proteins, separation of the negatively charged complexes according to molecular mass is possible. After electrophoresis the structure and function of the isolated protein complexes can be investigated. PMID:18369880

Reisinger, Veronika; Eichacker, Lutz A

2008-01-01

19

Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation  

Microsoft Academic Search

A strength of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is its ability to resolve and investigate the abundance of several thousand proteins in a single sample. This enables identification of the major proteins in a tissue or subcellular fraction by mass spectrometric methods. In addition, 2D PAGE can be used to compare quantities of proteins in related samples, such as

Kathryn S Lilley; Azam Razzaq; Paul Dupree

2002-01-01

20

Differences in the spatial and quantitative reproducibility between two second-dimensional gel electrophoresis systems.  

PubMed

Two-dimensional polyacrylamide gel electrophoresis (PAGE), together with 2-D gel electrophoresis (GE) analysis software, is a common technique to analyze a complex proteome. In order to accurately locate the differentially expressed proteins in human pituitary macroadenoma tissues in our long-term research program to clarify the molecular mechanisms of macroadenoma formation, a reproducible separation system is needed. An immobilized pH-gradient dry gel-strip (IPG strip) has been extensively used for first-dimensional isoelectric focusing (IEF), and has achieved a high degree of reproducibility in the IEF direction. For the second dimension (SDS-PAGE), different types of gel systems are available, including horizontal vs. vertical gel systems, and gradient vs. constant-percentage gels. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm), and a typical vertical system is the Dodeca system, which analyzes up to 12 gels at a time, using usually a single-concentration gel (190 x 205 x 1 mm). The present study evaluated the spatial and quantitative reproducibility of the two systems for the separation of the complex human pituitary proteome. PDQuest software was used to analyze the digitized gel-image data, and SPSS statistical software was used to analyze the data. The results demonstrated a high percentage (>99%) of protein-spot matches within each electrophoretic system. The Dodeca gel system demonstrated better between-gel reproducibility for spot position, higher resolution in the Sodium dodecyl sulfate (SDS)-PAGE direction, lower gel background, better spot quality, and higher reproducibility of the spot volume. PMID:12783460

Zhan, Xianquan; Desiderio, Dominic M

2003-06-01

21

Capillary Electrophoresis-Based Immunoassays: Principles & Quantitative Applications  

PubMed Central

The use of capillary electrophoresis as a tool to conduct immunoassays has been an area of increasing interest over the last decade. This approach combines the efficiency, small sample requirements, and relatively high speed of CE with the selectivity of antibodies as binding agents. This review examines the various assay formats and detection modes that have been reported for these assays, along with some representative applications. Most CE immunoassays in the past have employed homogeneous methods in which the sample and reagents are allowed to react in solution. These homogeneous methods have been conducted as both competitive binding immunoassays and as non-competitive binding immunoassays. Fluorescent labels are most commonly used for detection in these assays, but enzyme labels have also been utilized for such work. Some additional work has been performed in CE immunoassays with heterogeneous methods in which either antibodies or an analog of the analyte is immobilized to a solid support. These heterogeneous methods can be used for the selective isolation of analytes prior to their separation by CE or to remove a given species from a sample/reagent mixture prior to analysis by CE. These CE immunoassays can be used with a variety of detection modes, such as fluorescence, UV/visible absorbance, chemiluminescence, electrochemical measurements, mass spectrometry, and surface plasmon resonance.

Moser, Annette C.; Hage, David S.

2013-01-01

22

Efficient Quantitative Information Extraction from PCR-RFLP Gel Electrophoresis Images  

Microsoft Academic Search

For the purpose of PCR-RFLP analysis, as in the case of human papillomavirus (HPV) typing, quantitative information needs to be extracted from images resulting from one-dimensional gel electrophoresis by associating the image intensity with the concentration of biological material at the corresponding position on a gel matrix. However, the background intensity of the image stands in the way of quantifying

Christos Maramis; Anastasios Delopoulos

2010-01-01

23

Chapter 8 Quantitation of Protein  

Microsoft Academic Search

The measurement of protein concentration in an aqueous sample is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction

James E. Noble; Marc J. A. Bailey

2009-01-01

24

Quantitation and characterization of rat tissue metallothioneins by gel electrophoresis  

SciTech Connect

A discontinuous gradient gel electrophoretic system was developed to quantitate and characterize metallothionein (MT) in rat tissue. Vertical slab separating gels (1.5 mm x 14 cm x 12 cm) consisted of a linear polyacrylamide gradient 7.5 to 30% T and 5% Bis. The stacking gels (3% T and 20% Bis) were photopolymerized using riboflavin as the catalyst. Liver cytosols were prepared from rats which received (i.p.) various amounts of Zn (5 mg/kg BW) or Cd (2.5 mg/kg BW). Purified MT was prepared by gel filtration and DEAE ion-exchange chromatography. Cytosols were heated (80/sup 0/C, 2 min) and centrifuged to obtain a supernatant. An appropriate amount of supernatant and various amounts of MT standard were electrophoresed (constant current, 20 mA per slab) for 9 hours. Gels were stained with Commassie Blue (R-250, 0.25%) for 12 hours and destained. Gels were scanned by densitometer and peaks heights were determined. Significantly linear standard curves (..mu..g MT vs. peak height) were established for both MTI and MTII. (Cd, Zn)-MTI migrated slower than Zn-MTI while mobilities for both (Cd, Zn)- and Zn-MTII were the same. The accumulation of MTI was consistently less than MTII in liver from both Zn- and Cd-injected rats. Their results suggest that electrophoretic analysis is an excellent system not only for quantitation but also for characterization of MT in rat tissue.

Lin, L.Y.; McCormick, C.C.

1986-03-05

25

Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin.  

PubMed

The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms. PMID:24120174

Marie, Anne-Lise; Przybylski, Cédric; Gonnet, Florence; Daniel, Régis; Urbain, Rémi; Chevreux, Guillaume; Jorieux, Sylvie; Taverna, Myriam

2013-10-24

26

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.

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

2011-01-01

27

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.

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

2008-01-01

28

Abnormal protein patterns in blood serum and cerebrospinal fluid detected by capillary electrophoresis  

Microsoft Academic Search

Capillary zone electrophoresis and high-resolution agarose gel electrophoresis were compared to detect protein components in serum and cerebrospinal fluid of patients. Both electrophoretic methods proved to be useful for detection of protein abnormalities (e.g., mono- and oligoclonal bands) in biological fluids, but capillary electrophoresis offered several important advantages, such as sample application without preliminary concentration, lack of staining procedures, and

Mariella Ivanova; Elena Tzvetanova; Veska Jetcheva; Ferenc Kilár

2002-01-01

29

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.

1996-10-01

30

Protein separation by electrophoresis in a nonsieving amphoteric medium.  

PubMed

A numerical model has been used to study the separation of protein mixtures by zone electrophoresis in a nonsieving amphoteric medium. An amphoteric buffer fixes the pH of the solution close to its isoelectric point, where the buffer molecules are unchanged: they thus contribute very little to the conductivity of the solution. This means that high field strengths can be used for rapid separation without sacrificing resolution. The numerical study shows that in this process the band spreading that can reduce resolution is essentially due to differences in migration rate between protein-rich and low-protein zones: both differences in solution conductivity and in protein mobility are involved. Rules for judging the buffer capacity of amphoteric molecules are presented and it is shown how, for a given protein, the effectiveness of this technique varies with the range of pH in which it is applied. PMID:8832182

Blanco, S; Clifton, M J; Joly, J L; Peltre, G

1996-06-01

31

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 ..mu..m) 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.

1989-05-01

32

Quantitative analysis and chromatographic fingerprinting for the quality evaluation of Scutellaria baicalensis Georgi using capillary electrophoresis  

Microsoft Academic Search

Quantitative analysis and chromatographic fingerprinting for the quality evaluation of a Chinese herb Scutellaria baicalensis Georgi using capillary electrophoresis (CE) technique was developed. The separation was performed with a 50.0cm (42.0cm to the detector window)×75?m i.d. fused-silica capillary, and the CE fingerprint condition was optimized using the combination of central composite design and multivariate analysis. The optimized buffer system containing

Ke Yu; Yifei Gong; Zhongying Lin; Yiyu Cheng

2007-01-01

33

Method for quantitating cholesterol in subfractions of serum lipoproteins separated by gradient gel electrophoresis  

Microsoft Academic Search

Extensive heterogeneity in particle size distribution of serum lipoproteins of baboons was resolved by a procedure that combined\\u000a Sudan black B prestaining, polyacrylamide gradient gel electrophoresis (GGE), and quantitative densitometry. Each densitometric\\u000a scan represented a continuous distribution of the relative amount of cholesterol in a serum sample, as a function of the lipoprotein\\u000a particle size. For analytical purposes, each scan

M.-L. Cheng; C. M. Kammerer; W. F. Lowe; B. Dyke; J. L. VandeBerg

1988-01-01

34

Quantitative aspects of rare earth metal determinations using capillary electrophoresis with indirect absorbance detection  

SciTech Connect

The practical utility of capillary zone electrophoresis with indirect absorbance detection is examined for the separation and quantitation of rare earth metals. Various imidazole derivatives are investigated as to their suitability as running buffer (displaceable) detection ions with {alpha}-hydroxyisobutyric acid functioning as a chelating agent to enhance separations. Parameters important for quantitative analysis, such as limits of detection, relative standard deviation of peak areas, efficiency, resolution, peak shape and linear dynamic range are presented. The influence of sample matrix, method of injection, and background ion identity on these parameters are investigated and discussed.

Colburn, B.A.; Starnes, S.D.; Sepaniak, M.J. [Univ. of Tennessee, Knoxville, TN (United States)] [and others

1995-04-01

35

Photopatterned free-standing polyacrylamide gels for microfluidic protein electrophoresis.  

PubMed

Designed for compatibility with slab-gel polyacrylamide gel electrophoresis (PAGE) reagents and instruments, we detail development of free-standing polyacrylamide gel (fsPAG) microstructures supporting electrophoretic performance rivalling that of microfluidic platforms. For the protein electrophoresis study described here, fsPAGE lanes are comprised of a sample reservoir and contiguous separation gel. No enclosed microfluidic channels are employed. The fsPAG devices (120 ?m tall) are directly photopatterned atop of and covalently attached to planar polymer or glass surfaces. Leveraging the fast <1 h design-prototype-test cycle - significantly faster than mold based fabrication techniques - we optimize the fsPAG architecture to minimize injection dispersion for rapid (<1 min) and short (1 mm) protein separations. The facile fabrication and prototyping of the fsPAGE provides researchers a powerful tool for developing custom analytical assays. We highlight the utility of assay customization by fabricating a polyacrylamide gel with a spatial pore-size distribution and demonstrate the resulting enhancement in separation performance over a uniform gel. Further, we up-scale from a unit separation to an array of 96 concurrent fsPAGE assays in 10 min run time driven by one electrode pair. The fsPAG array layout matches that of a 96-well plate to facilitate integration of the planar free standing gel array with multi-channel pipettes while remaining compatible with conventional slab-gel PAGE reagents, such as staining for label-free protein detection. Notably, the entire fsPAGE workflow from fabrication, to operation, and readout uses readily available materials and instruments - making this technique highly accessible. PMID:23609800

Duncombe, Todd A; Herr, Amy E

2013-06-01

36

Protein Electrophoresis in the Biology Classroom Using "Safe" Gels.  

ERIC Educational Resources Information Center

Describes the use of electrophoresis in the high school utilizing two new gels that are easy to pour, do not require degassing, can be used on a horizontal gel electrophoresis, and are not neurotoxic or carcinogenic health hazards. Large diagrams and written instructions are used to present the protocol of setting up the electrophoresis. (PR)

Atkins, Thomas

1991-01-01

37

Quantitation of pyrimidine dimer contents of nonradioactive deoxyribonucleic acid by electrophoresis in alkaline agarose gels  

SciTech Connect

We have developed a method of quantitating the pyrimidine dimer content of nonradioactive DNAs. DNA samples are treated with the UV-endonuclease from Micrococcus luteus and then separated according to molecular weight by electrophoresis on alkaline agarose gels. From their migration relative to known molecular weight standards, their median molecular weight and thus the number of dimers per DNA molecule in each sample can be calculated. Results of action spectra for dimer formation in T7 bacteriophage measured by this method agree well with action spectra for T7 killing. In addition, the method gives dimer yields in good agreement with those obtained by others using alkaline sucrose gradient sedimentation.

Sutherland, B.M.; Shih, A.G.

1983-02-15

38

Quantitative determination of vasicine and vasicinone in Adhatoda vasica by high performance capillary electrophoresis.  

PubMed

A new method of capillary electrophoresis was developed for the quantitative determination of vasicine and vasicinone from Adhatoda vasica (L.) Nees. The electrophoretic separation was performed using a 47 cm x 50 microm ID (38.5 cm effective length) fused silica capillary. The samples were injected by pressure for 3 s at 50 mbar and the running voltage was 19 kV at the injector end of the capillary. The capillary temperature was maintained at 40 degrees C. The separation of the two alkaloids has been achieved within 11 min with good repeatability. The method was validated in terms of reproducibility, linearity, accuracy and applied for the quantitative determination of vasicine and vasicinone in A. vasica plant samples/extracts. Parameters affecting the resolution such as pH, temperature, organic modifier, buffer concentration and capillary dimensions were reported. PMID:18271297

Avula, B; Begum, S; Ahmed, S; Choudhary, M I; Khan, I A

2008-01-01

39

Quantitative Experimental Determination of Primer-Dimer Formation Risk by Free-Solution Conjugate Electrophoresis  

PubMed Central

DNA barcodes are short, unique ssDNA primers that “mark” individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis (FSCE) approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer-barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 basepairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive basepairs formed, yet non-consecutive basepairs did not create stable dimers even when 20 out of 30 possible basepairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation.

Desmarais, Samantha M.; Leitner, Thomas; Barron, Annelise E.

2012-01-01

40

Evaluation of serum protein separation by capillary electrophoresis: prospective analysis of 1000 specimens  

Microsoft Academic Search

To critically assess the method of capillary electrophoresis (CE) we examined 1000 prospective serum samples submitted for protein electrophoresis by both high-resolution agarose gel electrophoresis (HRAGE) and CE. CE was performed using a 72 cm (50 cm to detector) × 50 ?m I.D. fused-silica capillary with detection of absorbance at 200 nm. The 1000 samples examined contained 362 monoclonal paraproteins

Margaret A. Jenkins; Elena Kulinskaya; Helen D. Martin; Michael D. Guerin

1995-01-01

41

Compatibility of interspecific Manihot crosses presaged by protein electrophoresis .  

PubMed

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

2010-01-01

42

Capillary electrophoresis methods for the determination of covalent polyphenol-protein complexes.  

PubMed

The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein-polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (-)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein-polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine. PMID:21400190

Trombley, John D; Loegel, Thomas N; Danielson, Neil D; Hagerman, Ann E

2011-09-01

43

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

2004-01-01

44

Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins.  

PubMed

Solubilization of membrane proteins for two-dimensional electrophoresis (2DE) is very difficult. In this study, we report the use of 1,2-diheptanoyl-sn-glycero-3-phosphatdiyl choline (DHPC) as a detergent to solubilize integral membrane proteins for 2DE. Rat ventricular microsomal fractions enriched with sarco(endo)plasmic reticulum (SR) membrane proteins were used as a model system. Compatibility of DHPC with a high concentration of urea increases the solubility of proteins compared with sulphobetaines or ASB-14. Peptide mass analysis assisted in the identification of key SR membrane proteins including SR Ca(2+) ATPase and other membrane proteins, which have not previously been reported on 2DE. These results suggest that DHPC is a better detergent for solubilizing membrane proteins and may be useful in generating proteomic maps for most complex organelles including SR. PMID:14715292

Babu, Gopal J; Wheeler, Debra; Alzate, Oscar; Periasamy, Muthu

2004-02-01

45

THERMAL DETECTION OF DNA AND PROTEINS DURING GEL ELECTROPHORESIS  

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.

R. JOHNSTON

2000-08-01

46

Quantitative high-throughput measurement of gene expression with sub-zeptomole sensitivity by capillary electrophoresis.  

PubMed

Microarray technologies have provided the ability to monitor the expression of whole genomes rapidly. However, concerns persist with regard to quantitation and reproducibility, and the detection limits for individual genes in particular arrays are generally unknown. This article describes a semiautomated PCR-based technology, Q-RAGE, which rapidly provides measurements of mRNA abundance with extremely high sensitivity using fluorescent detection of specific products separated by capillary electrophoresis. A linear relationship between template concentration and fluorescent signal can be demonstrated down to template concentrations in the low aM region, corresponding to approximately 0.04 zmol (24 molecules) per reaction. The technique is shown to be quantitative over five orders of magnitude of template concentration, and average mRNA abundances of approximately 0.01 molecule per cell can be detected. A single predefined set of 320 primers provides 90-95% coverage of all eukaryotic genomes. Analysis of a set of 19 p53-regulated genes in untreated cultures of normal human epithelial cells, derived from three different tissues, revealed a 600-fold range of apparent constitutive expression levels. For most of the genes assayed, good correlations were observed among the expression levels in normal mammary, bronchial, and epidermal epithelial cells. PMID:16125665

Spyres, Lea; Gaddis, Sally; Bedford, Ella; Arantes, Stacey; Liburd, Nikki; Powell, K Leslie; Thames, Howard; Mitchell, David; Walborg, Earl; Rouabhia, Mahmoud; Aldaz, C Marcelo; MacLeod, Michael C

2005-10-15

47

Minimizing adsorption of histidine-tagged proteins for the study of protein-deoxyribonucleic acid interactions by kinetic capillary electrophoresis.  

PubMed

Affinity interactions between DNA and proteins play a crucial role in many cellular processes. Kinetic Capillary Electrophoresis is a highly efficient tool for kinetic and equilibrium studies of protein-DNA interactions. Recombinant proteins, which are typically used for in vitro studies of protein-DNA interactions, are often expressed with a His tag to aid in their purification. In this work, we study how His tags affect Kinetic Capillary Electrophoresis analysis of protein-DNA interactions. We found that the addition of a His tag can increase or decrease protein adsorption to a bare-silica capillary wall, dependent on the protein. For Kinetic Capillary Electrophoresis measurements, it is essential to have as little protein adsorption as possible. We screened a number of capillary coatings to reduce adsorption of the His-tagged DNA mismatch repair protein MutS to the capillary wall and found that UltraTrol LN was the most effective coating. The effectiveness of the coating was confirmed with the prevention of adsorption of His-tagged fat mass and obesity-associated protein. Under typical conditions, the coating reduced protein adsorption to a level at which accurate Kinetic Capillary Electrophoresis analysis of protein-DNA interactions was possible. We further used Kinetic Capillary Electrophoresis to study how the His tag affected Kd of protein-DNA interactions for the MutS protein. Using UltraTrol LN, we found that the effect of the His tag was insignificant. PMID:24275486

Liyanage, Ruchi; Krylova, Svetlana M; Krylov, Sergey N

2013-12-27

48

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

1977-01-01

49

Segmentation of Protein Spots in 2D Gel Electrophoresis Images with Watersheds Using Hierarchical Threshold  

Microsoft Academic Search

\\u000a 2D Gel Electrophoresis (2DGE) image is the most widely used method for the isolation of the objective protein by comparative\\u000a analysis of the protein spot pattern in the gel plane. The process of protein analysis is the method, which compares the protein\\u000a pattern that is spread in the gel plane with the contrast group and finds interesting protein spot by

Youngho Kim; Jung-ja Kim; Yonggwan Won; In Yongho

2003-01-01

50

Identification of intrinsically disordered proteins by a special 2D electrophoresis.  

PubMed

Intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure under physiological conditions. They constitute a significant fraction of various proteomes and have significant roles in key cellular processes. Here we report the development of a two-dimensional electrophoresis technique for their de novo recognition and characterization. This technique consists of the combination of native and 8 M urea electrophoresis of heat-treated proteins where IDPs are expected to run into the diagonal of the gel, whereas globular proteins either precipitate upon heat treatment or unfold and run off the diagonal in the second dimension. PMID:22821526

Tantos, Agnes; Tompa, Peter

2012-01-01

51

Evaluation of total protein content in tears of dogs by polyacrylamide gel disk electrophoresis.  

PubMed

Concentration of total proteins was measured and sodium dodecyl sulfate-polyacrylamide gel disk electrophoresis was performed on tear and plasma samples obtained from 26 healthy dogs, and the results were compared. Mean +/- SEM concentration of total proteins in tears was 0.63 +/- 0.04 g/dl, and significant effects of age or gender were not found. The protein composition of tears in dogs was complex, and bands from light and heavy chains of immunoglobulins were identified by electrophoresis. PMID:1586012

Barrera, R; Jiménez, A; López, R; Mañé, M C; Rodríguez, J F; Molleda, J M

1992-04-01

52

Capillary electrophoresis coupled to fluorescence spectroscopy for protein characterisation  

Microsoft Academic Search

Proteins are essential molecules in all living organisms. Their involvement in numerous biological processes has led to the development of protein-based medicines (biopharmaceuticals). For good understanding of the properties and function of endogenous proteins and biopharmaceuticals, extensive protein characterisation is required. This involves assessment of features such as purity, heterogeneity, stability, activity and conformation of the protein. For instance, an

B. J. de Kort

2012-01-01

53

Bence-Jones protein - quantitative  

MedlinePLUS

Immunoglobulin light chains - urine; Urine Bence-Jones protein ... Bence-Jones proteins are a part of regular antibodies, called light chains. Sometimes, when your body makes too many antibodies, the ...

54

[Urinary protein electrophoresis, which analysis to select? A simplified interpretation].  

PubMed

Permanent proteinuria is an early marker of the kidney dysfunction. Tracking by urinary strip, imposes a precise quantification by the laboratory. In front of the difficulties of urine collection during 24 hours, protein determination can be carried out on the urine of a miction and can be expressed as g per g of creatinine (uPCR). We analysed the impact of the expression of the proteinuria in g/L (uP) as compared to uPCR on the urinary electrophoretic profiles. A revision and a simplification of this in practice clinical interpretation is proposed. The proteinuria of 696 in-patients was quantified on an Olympus AU2700. The urinary electophoretic profiles (SDS-AGE) were interpreted by two biologists. uP and uPCR are well correlated (r=0.847, p< 0.0001). Data agreed for proteinuria > 1 g/L but concordance was obtained only in 74% of the subjects and 55% of the pathological patients. A repetition of the analyses is suggested. The interpretative diagram suggested with simplified comments improved interpretation. We advise an interpretation by two biologists. In conclusion, interpretation of the urinary electrophoretic profile rests on the rate of total proteinuria. Expression of the proteinuria as g/g of creatinine must be associated with the expression in g/L because of the analytical conditions. The SDS-AGE Technique does not allow the identification of the monoclonal compound but allows a quantitative follow-up under treatment and especially an early tracking of the type of renal dysfunction. PMID:24342788

Camaré, Caroline; Caussé, Elizabeth

2013-01-01

55

Electrophoresis studies for determining wheat–soy protein interactions in dough and bread  

Microsoft Academic Search

The interactions between soy and wheat proteins after dough mixing and bread baking were studied. Protein extractions from gluten and breadcrumbs elaborated with mixtures of wheat flour and enzyme-active full-fat, heat-treated full-fat, and enzyme-active defatted soy flours, and commercial isolated soy proteins were analyzed by electrophoresis. Different buffer systems with specific chemical action on proteins were used to investigate the

Pablo D. Ribotta; Alberto Edel León; Gabriela T. Pérez; María Cristina Añón

2005-01-01

56

Detection of serum proteins by native polyacrylamide gel electrophoresis using Blue Sepharose CL6B-containing stacking gels  

Microsoft Academic Search

Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and interferes with the resolution of other proteins, especially ?-antitrypsin which has mobility that is very similar to that of albumin. We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels containing Blue Sepharose CL-6B,

Haruhiro Muratsubaki; Kaoru Satake; Yasuhisa Yamamoto; Keiichiro Enomoto

2002-01-01

57

A quantitative structure property relationship study of electrophoretic mobility of analytes in capillary zone electrophoresis.  

PubMed

A quantitative structure property relationship (QSPR) is proposed to calculate the electrophoretic mobility of analytes in capillary zone electrophoresis. The proposed model employs logarithm of the electrophoretic mobility (ln micro) as dependent variable and partial charge (PQ), surface area (V(2/3)), total energy (TE), heat of formation (DeltaH(f)) and molecular refractivity (MR) as independent variables whose calculated using AM1 (Austin model 1) semi-empirical quantum mechanics method by HyperChem 7.0 software. The general form of the model is: ln micro =K(0)+K(1)PQ+K(2)V(2/3)+K(3)TE+K(4)DeltaH(f)+K(5)MR, where K(0)-K(5) are the model constants computed using a least-square method. The applicability of the model on real mobility data has been studied employing five experimental data sets of beta-blockers, benzoate derivatives, non-steroidal anti-inflammatory drugs, sulfonamides and amines in different buffers. The accuracy of the model is assessed using absolute average relative deviation (AARD) and the overall AARD value. The obtained AARD for the sets studied are 1.0 (N=10), 2.1 (N=26), 0.8 (N=11), 0.6 (N=13) and 2.7% (N=18), respectively, and the overall AARD is 1.4%. The model is cross-validated using one leave out technique and the obtained overall AARD is 1.8%. To further investigate on the applicability of the proposed model, the prediction capability of the model is evaluated by employing a minimum number of six experimental data points as training set, and predicting the mobility of other data points using trained models. The obtained overall AARD (for 48 predicted data points) is 5.6%. PMID:12927104

Jouyban, A; Yousefi, B H

2003-07-01

58

Pneumatic microvalve-based hydrodynamic sample injection for high-throughput, quantitative zone electrophoresis in capillaries.  

PubMed

A hybrid microchip/capillary electrophoresis (CE) system was developed to allow unbiased and lossless sample loading and high-throughput repeated injections. This new hybrid CE system consists of a poly(dimethylsiloxane) (PDMS) microchip sample injector featuring a pneumatic microvalve that separates a sample introduction channel from a short sample loading channel, and a fused-silica capillary separation column that connects seamlessly to the sample loading channel. The sample introduction channel is pressurized such that when the pneumatic microvalve opens briefly, a variable-volume sample plug is introduced into the loading channel. A high voltage for CE separation is continuously applied across the loading channel and the fused-silica capillary separation column. Analytes are rapidly separated in the fused-silica capillary, and following separation, high-sensitivity MS detection is accomplished via a sheathless CE/ESI-MS interface. The performance evaluation of the complete CE/ESI-MS platform demonstrated that reproducible sample injection with well controlled sample plug volumes could be achieved by using the PDMS microchip injector. The absence of band broadening from microchip to capillary indicated a minimum dead volume at the junction. The capabilities of the new CE/ESI-MS platform in performing high-throughput and quantitative sample analyses were demonstrated by the repeated sample injection without interrupting an ongoing separation and a linear dependence of the total analyte ion abundance on the sample plug volume using a mixture of peptide standards. The separation efficiency of the new platform was also evaluated systematically at different sample injection times, flow rates, and CE separation voltages. PMID:24865952

Kelly, Ryan T; Wang, Chenchen; Rausch, Sarah J; Lee, Cheng S; Tang, Keqi

2014-07-01

59

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.

2013-01-01

60

Quantitative in vivo measurements using microdialysis on-line with capillary zone electrophoresis  

Microsoft Academic Search

A system which couples microdialysis with capillary zone electrophoresis (CZE) on-line is used to monitor ascorbate and lactate in the caudate nucleus of rat brain. On-line interface of microdialysis probe and electrophoresis capillary, along with the high mass sensitivity of CZE, allows the probe to be operated at flow rates as low as 40 nl\\/min. Under these conditions, the relative

Mark W. Lada; Robert T. Kennedy

1995-01-01

61

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

PubMed

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

2013-01-01

62

Analysis of protein changes using two-dimensional difference gel electrophoresis.  

PubMed

A protocol for protein analysis using two-dimensional difference gel electrophoresis (2D-DIGE) is described. 2D-DIGE is one of the most popular and versatile methods of protein separation among rapidly increasing proteomics technologies. Similar to traditional two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the proteins are separated based on their charges and molecular weight by 2D-DIGE. Different from 2D-PAGE, proteins are pre-labeled with different fluorescent and different protein samples are run in one gel by this method. Therefore, 2D-DIGE not only carries the advantages of 2D-PAGE but also eliminates gel-to-gel variation and achieves high resolution, sensitivity, and reproducibility. PMID:24623216

Gao, Weimin

2014-01-01

63

Rapid (ten-minute) pore-gradient electrophoresis of proteins and peptides in Micrograd gels.  

PubMed

Precast gradient gels of short migration length (25 mm) have been developed to provide rapid electrophoretic separation without loss of resolution. These Micrograd gels have been prepared in gel ranges (conventional and unique) to match pore-gradient electrophoresis conditions to proteins/peptides ranging in size from several hundreds to millions. The Hylinx Micrograd gel combines an extreme gel range (6 to 48% polyacrylamide) with a novel crosslinker to provide sieving of polypeptides, and pore-limit electrophoresis of the smallest proteins (e.g. insulin monomer). All gel ranges (such as 3 to 30%) provide zone sharpening in routine analysis of conventional protein mixtures (e.g. serum) within 10 min electrophoresis at 200 to 300 volts. The gels are thin (1 mm) and thus stain quickly, but the gel cassette is of conventional overall width (83 mm), thus fitting many apparatus designs and accommodating 12 samples. The gels are finding valuable use in screening applications, requiring the electrophoretic analysis of many samples, and in cases where a rapid answer is needed, such as monitoring protein purification. The gels have proved particularly useful, in-house, for the latter application in developing Gradipore's new large-scale preparative electrophoresis system, the Gradiflow. PMID:1599958

Wrigley, C W; Margolis, J

1992-01-01

64

Spot volume vs. amount of protein loaded onto a gel: a detailed, statistical comparison of two gel electrophoresis systems.  

PubMed

The long-term goal of this research program is to clarify the molecular mechanisms that participate in the formation of human pituitary macroadenomas. One approach to that goal is to characterize the differentially expressed proteins that are found by a comparison of the proteomes of control pituitary vs. macroadenoma tissues. In order to accurately perform a comparative proteomics study, based on the combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and PDQuest 2-D analysis software, a reproducible 2-DE separation system with a wide linear dynamic measure range is needed. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm); a typical vertical system is the Dodeca system that analyzes up to 12 gels at a time on a single-concentration gel (190 x 205 x 1.0 mm). We have evaluated (Zhan and Desiderio, Electrophoresis 2003, 24, 1834-1846) the spatial and quantitative reproducibility of the two second-dimensional gel systems to separate a human pituitary proteome; that study showed a higher reproducibility for the Dodeca gel system. This present study investigated the relationship between the spot volume and the amount of protein loaded onto the gel for those two 2-D systems. The results demonstrated that the Dodeca gel system provides a wider linear dynamic range to measure the changes in the protein abundance in pituitary proteome. PMID:12783459

Zhan, Xianquan; Desiderio, Dominic M

2003-06-01

65

Accurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry  

PubMed Central

Quantitation of human dystrophin protein in muscle biopsies is a clinically relevant endpoint for both diagnosis and response to dystrophin-replacement therapies for dystrophinopathies. A robust and accurate assay would enable the use of dystrophin as a surrogate biomarker, particularly in exploratory Phase 2 trials. Currently available methods to quantitate dystrophin rely on immunoblot or immunohistochemistry methods that are not considered robust. Here we present a mass spectrometry based approach to accurately quantitate dystrophin protein in a total protein extract from human muscle biopsies. Our approach uses a combination of stable isotope labeled dystrophin as a spike-in standard, gel electrophoresis and high precision mass spectrometry to detect and quantitate multiple peptides of dystrophin within a complex protein mixture. The method was found highly reproducible and linear over a wide dynamic range, detecting as low as 5% of dystrophin relative to the normal amount in healthy individuals.

Brown, Kristy J.; Marathi, Ramya; Fiorillo, Alyson A.; Ciccimaro, Eugene F.; Sharma, Seema; Rowlands, David S.; Rayavarapu, Sree; Nagaraju, Kanneboyina; Hoffman, Eric P.; Hathout, Yetrib

2013-01-01

66

Accurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry.  

PubMed

Quantitation of human dystrophin protein in muscle biopsies is a clinically relevant endpoint for both diagnosis and response to dystrophin-replacement therapies for dystrophinopathies. A robust and accurate assay would enable the use of dystrophin as a surrogate biomarker, particularly in exploratory Phase 2 trials. Currently available methods to quantitate dystrophin rely on immunoblot or immunohistochemistry methods that are not considered robust. Here we present a mass spectrometry based approach to accurately quantitate dystrophin protein in a total protein extract from human muscle biopsies. Our approach uses a combination of stable isotope labeled dystrophin as a spike-in standard, gel electrophoresis and high precision mass spectrometry to detect and quantitate multiple peptides of dystrophin within a complex protein mixture. The method was found highly reproducible and linear over a wide dynamic range, detecting as low as 5% of dystrophin relative to the normal amount in healthy individuals. PMID:23646235

Brown, Kristy J; Marathi, Ramya; Fiorillo, Alyson A; Ciccimaro, Eugene F; Sharma, Seema; Rowlands, David S; Rayavarapu, Sree; Nagaraju, Kanneboyina; Hoffman, Eric P; Hathout, Yetrib

2012-12-18

67

Proteoliposome-based capillary electrophoresis for screening membrane protein inhibitors.  

PubMed

A method for screening of monoamine oxidase (MAO) inhibitor was carried out using capillary electrophoresis (CE) based on the interaction of MAO and its substrate kynuramine (Kyn). Bioactive proteoliposome was reconstituted by liposome and MAO and then was applied as the pseudostationary phase (PSP) of CE to mimic the interaction between the enzyme and its substrate. N-prolmrgyl-R-2-heptylamine (R-2-HPA) and rasagiline [N-propargyl-1-(R)-aminoindan], which are two kinds of MAO inhibitors, were added into the running buffers containing proteoliposome. The results showed that the relative migration time ratio (RMTR × 10(-1)) values of Kyn were enhanced from 8.88 to 9.31 with an increase of the concentrations of rasagiline from 10(-6) to 1 mM. However, the RMTR values of Kyn were enhanced from 8.83 to 9.14 with an increase of the concentrations of R-2-HPA from 10(-6) to 1 mM. The RMTR value of Kyn in the presence of rasagiline was larger than that in the presence of R-2-HPA when rasagiline and R-2-HPA were at the same concentration. The results indicated that the interaction between Kyn and MAO was weakened with the increase of the inhibitors. In addition, the results of offline incubation showed that the inhibitions of rasagiline were 100.0, 72.1, 51.8 and 5.4% at the concentration of 1, 10(-2), 10(-4) and 10(-6) mM; moreover, the inhibitions of R-2-HPA were 70.0, 44.9, 4.1 and 0.9% at the concentrations of 1, 10(-2), 10(-4) and 10(-6) mM. The inhibition efficiency of rasagiline was stronger than that of R-2-HPA at the same concentration. Additionally, the interaction between Kyn and liposome was also investigated. This newly developed method might provide a potential tool for screening MAO inhibitor. PMID:22547660

Li, Bing; Lv, Xuefei; Geng, Lina; Qing, Hong; Deng, Yulin

2012-08-01

68

Identification of methanococcus jannaschii proteins in 2-D gel electrophoresis patterns by mass spectrometry.  

SciTech Connect

The genome of Methanococcus jannaschii has been sequenced completely and has been found to contain approximately 1,770 predicted protein-coding regions. When these coding regions are expressed and how their expression is regulated, however, remain open questions. In this work, mass spectrometry was combined with two-dimensional gel electrophoresis to identify which proteins the genes produce under different growth conditions, and thus investigate the regulation of genes responsible for functions characteristic of this thermophilic representative of the methanogenic Archaea.

Liang, X.

1998-06-10

69

Methodological Challenges of Protein Analysis in Blood Serum and Cerebrospinal Fluid by Capillary Electrophoresis  

Microsoft Academic Search

Summary High-performance capillary electrophoresis (HPCE or CE) is an ultrasensitive analytical technique with high resolving power and a wide area of applications including peptide\\/protein analysis. Its applicability is greatly enhanced by the short separation times, the ease of method development and the minimum sample and organic solvent requirements. Various HPCE modes have been developed for peptide\\/protein analysis, including capillary zone

F. N. Lamari; N. K. Karamanos

2003-01-01

70

Comparison of gradient gel electrophoresis and zonal ultracentrifugation for quantitation of high density lipoproteins  

Microsoft Academic Search

The study was conducted to compare gradient gel electrophoresis (GGE) and zonal ultracentrifugation for quanti- tation of human plasma high density lipoproteins (HDL). Plasma samples were obtained from seven normal subjects consuming a high fat diet (65% total calories) followed by a high carbohydrate diet (65% total calories). HDL were fractionated into HDL2 and HDLS by zonal ultracentrifugation and lipid

Constance A. McNerney; Moti L. Kashyap; Roger L. Barnhart

71

Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis.  

PubMed

Blue native gel electrophoresis (BN-PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN-PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN-PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed. PMID:24662748

Raghupathy, V; Oommen, Anna; Ramachandran, Anup

2014-06-15

72

Lymphocyte, monocyte, and granulocyte proteins compared by use of two-dimensional electrophoresis  

SciTech Connect

Cellular proteins were compared from normal human blood lymphocytes, monocytes, and granulocytes, using high-resolution two-dimensional electrophoresis. The leukocytes were isolated from peripheral blood by centrifugation on density step gradients (yielding a fraction of purified granulocytes and a lymphocyte/monocyte mixture) and monocytes were subsequently separated from lymphocytes by virtue of their adherence to plastic. The cells were labeled with (/sup 35/S)-methionine after various intervals in culture, then solubilized and analyzed by two-dimensional electrophoresis. Although most proteins in each cell type are common to all three, there are nevertheless several specific marker proteins that distinguish one cell type from another. We also examined the appearance of these markers in three lines of cultured cells from humans (GM607, a B-lymphoblastoid line; HL-60, a promyelocytic leukemic line; and 1494, a normal skin fibroblast).

Gemmell, M.A.; Anderson, N.L.

1982-04-01

73

Speciation of protein-bound trace elements by gel electrophoresis and atomic spectrometry.  

PubMed

The metabolism of trace elements, in particular their binding to proteins in biological systems is of great importance in biochemical, toxicological, and pharmacological studies. As a result there has been a sustained interest over the last two decades in the speciation of protein-bound metals. Various analytical approaches have been employed, combining efficient separation of metalloproteins by liquid chromatography or electrophoresis with high-sensitivity elemental detection. Slab-gel electrophoresis (GE) is a key platform for high-resolution protein separation, and has been combined with autoradiography and various atomic spectrometric techniques for in-gel determination of protein-bound metals. Recently, the combination of GE with state-of-the-art inductively coupled plasma-mass spectrometry (ICP-MS), particularly when linked to laser ablation (LA) for direct gel interrogation, has opened up new opportunities for rapid characterization of metalloproteins. The use of GE and atomic spectrometry for the speciation of protein-bound trace elements is reviewed in this paper. Technical requirements for gel electrophoresis/atomic spectrometric measurement are considered in terms of method compatibilities, detection capability and potential usefulness. The literature is also surveyed to illustrate current status and future trends. PMID:15300764

Ma, Renli; McLeod, Cameron W; Tomlinson, Kerry; Poole, Robert K

2004-08-01

74

Capillary zone electrophoresis for separation and quantitative determination of mexiletine and its main phase I metabolites.  

PubMed

The simultaneous separation and quantification of the analytes within the minimum analysis time and the maximum resolution and efficiency are the main objectives in the development of a capillary electrophoretic method for the determination of solutes. In this paper we describe a specific, sensitive and robust method, using capillary zone electrophoresis with internal standard and UV detection, for the separation and quantification of the anti-arrhythmic drug mexiletine, its main phase I metabolites, and its main nitrogenous degradation product. PMID:23826880

Bruno, Claudio; Cavalluzzi, Maria Maddalena; Carocci, Alessia; Catalano, Alessia; Franchini, Carlo; Lentini, Giovanni

2013-03-01

75

Precipitation of champagne base wine proteins prior to 2D electrophoresis.  

PubMed

Numerous methods have been employed to depict the protein content of wines. Among them, two-dimensional electrophoresis (2D-E) presents a powerful resolution, but has been poorly applied to wine. Furthermore, 2D-E was coupled with various extraction methods of proteins without any reference method for wine. Here, we describe a rapid method to extract proteins from a champagne base wine through ultrafiltration followed by precipitation with ethanol and trichloroacetic acid. More than 50 spots were visualized on 2D-gels (7 cm, pH 3-6) by colloidal Coomassie Brilliant Blue staining. PMID:24136561

Cilindre, Clara

2014-01-01

76

[Clusterization of halophilic and halotolerant eubacteria using whole-cell protein electrophoresis data].  

PubMed

Total cell proteins of the nineteen halophilic and halotolerant eubacteria isolated from marine sediments and highly mineralized formation waters of oil fields were investigated by SDS gel electrophoresis. The microorganisms studied, phenotypically identified as belonging to the genera Dietzia, Rhodococcus, Staphylococcus, Cytophaga, Brevibacterium, and Archangium, were found to form clearly distinguishable clusters (20-30% similarity at the generic level) on the dendrogram derived from electrophoretic protein patterns. Protein similarity data confirmed the heterogeneity of Rhodococcus maris and its relatedness to the genus Dietza. PMID:11315673

Bykova, S A; Zviagintseva, I S; Akhlynin, D S; Beliaev, S S; Gal'chenko, V F

2000-01-01

77

A technique for detecting antifungal activity of proteins separated by polyacrylamide gel electrophoresis.  

PubMed

A technique was developed for the detection of antifungal activity of proteins after discontinuous polyacrylamide gel electrophoresis under native conditions. The antifungal activity is detected as growth inhibition zones in a homogeneous fungal lawn, grown in an agar layer spread on top of the polyacrylamide gel. The position of proteins with antifungal activity can be determined on a diffusion blot prepared from the same gel. The technique is illustrated for three antifungal plant proteins, i.e. alpha-purothionin, Urtica dioica agglutinin, and tobacco chitinase. PMID:1889394

De Bolle, M F; Goderis, I J; Terras, F R; Cammue, B P; Broekaert, W F

1991-06-01

78

Southern elephant seal (Mirounga leonina)II. Studies of milk protein fractions by gel electrophoresis  

Microsoft Academic Search

Milk protein fractions during various stages of lactation in the southern elephant seal Mirounga leonina were analysed. Twelve milk samples were taken from ten females throughout the lactation period during 1990 and 1991 at Stranger\\u000a Point, King George Island, South Shetland Islands. Milk samples were subjected to polyacrylamide gel electrophoresis (PAGE).\\u000a Samples from different days of lactation gave similar qualitative

P. A. Ronayne de Ferrer; R. A. Gonzalez Colaso; M. E. I. Marquez; A. R. Carlini; D. F. Vergani; G. A. Daneri

1996-01-01

79

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

2006-01-01

80

Clusterization of halophilic and halotolerant eubacteria using whole-cell protein electrophoresis data  

Microsoft Academic Search

Total cell proteins of the nineteen halophilic and halotolerant eubacteria isolated from marine sediments and highly mineralized\\u000a formation waters of oil fields were investigated by SDS gel electrophoresis. The microorganisms studied, phenotypically identified\\u000a as belonging to the generaDietzia, Rhodococcus, Staphylococcus, Cytophaga, Brevibacterium, andArchangium, were found to form clearly distinguishable clusters (20–33% similarity at the generic level) on the dendrogram derived

S. A. Bykova; I. S. Zvyagintseva; D. S. Akhlynin; S. S. Belyaev; V. F. Gal’chenko

2000-01-01

81

Application of difference gel electrophoresis to the identification of inner medullary collecting duct proteins  

Microsoft Academic Search

In this study, we present a standardized approach to purification of native inner medullary collecting duct (IMCD) cells from rat kidney for proteomic analysis and apply the approach to identification of abundant proteins utilizing two-dimensional difference gel electrophoresis (DIGE) coupled with matrix-assisted laser desorption-ionization-time of flight mass spectrometry. Fractionation of inner medullary cell suspensions by low-speed centrifugation gave a highly

J. D. Hoffert; B. W. M. van Balkom; C. L. Chou; M. A. Knepper

2004-01-01

82

Isolation of Spiroplasma citri membranes and characterization of membrane proteins by two-dimensional gel electrophoresis  

Microsoft Academic Search

This paper describes a method for isolating plasma membranes fromSpiroplasma citri and for comparing membrane and cytoplasmic proteins by two-dimensional gel electrophoresis. Plasma membranes ofS. citri were stabilized against fragmentation by coating cell with Concanavalin A just prior to lysis. After lysis of the cells by ultrasonic irradiation, membranes were purified by differential centrifugation and step gradients. The purified fraction,

Philippe Simoneau; Jacques Labarère

1988-01-01

83

Completely monodisperse, highly repetitive proteins for bioconjugate capillary electrophoresis: development and characterization.  

PubMed

Protein-based polymers are increasingly being used in biomaterial applications because of their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for free-solution conjugate electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis. PMID:21553840

Lin, Jennifer S; Albrecht, Jennifer Coyne; Meagher, Robert J; Wang, Xiaoxiao; Barron, Annelise E

2011-06-13

84

Separation and relative quantitation of mouse plasma esterases with disc electrophoresis  

Microsoft Academic Search

A technique for the qualitative and quantitative study of mouse plasma esterases using disc elect rophoresis in acrylamide gel is described. This technique has been employed tK) study sub- strate specificity and quantitative relationships among various mouse strain and sex dependent esterases. Properties of acrylamide gels are discussed relative to their effect on the quaiitita- tive estimation of plasma esterases.

R. C. ALLEN; R. A. POPP; D. J. MOORE

1965-01-01

85

Qualitative and quantitative assessment of marketed erythropoiesis-stimulating agents by capillary electrophoresis.  

PubMed

Formulated erythropoiesis stimulating agents (ESAs) containing erythropoietin (EPO)-alpha, EPO-beta or darbepoetin-alpha were analyzed by capillary electrophoresis with a previously published method requiring no sample pre-treatment [1]. In this study, the method proved to be applicable to all formulations encountered, that is, in the presence of polysorbate 80, polysorbate 20 or human serum albumin as major excipients, thus extending the range of products that can be analyzed without pre-treatment. Method performance was evaluated and showed good linearity, range, precision and sensitivity. No significant matrix effects were observed for the various formulations. The ability of the method to resolve isoforms of each of the three active ingredients enabled comparison of the isoform distribution of finished products with that of the respective drug substance. In general, finished products and their corresponding drug substances showed similar isoform distribution and all were within manufacturer specifications. In addition, the content in active ingredient in the various dosage strengths was found to be in close agreement with the label claims with the exception of 2 out of 131 containers analyzed. Overall, this study demonstrated that the capillary zone electrophoresis method could be successfully applied to the analysis of most of the ESA products currently on the market in North America and Europe and that all products were found to have good batch-to-batch consistency. PMID:22954449

Boucher, Sylvie; Kane, Anita; Girard, Michel

2012-12-01

86

Electrophoresis-tutor: an image-based personal computer program that teaches clinical interpretation of protein electrophoresis patterns of serum, urine, and cerebrospinal fluid.  

PubMed

High-resolution protein electrophoresis of serum, urine, and cerebrospinal fluid (CSF) can aid in the diagnosis of multiple myeloma, amyloidosis, macroglobulinemia, multiple sclerosis, and other diseases. Electrophoresis-Tutor is a personal computer program based on approximately 150 digital images that teaches the clinical interpretation of agarose gel electrophoretic patterns. The program is divided into the following sections: introduction, CSF, serum, urine, review of disease states, program navigator, and final exam. The CSF section describes normal and abnormal CSF findings with emphasis on oligoclonal banding, as seen in the CSF of patients with multiple sclerosis. The serum section emphasizes monoclonal gammopathy patterns but also has detailed descriptions of inflammation, liver disease, protein-losing disorders, genetic deficiencies, and other patterns. Monoclonal gammopathy is described in the context of specific associated clinical conditions (e.g., myeloma, amyloidosis). For each monoclonal gammopathy example, results of standard electrophoresis, densitometry, and immunofixation are presented. The review of disease states uses animation to illustrate the development and remission of a variety of pathological patterns. The program navigator allows the user to jump quickly to any place in the program. The optional exam contains 20 questions, and detailed feedback is given after each question. Electrophoresis-Tutor can be used as a stand-alone teaching tool, a companion to traditional instruction, or a reference source. PMID:7656448

Astion, M L; Rank, J; Wener, M H; Torvik, P; Schneider, J B; Killingsworth, L M

1995-09-01

87

The Use of Wool Fast Blue Bl for the Electrophoresis of Human Parotid Saliva Proteins in Acrylamide Gel.  

National Technical Information Service (NTIS)

Wool Fast Blue BL was examined as a stain to delineate the protein components separated by the acrylamide gel electrophoresis of parotid saliva. The tests were performed on samples of parotid saliva collected from young male naval personnel by stimulation...

T. S. Meyer B. L. Lamberts

1968-01-01

88

Quantitative affinity electrophoresis of RNA-small molecule interactions by cross-linking the ligand to acrylamide.  

PubMed

We show that the affinity electrophoresis analysis of RNA-small molecule interactions can be made quantifiable by cross-linking the ligand to the gel matrix. Using an RNA-aminoglycoside model system to verify our method, we attached an acryloyl chloride molecule to the aminoglycosides paromomycin and neomycin B to synthesize an acrylamide-aminoglycoside monomer. This molecule was then used as a component in gel polymerization for affinity electrophoresis, covalently attaching an aminoglycoside molecule to the gel matrix. To test RNA binding to the cross-linked aminoglycosides, we used the aminoglycoside binding RNA molecule derived from thymidylate synthase messenger RNA (mRNA) that contains a C-C mismatch. Binding is indicated by the difference in RNA mobility between gels with cross-linked ligand, with ligand embedded during polymerization, and with no ligand present. Critically, the predicted straight line relationship between the reciprocal of the relative migration of the RNA and the ligand concentration is obtained when using cross-linked aminoglycosides, whereas a straight line is not obtained using embedded aminoglycosides. Average apparent dissociation constants are determined from the slope of the line from these plots. This method allows an easy quantitative comparison between different nucleic acid molecules for a small molecule ligand. PMID:23928050

Boodram, Sherry N; McCann, Lucas C; Organ, Michael G; Johnson, Philip E

2013-11-15

89

Routine diagnosis with PhastSystem compared to conventional electrophoresis: automated sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining and western blotting of urinary proteins.  

PubMed

The recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient-gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional "macro"-method with self-cast SDS-polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 microL sample volumes and an 8-25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS-PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha-1-antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS-PAGE and staining allows easy standardization of urine SDS-PAGE among clinical routine laboratories. PMID:2469571

Scherberich, J E; Fischer, P; Bigalke, A; Stangl, P; Wolf, G B; Haimerl, M; Schoeppe, W

1989-01-01

90

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

PubMed

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

2014-12-01

91

Quantitative determination of sulfur-containing anions in complex matrices with capillary electrophoresis and conductivity detection.  

PubMed

The analysis of sulfur species in complex matrices, like environmentally related samples, requires selective and sensitive as well as robust determination methods. As many as possible different anions need to be quantified in a reasonable analysis time. Besides ion chromatography, capillary electrophoresis has proven to be a very efficient technique for the separation and determination of ionic compounds. With the advantages of less sample and solvent consumption compared to conventional LC, short separation time, inexpensive and robust capillaries, CE was used to separate the anions sulfate, sulfite, thiosulfate, thiocyanate and sulfide. Detection and injection modes and the composition of the separation buffers have been varied to find the most suitable conditions. Conductivity detection after electrokinetic sample injection and improvement of calibration linearity allowed the determination of sulfur containing anions with low limits of detection (8 to 50 micrograms/l). The developed CE method was applied to the analysis of water from an open-pit mining lake. PMID:10427765

Hissner, F; Mattusch, J; Heinig, K

1999-07-01

92

Western blotting of basic proteins after nondenaturing electrophoresis in acid conditions using the PhastSystem.  

PubMed

Electroblotting of basic proteins was performed from minigels after electrophoresis, under nondenaturing acidic conditions, by using the automated PhastSystem. Depending on the molecular masses of the proteins to be studied, various precast gel media were chosen. The transfer membranes with various types (nitrocellulose and polyvinylidene difluoride) and pore sizes (0.45 and 0.2 micron) were chosen accordingly. For the semidry electric transfer, a simple, discontinuous two-buffer system was used. The anode solution contained 0.3 M Tris, pH 10.4, and the cathode solution, 40 mM 6-amino-n-hexanoic acid, pH 7.6, with 20% v/v methanol each. The addition of 0.1% sodium dodecyl sulfate (SDS) in the cathode solution facilitated the elution of proteins from the gels and directed the migration of the negative SDS-protein complexes towards the anode membranes. The transfer conditions following native polyacrylamide gel electrophoresis allowed the visualization of basic proteins, with molecular weights ranging from 29,000 to 5,000, for which isoforms could be resolved and which retained their biological properties. PMID:8223396

Davril, M; Ducourouble, M P; Van-Seuningen, I

1993-09-01

93

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods  

PubMed Central

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

Gauci, Victoria J.; Wright, Elise P.

2010-01-01

94

Quantitative Multiple Reaction Monitoring of Peptide Abundance Introduced via a Capillary Zone Electrophoresis-Electrospray Interface  

PubMed Central

We demonstrate the use of capillary zone electrophoresis with an electrokinetic sheath-flow electrospray interface coupled to a triple quadrupole mass spectrometer for the accurate and precise quantification of leu-enkaphalin in a complex mixture using multiple-reaction monitoring (MRM). Assay time is <6 minutes, with no re-equilibration required between runs. A standard curve of Leu-enkephalin was performed in the presence of a background tryptic digest of bovine albumin. We demonstrate reasonably reproducible peak heights (21% relative standard deviation), retention times (better than 1% relative standard deviation), and robust electrospray quality. Our limit-of detection (3?) was 60 pM, which corresponds to the injection of 115 zeptomole of peptide. This is a 10–20-fold improvement in mass sensitivity than we have obtained by nano HPLC/MRM and substantially better than reported for LC/MS/MS. Further quantification was performed in the presence of stable-isotope labeled versions of the peptides; under these conditions, linearity was observed across nearly four orders of magnitude. The concentration detection limit was 240 pM for the stable-isotope labeled quantification.

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

2013-01-01

95

High-throughput separation of DNA and proteins by three-dimensional geometry gel electrophoresis: feasibility studies.  

PubMed

We report a novel separation method that is applicable to both DNA and protein samples, based on electrophoresis in a three-dimensional (3-D) geometry. In contrast to conventional electrophoresis, samples are applied in a two-dimensional, planar array to one of the surfaces of a 3-D geometry separation medium. Loading onto a plane results in a very high sample capacity. Sample migration and separation occur along the third spatial dimension, which is perpendicular to the loading plane. The key problem of electrophoresis in a 3-D geometry separation setup is that temperature gradients are caused by Joule's heat, affecting the electrical conductivity and viscosity of the separation medium. A means of achieving straight sample migration under these circumstances is to force heat flow through the separation medium parallel to the axis of sample migration. This can be done by dissipating the heat via the electrode sides of the gel and blocking any other heat transfer. The separation of DNA and proteins by this method has been tested using agarose gel electrophoresis, polyacrylamide gel electrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Data were acquired off-line by conventional staining methods as well as on-line by detection of laser-induced fluorescence. We describe how to excise samples from the separation medium for preparative purposes. Possible unique applications of this 3-D geometry electrophoresis separation method are also discussed. PMID:14679562

Ventzki, Robert; Stegemann, Josef

2003-12-01

96

Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots  

DOEpatents

After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

Zhang, Jian-Shi (Shanghai, CN); Giometti, Carol S. (Glenview, IL); Tollaksen, Sandra L. (Montgomery, IL)

1989-01-01

97

Quantitative high-throughput measurement of gene expression with sub-zeptomole sensitivity by capillary electrophoresis  

Microsoft Academic Search

Microarray technologies have provided the ability to monitor the expression of whole genomes rapidly. However, concerns persist with regard to quantitation and reproducibility, and the detection limits for individual genes in particular arrays are generally unknown. This article describes a semiautomated PCR-based technology, Q-RAGE, which rapidly provides measurements of mRNA abundance with extremely high sensitivity using fluorescent detection of specific

Lea Spyres; Sally Gaddis; Ella Bedford; Stacey Arantes; Nikki Liburd; K. Leslie Powell; Howard Thames; David Mitchell; Earl Walborg; Mahmoud Rouabhia; C. Marcelo Aldaz; Michael C. MacLeod

2005-01-01

98

HEMATOLOGICAL, PROTEIN ELECTROPHORESIS AND CHOLINESTERASE VALUES OF FREE-LIVING NESTLING PEREGRINE FALCONS IN SPAIN  

Microsoft Academic Search

Protein electrophoresis, hematological and cholinesterase values were determined in 32 nestling free-living peregrine falcons (Falco peregrinus) (15- to 27-days-old) in order to estab- lish normal reference values for this population. The following values (mean SD) were ob- served: prealbumin 0.31 0.04 g\\/dl, albumin 1.25 0.06 g\\/dl, 1 and 2-globulin 0.23 0.02 and 0.16 0.02 g\\/dl respectively, -globulin 1.02 0.05 g\\/dl,

Maria del Pilar Lanzarot; Andres Montesinos; Manuel Ignacio San Andres; Casilda Rodrõ ´ guez; Marõ ´ a; Victoria Barahona

99

Using affinity capillary electrophoresis to determine binding stoichiometries of protein-ligand interactions.  

PubMed

We have developed a new method utilizing affinity capillary electrophoresis (ACE) for the determination of binding stoichiometries in biochemical systems. Using the same concentration of a ligand in the sample and the electrophoresis buffer, the appearance of an inverted peak corresponding to the free ligand in the resulting electropherogram provides a criterion of binding of a ligand to its receptor protein. For both low (fast off rates) and high (slow off rates) affinity systems, analysis of the integration of free ligand peak in electropherograms as a function of the total concentration of a ligand in samples at constant concentration of receptor protein yields the binding stoichiometry of the ligand to the protein. Applications of this technique to studies of (i) the inhibition of carbonic anhydrases (CA, EC 4.2.1.1, from human and bovine erythrocytes) by 4-alkylbenzenesulfonamide 1, (ii) the interaction of a monoclonal antibody to human serum albumin (anti-HSA) with its antigen HSA, and (iii) the binding of streptavidin (from Streptomyces avidinii) to biotin derivatives (monobiotinylated oligodeoxyribonucleotide 2, fluorescein biotin, or Lucifer Yellow biotin) yield stoichiometries of 1:1, 1:2, and 1:4, respectively. For multivalent, tight-binding systems, this ACE method can readily separate stable intermediate species. This method is generally applicable to both tight- and weak-binding systems, requires only nanograms of proteins and ligands, involves no radioactive materials, and does not require changes in electrophoretic mobilities of receptor proteins upon binding with ligands. It thereby provides a rapid, sensitive, and convenient method for measuring binding stoichiometries of ligands to proteins. PMID:8075061

Chu, Y H; Lees, W J; Stassinopoulos, A; Walsh, C T

1994-09-01

100

Aptamers in Affinity Separations:Capillary Electrophoresis  

Microsoft Academic Search

Assays employing aptamers in capillary electrophoresis (CE), including competitive and noncompetitive assays, fluorescence polarization (FP) assays, nonequilibrium capillary electrophoresis of equilibrium mixtures, and affinity-polymerase chain reaction-CE assays, are summarized. These assays can be used to estimate dissociation rate and equilibrium binding constants, determine binding stoichiometries, study molecular interactions, and quantitatively determine specific analytes (e.g., proteins) in complex media. They can

Jeffrey W. Guthrie; Yuanhua Shao; X. Chris Le

2009-01-01

101

Capillary affinity electrophoresis for the screening of post-translational modification of proteins with carbohydrates.  

PubMed

Glycosylation is one of the most important post-translational events for proteins, affecting their functions in health and disease, and plays significant roles in various information traffics for intracellular and intercellular biological events (Hancock, W. S. J. Proteome Res. 2002, 1, 297). We have attempted to obtain the information on the numbers and amounts of carbohydrate chains. Interaction between carbohydrate chains and proteins that recognize them is a target to understand the biological roles of glycosylation. To date, there have been a few strategies for simultaneous analysis of the interactions between complex mixtures of carbohydrates and proteins. Here, we report an approach to categorize carbohydrate chains using a few glycoprotein samples as models for the studies on the analysis of post-translational modification of proteins with carbohydrates. A combination of some specific lectins was used as carbohydrate-binding proteins. The method is based on high-resolution separation of fluorescent-labeled carbohydrates by capillary electrophoresis with laser-induced fluorescent detection in the presence of carbohydrate-binding proteins at different concentrations. The present technique affords (1) simultaneous determination of carbohydrate chains, (2) binding specificity of the constituent carbohydrate chains to specific proteins, and (3) kinetic data such as the association constant of each carbohydrate. We found that the lectins employed in the present study could discriminate subtle difference in linkages and resolved the carbohydrate mixtures. The results will be useful, for example, to understand the biological events expressed with carbohydrate changes on the cell surface. PMID:12643546

Nakajima, Kazuki; Oda, Yasuo; Kinoshita, Mitsuhiro; Kakehi, Kazuaki

2003-01-01

102

Use of capillary sodium dodecyl sulfate gel electrophoresis to detect the prion protein extracted from scrapie-infected sheep  

Microsoft Academic Search

Scrapie in sheep and in goats is the prototype of a group of transmissible spongiform encephalopathies (TSE). A feature of these diseases is the accumulation in the brain of rod shaped fibrils that form from an aggregated protein that is a protease-resistant form of a modified normal host cell protein. In this study, we compared SDS gel capillary electrophoresis to

Mary Jo Schmerr; Allen Jenny; Randall C. Cutlip

1997-01-01

103

Advances in the measurement of protein mobility using laser Doppler electrophoresis - the diffusion barrier technique.  

PubMed

A new technique for the measurement of protein mobility using laser Doppler electrophoresis (LDE) is introduced and characterised. The diffusion barrier approach loads a tiny protein sample volume into a much larger volume of dispersant, which contains the electrodes; the LDE measurement is then recorded before the sample can diffuse to the electrodes. We demonstrate that sample volumes are reduced by up to two orders of magnitude to volumes typically associated with separation techniques (?50 ?L), no reduction in measurement sensitivity occurs, samples can be retrieved usefully intact, post-measurement and typical measurement times are of the order of minutes. Measurements of BSA mobility up to 75°C and 1 M buffer concentration and lysozyme at a concentration as low as 0.5 mg/mL are demonstrated using the technique with good agreement with literature values. PMID:21769888

Corbett, Jason C W; Connah, Malcolm T; Mattison, Kevin

2011-07-01

104

Comparison of protein mixtures in aqueous humor by membrane preconcentration - capillary electrophoresis - mass spectrometry.  

PubMed

The significance of proteomic research is coupled with the recent exponential growth of these investigations. Currently, the most popular techniques used for these studies include the coupling of 1- and 2-dimensional electrophoresis with mass spectrometric analysis of the extracted and digested proteins. However, detection limits of gel staining methods have led to a search for complimentary techniques that afford the detection of lower concentrations of biologically relevant proteins. In the present studies, we have evaluated the applicability of on-line capillary electrophoresis - mass spectrometry (CE-MS) for this application. Specifically, we used membrane preconcentration-CE-MS (mPC-CE-MS) to analyze 13 samples of human aqueous humor (AH) from patients with various ocular pathologies (cataract, cataract plus glaucoma, and cataract plus pseudoexfoliation syndrome). This approach enabled rapid analysis of a relatively large volume (1 microL of each specimen, and a protein map for each was created. Measured average molecular weights (Mr) were used to tentatively identify proteins after search of the SWISS-PROT database using TagIdent from ExPaSy. Among those proteins tentatively identified are beta-2 microglobulin (Mr 11731.2), apolipoprotein A1 (Mr 28078.6) and serum albumin (Mr 66400). Proteins with Mr of 4349 (unidentified), 11731.2 (beta-2 microglobulin), 13400-14100 (immunoglobulin fragments), 28078.2 (apolipoprotein A1) and approximately 68000 (serum albumin) were observed in the majority of specimens. Generally no significant differences were noted in the protein composition of aqueous humor samples from different pathologies. However, the absence of an Mr 13345 protein and its oxidized form (Mr 13361) in samples from patients with pseudoexfoliation syndrome was noted. Occasionally the alpha-and beta-chains of hemoglobin, a contaminant in aqueous humor introduced during sampling, were also detected. We conclude from these studies that mPC-CE-MS is an attractive complimentary technique for proteome research, as this approach enables direct mapping and characterization of low concentrations of proteins that are present in complex physiologically derived fluids. PMID:9788321

Rohde, E; Tomlinson, A J; Johnson, D H; Naylor, S

1998-10-01

105

Quantitative capillary electrophoresis determination of oversulfated chondroitin sulfate as a contaminant in heparin preparations  

PubMed Central

A simple, accurate and robust quantitative CE method for the determination of oversulfated chondroitin sulfate (OSCS) as a contaminant in heparin (Hep) preparations is described. After degradation of the polysaccharides by acidic hydrolysis, the hexosamines produced, i.e., GlcN from Hep and GalN from OSCS, were derivatized with anthranilic acid (AA) and separated by means of CE in approx. 10 min with high sensitivity detection at 214 nm (limit of detection (LOD) of approx. 200 pg). Furthermore, AA-derivatized GlcN and GalN showed quite similar molar absorptivity allowing for direct and simple quantification of OSCS in Hep samples. Moreover, a preliminary step of specific enzymatic treatment by using chondroitin ABC lyase may be applied for the specific elimination of interference in the analysis due to the possible presence in Hep samples of natural chondroitin sulfate and dermatan sulfate impurities, making this analytical approach highly specific for OSCS contamination, since chondroitin ABC lyase is unable to act on this semi-synthetic polymer. The CE method was validated for specificity, linearity, accuracy, precision, LOD and limit of quantification (LOQ). Due to the very high sensitivity of CE, as little as 1% OSCS contaminant in Hep sample could be detected and quantified. Finally, a contaminated raw Hep sample was found to contain 38.9% OSCS while a formulated contaminated Hep was calculated to have 39.7% OSCS.

Volpi, Nicola; Maccari, Francesca; Linhardt, Robert J.

2014-01-01

106

Seasonal influence on biochemical profile and serum protein electrophoresis for Boa constrictor amarali in captivity.  

PubMed

Similarly to other reptiles, snakes are ectothermic animals and depend exclusively on the environment for the maintenance of their physiological, biochemical and immunological processes. Thus, changes in biochemical values can be expected due to seasonal influence. Twenty-two adult specimens of Boa constrictor amarali kept in captivity were used. Blood collections were done in two different seasons: winter (July 2004) and summer (January 2005) for the following assays: uric acid, aspartate aminotransferase (AST), glucose, cholesterol, total protein, and serum protein electrophoresis. The mean biochemical results found in summer and winter, respectively, were: 6.3 ± 3.4 and 11.3 ± 6.2 mg/dL for uric acid; 28.7 ± 12.4 and 20.7 ± 16.2 UI/L for AST; 26.3 ± 17 and 17.4 ± 6.8 mg/dL for glucose; 67.3 ± 30.2 and 69.7 ± 38.5 mg/dL for cholesterol; and 5.9 ± 1.6 and 5.9 ± 1.4 g/dL for total protein. Results regarding electrophoresis in summer and winter, respectively, were: 1.9 ± 0.7 and 2.4 ± 0.6 g/dL for albumin; 0.7 ± 0.2 and 0.5 ± 0.2 g/dL for ?-globulin; 1.5 ± 0.5 and 1.7 ± 0.6 g/dL for ?-globulin; and 1.8 ± 0.5 and 1.5 ± 0.5 g/dL for ?-globulin. In the summer, there was a significant increase in AST and a decrease in uric acid (p < 0.05). Serum protein electrophoresis showed a significant increase in ?-globulin fraction (p < 0.05) in the same season. There were not significant differences between seasons for the remaining variables. Based on these results, the period of the year must be considered in the interpretation of some biochemical values for these animals. PMID:21755171

Silva, L F N; Riani-Costa, C C M; Ramos, P R R; Takahira, R K

2011-05-01

107

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

PubMed

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

2012-12-01

108

Development and validation of a method for quantitative determination of econazole nitrate in cream formulation by capillary zone electrophoresis.  

PubMed

A simple, fast, inexpensive and reliable capillary zone electrophoresis (CZE) method for the determination of econazole nitrate in cream formulations has been developed and validated. Optimum conditions comprised a pH 2.5 phosphate buffer at 20 mmol L(-1) concentration, +30 kV applied voltage in a 31.5 cm x 50 microm I.D. capillary. Direct UV detection at 200 nm led to an adequate sensitivity without interference from sample excipients. A single extraction step of the cream sample in hydrochloric acid was performed prior to injection. Imidazole (100 microg mL(-1)) was used as internal standard. Econazole nitrate migrates in approximately 1.2 min. The analytical curve presented a coefficient of correlation of 0.9995. Detection and quantitation limits were 1.85 and 5.62 microg mL(-1), respectively. Excellent accuracy and precision were obtained. Recoveries varied from 98.1 to 102.5% and intra- and inter-day precisions, calculated as relative standard deviation (RSD), were better than 2.0%. The proposed CZE method presented advantageous performance characteristics and it can be considered suitable for the quality control of econazole nitrate cream formulations. PMID:18406411

Gaona-Galdos, Angel Arturo; Zanolli Filho, Luiz Antonio; Tavares, Marina Franco Maggi; Aurora-Prado, María Segunda; Santoro, Maria Inês Rocha Miritello; Kedor-Hackmann, Erika Rosa Maria

2008-05-30

109

Programed nebulizing-gas pressure mode for quantitative capillary electrophoresis-mass spectrometry analysis of endocrine disruptors in honey.  

PubMed

The application of programed nebulizing-gas pressure (PNP) has been previously described to be a simple strategy for the separation of anions by capillary electrophoresis-electrospray-mass spectrometry (CE-ESI-MS). The PNP mode provided high resolution and stable analyses and also had the advantage of allowing the use of capillaries wider than the 50-75 ?m conventional ones. Here, the application of the PNP approach to the quantitative analysis of pollutants in real samples by CE-ESI-MS is described for the first time; in particular, for the determination several endocrine disruptors (2,4-dichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, bisphenol-A, 4-tert-butyl-phenol, and 4-tert-butyl benzoic acid) in honey. For sample pretreatment, different liquid-liquid extraction (LLE) procedures were assayed and compared to the QuEChERS(©) methodology prior to electrophoretic analysis. With the application of the PNP approach to CE-ESI-MS, the limits of detection achieved were in the 1-4 ng/g range with a simple liquid-liquid procedure without any further clean-up step; relative standard deviation values in the 2-9% range were found. The analytical characteristics allow the proposed method to be used in the control analysis of these compounds in honey. PMID:22887158

Domínguez-Álvarez, Javier; Rodríguez-Gonzalo, Encarnación; Hernández-Méndez, Jesús; Carabias-Martínez, Rita

2012-08-01

110

Protein binding of procyanidins: studies using polyacrylamide gel electrophoresis and French maritime pine bark extract.  

PubMed

The application of PAGE to determine the interaction between procyanidins and proteins, as presented here, enables one to directly determine the binding of either a pure of a complex mixture of flavonoids to a particular protein. If the protein of interest is an enzyme, the combination of PAGE with quantitative activity measurements allows identifying whether a change in the enzyme activity is related to the binding. Data presented suggest that PBE and EGb 761 have protein-binding properties, which, in addition to their redox-based effects, could provide a biochemical basis for their action in biological systems. PMID:11400382

Moini, H; Guo, Q; Packer, L

2001-01-01

111

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.

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

2012-01-01

112

Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots  

DOEpatents

After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a dc power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. 8 figs.

Zhang, Jian-Shi; Giometti, C.S.; Tollaksen, S.L.

1987-09-04

113

In-gel staining of proteins in native poly acryl amide gel electrophoresis using tetrakis(4-sulfonato phenyl)porphyrin.  

PubMed

Protein identification in polyacrylamide gel electrophoresis (PAGE) requires post-electrophoretic steps like fixing, staining and destaining of the gel, which are time-consuming and cumbersome. We have developed a method for direct visualization of protein bands in PAGE using tetrakis(4-sulfonato phenyl)porphyrin (TPPS) as a dye without the need for any post electrophoretic steps, where separation and recovery of enzymes become much easier for further analysis. Activity staining was done to prove that the biochemical activity of the enzymes was preserved after electrophoresis. PMID:21233569

Divakar, Kalivarathan; Sujatha, Vijayan; Barath, Sridhar; Srinath, Krishnamurthy; Gautam, Pennathur

2011-01-01

114

Laser ablation inductively coupled plasma-mass spectrometry in combination with gel electrophoresis: a new strategy for speciation of metal binding serum proteins  

NASA Astrophysics Data System (ADS)

A new hyphenated technique-crossed immunoelectrophoresis in combination with laser ablation inductively coupled plasma (ICP)-mass spectrometry—for the identification and quantitation of metal binding proteins in blood serum is described. Human serum enriched with Co was subjected to electrophoresis and the agarose gels corresponding to the first and second dimensions were interrogated and analysed using a Nd Yag laser (1064 nm) interfaced to ICP-mass spectrometry. Comparison of the distribution map for Co with the protein distribution map obtained via Coommassie Brilliant Blue staining allowed identification of main Co binding serum proteins. Signals for Co (single ion monitoring, mle 59) were transient in nature and for gels enriched with increasing concentrations of Co, peak area response was linear with concentration. Precision for replicate analyses was 6% R.S.D. and the limit of detection was - 0.29 ng.

Neilsen, J. L.; Abildtrup, A.; Christensen, J.; Watson, P.; Cox, A.; McLeod, C. W.

1998-02-01

115

Separation of acidic and basic proteins by nanoparticle-filled capillary electrophoresis.  

PubMed

We present the first example of the analysis of acidic and basic proteins by nanoparticle-filled capillary electrophoresis. Compared to the didodecyldimethylammonium bromide (DDAB)-coated capillary, the DDAB-capped gold nanoparticles (AuNPs) as pseudostationary phase were found to form more stable coating on the capillary wall, thus leading to greater separation efficiency and high reproducibility. In addition to their advantages for protein separation, DDAB-capped AuNPs can generate high reversed electroosmotic flow, which is 75% greater than DDAB at pH 3.5. To allow strong interactions with proteins, the AuNPs were modified with poly(ethylene oxide) via noncovalent bonding to form gold nanoparticles/polymer composites (AuNPPs). Using a capillary dynamically coated with DDAB-capped AuNPs and filled with AuNPPs under acidic conditions (10 mM phosphate, pH 3.5), we have demonstrated the separation of acidic and basic proteins with peak efficiencies ranging from 71 000 to 1 007 000 plates/m and relative standard deviations of migration time less than 0.6%. Additionally, the proposed method has been applied to the analyses of biological samples, including saliva, red blood cells, and plasma. With simplicity, high resolving power, and high reproducibility, the proposed method has shown great potential for proteomics applications and clinical diagnosis. PMID:17134133

Yu, Cheng-Ju; Su, Chih-Lin; Tseng, Wei-Lung

2006-12-01

116

Western Blotting Using Microchip Electrophoresis Interfaced to a Protein Capture Membrane  

PubMed Central

Western blotting is a commonly used assay for proteins. Despite the utility of the method, it is also characterized by long analysis times, manual operation, and lack of established miniaturized counterpart. We report a new way to Western blot which addresses these limitations. In the method, sodium dodecyl sulfate (SDS)-protein complexes are separated by sieving electrophoresis in a microfluidic device or chip. The chip is interfaced to a moving membrane so that proteins are captured in discrete zones as they migrate from the chip. Separations of SDS-protein complexes in the molecular weight range of 11 to 155 kDa were completed in 2 min with 4 × 104 theoretical plates at 460 V/cm. Migration time and peak area relative standard deviations were 3–6% and 0.2% respectively. Detection limit for actin was 0.7 nM. Assays for actin, AMP-kinase, carbonic anhydrase, and lysozyme are shown to demonstrate versatility of the method. Total analysis time including immunoassay was 22–32 min for a single sample. Because processing membrane for immunoassay is the slow step of the assay, sequential injections from different reservoirs on the chip and capture in different tracks on the same membrane allow increased throughput. As a demonstration, 9 injections were collected on one membrane and analyzed in 43 min (~5 min/sample). Further improvements in throughput are possible with more reservoirs or parallel channels.

Jin, Shi; Anderson, Gwendolyn J.; Kennedy, Robert T.

2013-01-01

117

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis  

Microsoft Academic Search

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression

Katherine M Williams; Thomas Marshall

2001-01-01

118

Analysis of total proteins in pollen of Humulus scandens Lour in Wuhan Region of China by two-dimensional electrophoresis  

Microsoft Academic Search

Total proteins in the pollen of Humulus scandens Lour, one of the most popular aeroallergens in China, were analyzed by two-dimensional electrophoresis in the current study.\\u000a The proteins were extracted by Trichloracetic acid (TCA) method, and then separated by isoelectric focusing as the first dimension\\u000a and SDS-PAGE as the second dimension. The spots of proteins were visualized by staining with

Dongdong Li; Shaoheng He

2007-01-01

119

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.

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

2013-01-01

120

Capillary zone electrophoresis of proteins in semidilute polymer solutions: inter- and intra-polymer predictability of size-dependent retardation.  

PubMed

The retardation of three "spherical" proteins with Stokes' radii of 2.0, 2.4, and 3.0 nm (35-104 kDa) was studied in capillary zone electrophoresis (CZE), using semidilute solutions of polyethylene glycol (PEG), linear polyacrylamide (PA), and polyvinyl alcohol (PVA). The purpose was to test the models predicting that the ratio of particle radius, R, to the mesh size of polymer network (the correlation or screening length of a semidilute polymer solution), xi, directly governs the size-dependent retardation in the form: mu/muo = exp (-R/xi). Here xi = kc-0.75, where c is polymer concentration and the numerical factor kcan be calculated based on polymer molecular weight. In application to polymers in a "good solvent" (PA and PEG in the aqueous buffer) and to proteins of 2.4 and 3.0 nm radius, that relation between relative mobility and R/xi was found to be obeyed for PA, while for PEG the value of k derived from retardation experiments significantly exceeded that which was theoretically calculated. Thus, the retardation appears to be polymer-specific, rather than universal, even for polymers in a "good solvent". It is suggested that, in that case, retardation of proteins of R > 2 nm be quantitatively described in the form mu/muo = exp[-p(R/xi], where p is a parameter depending on monomer type and/or polymer polydispersity. For PVA, the logarithm of mu/muo was found to be linearly related to c (in line with the prediction that the aqueous buffer is a "poor solvent" for this polymer) and to be near-independent of R. PMID:10546824

Stastna, M; Radko, S P; Chrambach, A

1999-10-01

121

A quantitative measure for protein conformational heterogeneity  

NASA Astrophysics Data System (ADS)

Conformational heterogeneity is a defining characteristic of proteins. Intrinsically disordered proteins (IDPs) and denatured state ensembles are extreme manifestations of this heterogeneity. Inferences regarding globule versus coil formation can be drawn from analysis of polymeric properties such as average size, shape, and density fluctuations. Here we introduce a new parameter to quantify the degree of conformational heterogeneity within an ensemble to complement polymeric descriptors. The design of this parameter is guided by the need to distinguish between systems that couple their unfolding-folding transitions with coil-to-globule transitions and those systems that undergo coil-to-globule transitions with no evidence of acquiring a homogeneous ensemble of conformations upon collapse. The approach is as follows: Each conformation in an ensemble is converted into a conformational vector where the elements are inter-residue distances. Similarity between pairs of conformations is quantified using the projection between the corresponding conformational vectors. An ensemble of conformations yields a distribution of pairwise projections, which is converted into a distribution of pairwise conformational dissimilarities. The first moment of this dissimilarity distribution is normalized against the first moment of the distribution obtained by comparing conformations from the ensemble of interest to conformations drawn from a Flory random coil model. The latter sets an upper bound on conformational heterogeneity thus ensuring that the proposed measure for intra-ensemble heterogeneity is properly calibrated and can be used to compare ensembles for different sequences and across different temperatures. The new measure of conformational heterogeneity will be useful in quantitative studies of coupled folding and binding of IDPs and in de novo sequence design efforts that are geared toward controlling the degree of heterogeneity in unbound forms of IDPs.

Lyle, Nicholas; Das, Rahul K.; Pappu, Rohit V.

2013-09-01

122

[Computer-assisted findings on protein electrophoresis on cellulose acetate film].  

PubMed

Until a short time ago efficient computer assisted reporting of electrophoretograms was not possible owing to the lack of appropriate technology. By integrating a fully mechanized electrophoresis system into the laboratory data processing system of a centralized institute of clinical chemistry, the separation and the interpretation of results can be performed for 300 samples per day. The interpretation is based upon available patient data (specified clinical diagnoses, previous results), calculated fractions and analysis of the electrophoretic curve in the area of albumin and the beta- and gamma-globulin fraction. In this way it is possible to classify hereditary bisalbuminaemias and to detect transient bisalbuminaemias and monoclonal gammopathies. Diagnostic indications of specific dysproteinaemias and defect proteinaemias are printed on the report form if individual serum protein fractions and the total protein content are changed specifically. The results of the different electrophoretic fractions, the total protein and a set of possible alterations of the curve are numerically coded according to the scaled biochemical ranges in five definite sections or to a decision matrix (alteration 'given' or 'not given'), respectively. The composed 'result patterns' are matched with preassigned 'reference patterns' stored in the computer file. The procedure is efficient and adaptable to other areas of analysis. PMID:6207264

Knüppel, W; Neumeier, D; Fateh-Moghadam, A; Knedel, M

1984-06-01

123

Prototype for integrated two-dimensional gel electrophoresis for protein separation.  

PubMed

Two-dimensional gel electrophoresis practitioners have long waited for a fully automated system. This article presents an integrated platform that is capable of complete automation from sample introduction to spots detection. The strip gel for the first dimensional separation is fixed on the edge of a discrete planar stage before separation. A pair of platinum pin electrodes for isoelectric focusing (IEF) makes contact from underneath the stage. IEF is performed directly after rehydration and protein loading. After the first dimensional separation, sodium dodecyl sulfate (SDS) equilibration is done on the same stage without moving the gel. The IEF stage is then moved horizontally to couple with a precast second dimensional gel. The <0.5 mm gap between the two gels is filled with poly (ethylene oxide) solution. After SDS-polyacrylamide gel electrohporesis separation, a charge-coupled device camera is used to detect spots via protein native fluorescence excited by a Hg (Xe) lamp with the gel inside the running cell. Potential for full automation is demonstrated with 0.5 microg of Escherichia coli proteins on this miniaturized platform. More than 240 spots are detected in a total experiment time of <2.5 h. PMID:16130711

Xu, Aoshuang; Sluszny, Chanan; Yeung, Edward S

2005-09-16

124

Use of Two-Dimensional Gel Electrophoresis To Measure Changes in Synovial Fluid Proteins from Patients with Rheumatoid Arthritis Treated with Antibody to CD4  

Microsoft Academic Search

Synovial fluid proteins from microliter volumes of synovial fluid were resolved by two-dimensional poly- acrylamide gel electrophoresis and detected by silver staining to investigate the feasibility of using two- dimensional (2D) electrophoresis in the clinical research setting and provide global disease information of disease progression. Several hundred proteins could be resolved as spots, many of which displayed the characteristic pattern

MARJORIE A. SMITH; SATBINDER K. BAINS; JOANNA C. BETTS; ERNEST H. S. CHOY; E. D. Zanders

2001-01-01

125

Improved method for the simultaneous determination of whey proteins, caseins and para-?-casein in milk and dairy products by capillary electrophoresis  

Microsoft Academic Search

A capillary electrophoresis method for the simultaneous determination of whey proteins, caseins and their degradation products, such as para-?-casein, was proposed. The effect of several parameters (pH, ionic strength and concentration of urea in the electrophoresis buffer and applied voltage) on the analysis time and on the separation efficiency of the major milk proteins was studied. Using a hydrophilically coated

Beatriz Miralles; Volker Rothbauer; Mar??a A Manso; Lourdes Amigo; Ingolf Krause; Mercedes Ramos

2001-01-01

126

Changes in myofibrillar proteins during processing of salted cod ( Gadus morhua) as determined by electrophoresis and differential scanning calorimetry  

Microsoft Academic Search

The effects of salt-curing, drying and rehydration on muscle proteins in cod (Gadus morhua) were studied during the processing of heavily salted cod or “bacalhau”. The aim was to observe conformational stability and possible degradation or denaturation, with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and differential scanning calorimetry (DSC). The salting process significantly decreased the heat stabilities of both

Kristin Anna Thorarinsdottir; Sigurjon Arason; Margret Geirsdottir; Sigurdur G. Bogason; Kristberg Kristbergsson

2002-01-01

127

Rapid Quantitative Analysis of Proteins or Protein Function in Complex Mixtures.  

National Technical Information Service (NTIS)

Analytical reagents and mass spectrometry-based methods using these reagents for the rapid, and quantitative analysis of proteins or protein function in mixtures of proteins. The methods employ affinity labeled protein reactive reagents having three porti...

R. H. Aebersold M. H. Gelb S. P. Gygi C. R. Scott F. Turecek S. A. Gerber B. Rist

2004-01-01

128

Separation of acidic and basic proteins by capillary electrophoresis using gemini surfactants and gemini-capped nanoparticles as buffer additives  

Microsoft Academic Search

This paper demonstrated simultaneous separation of acidic and basic proteins using cationic gemini surfactants as buffer additives\\u000a in capillary electrophoresis. We showed that even at a low concentration (0.1 mmol·L?1) of alkanediyl-?,?-bis(dimethyloctadecylammonium bromide) (18-s-18), the wall adsorption of both acidic and basic proteins could be effectively suppressed under acidic conditions. Smaller\\u000a micelle size (e.g., s = 5–8) is more effective

Qian Liu; YanQing Li; YanMin Yang; ShouZhuo Yao

2009-01-01

129

Search for serum protein-binding disaccharides and disaccharide-binding serum proteins by affinity capillary electrophoresis.  

PubMed

The potential use of affinity capillary electrophoresis in a microscale search for mutually interacting substances in biological fluid is demonstrated. Some disaccharides, especially gentiobiose (Gen), derivatized with 1-phenyl-3-methyl-5-pyrazolone, caused peak retardation when electrophoresed in a neutral running buffer, containing human serum. Gen, the most significantly retarded disaccharide, was converted to its negatively charged bis-mercaptoethanesulfonate derivative (MerESGen), and a serum sample was analyzed in a neutral buffer containing the derivatized disaccharide. Two peaks, belonging to the beta-globulin fraction, were found to be remarkably retarded in the buffer containing MerES-Gen in a concentration-dependent way. These findings prove an interaction between disaccharides and serum proteins. PMID:15004849

Taga, Atsushi; Yamamoto, Yuka; Maruyama, Rie; Honda, Susumu

2004-03-01

130

OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins.  

PubMed

In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum-protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and post-separation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum-protein bond stability under the conditions studied. SDS-PAGE in the absence of ?-mercaptoethanol or dithiotreitol preserved the platinum-protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3-2.0 ?g were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated. PMID:21255782

Mena, Ma Luz; Moreno-Gordaliza, Estefanía; Moraleja, Irene; Cañas, Benito; Gómez-Gómez, Ma Milagros

2011-03-01

131

Specific and general stress proteins in Bacillus subtilis - a two-dimensional protein electrophoresis study  

Microsoft Academic Search

A computer-aided analysis of high resolution two-dimensional polyacrylamide gels was used to investigate the changes in the protein synthesis profile in B. subtilis wild-type strains and sigB mutants in response to heat shock, salt and ethanol stress, and glucose or phosphate starvation. The data provided evidence that the induction of at least 42 general stress proteins absolutely required the alternative

Jtjrg Bernhardt; U. Volker; A. Volker; Haike Antelmann; Roland Schmid; Hiltraut Mach; Michael Hecker

1997-01-01

132

Protein Reverse Staining: High-Efficiency Microanalysis of Unmodified Proteins Detected on Electrophoresis Gels  

Microsoft Academic Search

A methodology is presented for efficiently gaining structural information from electrophoresed proteins after on-gel detection by imidazole-sodium dodecyl sulfate-zinc reverse staining. As a consequence of reverse staining, (a) protein bands arise transparent against a deep white-stained background, limits of detection being in the femtomole range; (b) there is no loss of image when the gel is kept in distilled water

C. Fernandezpatron; M. Calero; P. R. Collazo; J. R. Garcia; J. Madrazo; A. Musacchio; F. Soriano; R. Estrada; R. Frank; L. R. Castellanosserra; E. Mendez

1995-01-01

133

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

PubMed

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

2006-11-17

134

Electrophoresis of DNA-protein conjugates: hydrodynamic end effects and electrostatic interactions  

NASA Astrophysics Data System (ADS)

DNA fragments can be separated by length in free solution by attaching them to neutral or positively charged ``drag-tags'' (e.g., proteins), a technique known as end-labeled free-solution electrophoresis (ELFSE). We first extend a previous theory of ELFSE for neutral drag-tags to the case of weakly charged drag-tags. The simplest variant of the theory assumes that all parts contribute equally to the mobility (no end effects) and that both the DNA and the drag-tag are fully flexible and do not interact. We analyze the influence of these assumptions. We obtain the exact (within the Kirkwood-Riseman approximation) form of the function describing the end effects for flexible polymers. The main significance of the end effects is the N-3/4 (instead of N-1) form of the correction to the mobility for large DNA lengths N. We also show that the end effects are weaker for semiflexible and stiff polymers. Using a simple model, we study how the conformation of the drag-tag changes due to the electrostatic interaction with the DNA and how this influences the mobility.

Chubynsky, Mykyta V.; Slater, Gary W.

2012-02-01

135

Influence of one- and two-dimensional gel electrophoresis procedure on metal–protein bindings examined by electrospray ionization mass spectrometry, inductively coupled plasma mass spectrometry, and ultrafiltration  

Microsoft Academic Search

Three independent methods, (i) electrospray ionization mass spectrometry (ESI-MS), (ii) carrying out the complete protein preparation procedure required for protein gel electrophoresis (GE) including extraction, precipitation, washing, and desalting with subsequent microwave digestion of the produced protein fractions for metal content quantification, and (iii) ultrafiltration for separating protein-bound and unbound metal fractions, were employed to elucidate the influences of protein

Anne-Christine Schmidt; Bianca Störr; Nicolai-Alexeji Kummer

2011-01-01

136

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis.  

PubMed

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE. PMID:11245891

Williams, K M; Marshall, T

2001-02-26

137

Horizontal polyacrylamide electrophoresis for the determination of serum protein (haptoglobin) and red cell enzyme polymorphisms  

Microsoft Academic Search

General and special procedures for the determination of serum (haptoglobin) and red cell enzyme polymorphisms by horizontal polyacrylamide (PAA) gel electrophoresis are given. As compared to starch gel techniques certain advantages could be established:1.Improved separation and reproducibility of the patterns using PAA.2.PAA gels are less sensitive to heating and desiccation during electrophoresis.3.PAA gels can be stored prior to use.4.PAA is

H. H. Hoppe; Windemut Hennig; B. Brinkmann

1972-01-01

138

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

PubMed

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

2012-10-16

139

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. Contribution to the Topical Issue "Nano-scale Insights into Ion-beam Cancer Therapy", edited by Andrey V. Solov'yov, Nigel Mason, Paulo Limão-Vieira and Malgorzata Smialek-Telega.

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

2014-06-01

140

Speciation of iodine-containing proteins in Nori seaweed by gel electrophoresis laser ablation ICP-MS.  

PubMed

An analytical approach providing an insight into speciation of iodine in water insoluble fraction of edible seaweed (Nori) was developed. The seaweed, harvested in the Galician coast (Northwestern Spain), contained 67.7±1.3?gg(-1) iodine of which 25% was water soluble and could be identifies as iodide. Extraction conditions of water insoluble residue using urea, NaOH, SDS and Triton X-100 were investigated. The protein pellets obtained in optimized conditions (after precipitation of urea extracts with acetone), were digested with trypsin and protease XIV. Size exclusion chromatography-ICP-MS of both enzymatic digests demonstrated the occurrence of iodoaminoacids putatively present in proteins. Intact proteins could be separated by gel electrophoresis after an additional extraction of the protein extract with phenol. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) with laser ablation ICP-MS detection of (127)I indicated the presence of iodine in protein bands corresponding to molecular masses of 110kDa, 40kDa, 27kDa, 20kDa and 10kDa. 2D IEF-SDS PAGE with laser ablation ICP-MS (127)I imaging allowed the detection of 5 iodine containing protein spots in the alkaline pI range. PMID:24913873

Romarís-Hortas, V; Bianga, J; Moreda-Piñeiro, A; Bermejo-Barrera, P; Szpunar, J

2014-09-01

141

Ultrafast quantitation of six quinolones in water samples by second-order capillary electrophoresis data modeling with multivariate curve resolution-alternating least squares.  

PubMed

This paper presents the development of a capillary electrophoresis method with diode array detector coupled to multivariate curve resolution-alternating least squares (MCR-ALS) to conduct the resolution and quantitation of a mixture of six quinolones in the presence of several unexpected components. Overlapping of time profiles between analytes and water matrix interferences were mathematically solved by data modeling with the well-known MCR-ALS algorithm. With the aim of overcoming the drawback originated by two compounds with similar spectra, a special strategy was implemented to model the complete electropherogram instead of dividing the data in the region as usually performed in previous works. The method was first applied to quantitate analytes in standard mixtures which were randomly prepared in ultrapure water. Then, tap water samples spiked with several interferences were analyzed. Recoveries between 76.7 and 125 % and limits of detection between 5 and 18 ?g L(-1) were achieved. PMID:24566760

Alcaráz, Mirta R; Vera-Candioti, Luciana; Culzoni, María J; Goicoechea, Héctor C

2014-04-01

142

Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Shigella dysenteriae proteome.  

PubMed

The in vitro stationary phase proteome of the human pathogen Shigella dysenteriae serotype 1 (SD1) was quantitatively analyzed in Coomassie Blue G250 (CBB)-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method APEX for absolute protein expression measurements. Of the 4502 genome-predicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A squared Pearson correlation analysis revealed a reasonably good correlation (R2 = 0.67) for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. Stoichiometric ratios of subunits of protein complexes characterized in E. coli were compared with APEX quantitative ratios of orthologous SD1 protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, demonstrating versatile applications of the APEX method in quantitative proteomics. PMID:19563668

Kuntumalla, Srilatha; Braisted, John C; Huang, Shih-Ting; Parmar, Prashanth P; Clark, David J; Alami, Hamid; Zhang, Quanshun; Donohue-Rolfe, Arthur; Tzipori, Saul; Fleischmann, Robert D; Peterson, Scott N; Pieper, Rembert

2009-01-01

143

Proteins synthesized in African swine fever virus-infected cells analyzed by two-dimensional gel electrophoresis.  

PubMed

At least 74 acidic and 37 basic proteins are synthesized in African swine fever virus (ASFV)-infected monkey cells not detected in uninfected cells analyzed by two-dimensional gel electrophoresis. Essentially all the proteins synthesized early during infection are also observed at late times. The use of inhibitors such as cycloheximide and phosphonoacetate has led to the identification of 34 immediate early and 13 delayed early polypeptides. Therefore 64 proteins were classified as late polypeptides. Several ASFV-induced proteins are phosphorylated as proteins a1, a4, a20, a41, a48, a49, a51, a52, a55, a58, a67, b2, b12, b28, and b32. PMID:3629977

Urzainqui, A; Tabarés, E; Carrasco, L

1987-09-01

144

Aptamers in Affinity Separations:Capillary Electrophoresis  

Microsoft Academic Search

\\u000a Assays employing aptamers in capillary electrophoresis (CE), including competitive and noncompetitive assays, fluorescence\\u000a polarization (FP) assays, nonequilibrium capillary electrophoresis of equilibrium mixtures, and affinity-polymerase chain\\u000a reaction-CE assays, are summarized. These assays can be used to estimate dissociation rate and equilibrium binding constants,\\u000a determine binding stoichiometries, study molecular interactions, and quantitatively determine specific analytes (e.g., proteins)\\u000a in complex media. They can

Jeffrey W. Guthrie; Yuanhua Shao; X. Chris Le

145

The detection and quantitation of protein oligomerization.  

PubMed

There are many different techniques available to biologists and biochemists that can be used to detect and characterize the self-association of proteins. Each technique has strengths and weaknesses and it is often useful to combine several approaches to maximize the former and minimize the latter. Here we review a range of methodologies that identify protein self-association and/or allow the stoichiometry and affinity of the interaction to be determined, placing an emphasis on what type of information can be obtained and outlining the advantages and disadvantages involved. In general, in vitro biophysical techniques, such as size exclusion chromatography, analytical ultracentrifugation, scattering techniques, NMR spectroscopy, isothermal titration calorimetry, fluorescence anisotropy and mass spectrometry, provide information on stoichiometry and/or binding affinities. Other approaches such as cross-linking, fluorescence methods (e.g., fluorescence correlation spectroscopy, FCS; Förster resonance energy transfer, FRET; fluorescence recovery after photobleaching, FRAP; and proximity imaging, PRIM) and complementation approaches (e.g., yeast two hybrid assays and bimolecular fluorescence complementation, BiFC) can be used to detect protein self-association in a cellular context. PMID:22949109

Gell, David A; Grant, Richard P; Mackay, Joel P

2012-01-01

146

A Nanogram Level Colloidal Gold Single Reagent Quantitative Protein Assay  

PubMed Central

We have developed a nanogram level quantitative protein assay based on the binding of colloidal gold to proteins adhered to nitrocellulose paper. The protein-gold complex produces a purple color proportional to the amount of protein present, and the intensity of the stain is quantified by densitometry. Typical assays require minimal starting material (10 – 20 ?l) containing 2 – 5 ?g protein. A small volume (2 ?l) of protein solution is applied to nitrocellulose paper in a grid array, and dried. The nitrocellulose is incubated in colloidal gold suspension with gentle agitation (4–16 h), rinsed with water and scanned. Densitometric analysis of the scanned images allows quantitation of the unknown sample protein concentration by comparison with protein standards placed on the same nitrocellulose grid. The assay requires significantly less sample than conventional protein assays. In this report, the Golddots assay is calibrated against weighed protein samples, and compared with the Pierce Micro BCA protein assay kit. In addition, the assay is evaluated with several known proteins with different physical properties.

Harrison, Gerald; Haffey, Patrick; Golub, Ellis E.

2008-01-01

147

[The byssus of Mytilus: electrophoresis of hydroxyproline-rich proteins from the "collagen gland"].  

PubMed

An hydroxyproline rich fraction has been extracted from the byssus gland of Mytilus. This fraction could be the intracellular precursor of the "secreted collagen" found in the byssus threads. Its molecular weight, estimated through SDS gel electrophoresis, appears lower than that of a chains of mesenchymal collagen. PMID:825306

Pujol, J P; Bocquet, J; Borel, J P

1976-09-20

148

Quantitative Proteomic profiling identifies protein correlates to EGFR kinase inhibition  

PubMed Central

Clinical oncology is hampered by a lack of tools to accurately assess a patient’s response to pathway-targeted therapies. Serum and tumor cell surface proteins whose abundance, or change in abundance in response to therapy, differentiates patients responding to a therapy from patients not-responding to a therapy could be usefully incorporated into tools for monitoring response. Here we posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. In this study we use Stable Isotope Labeling of Amino acids in Culture (SILAC) with proteomic technologies to quantitatively analyze the gefitinib-related protein changes in a model system for sensitivity to EGFR targeted tyrosine kinase inhibitors. We identified 3,707 intracellular proteins, 1,276 cell surface proteins, and 879 shed proteins. More than 75% of the proteins identified had quantitative information and a subset consisting of [400] proteins showed a statistically significant change in abundance following gefitinib treatment. We validated the change in expression profile in vitro and screened our panel of response markers in an in vivo isogenic resistant model and demonstrated that these were markers of gefitinib response and not simply markers of phospho-EGFR downregulation. In doing so, we also were able to identify which proteins might be useful as markers for monitoring response and which proteins might be useful as markers for a priori prediction of response.

Kani, Kian; Faca, Vitor M.; Hughes, Lindsey D.; Zhang, Wenxuan; Fang, Qiaojun; Shahbaba, Babak; Luethy, Roland; Erde, Jonathan; Schmidt, Joanna; Pitteri, Sharon J.; Zhang, Qing; Katz, Jonathan E.; Gross, Mitchell E.; Plevritis, Sylvia K.; McIntosh, Martin W.; Jain, Anjali; Hanash, Sam; Agus, David B.; Mallick, Parag

2014-01-01

149

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.

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

2013-01-01

150

Rapid protein separations in ultra-short microchannels: microchip sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing.  

PubMed

We have developed novel protein gel electrophoresis techniques, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) in short microchannels (approximately millimeters) that take less than a minute. A photopatterning technique was used to cast in situ crosslinked polyacrylamide gel in a microchannel to perform SDS-PAGE. A fluorescent protein marker sample (Mr range of 20,000-200,000) was separated in less than 30 s in less than 2 mm of channel length. Crosslinked polyacrylamide gel, patterned in channels using UV light, provides higher sieving power and sample stacking effect, therefore yielding faster and higher-resolution separation in a chip. IEF of proteins was also achieved in a microchannel, and several proteins were focussed within tens of seconds in mm-length channels. As resolution in IEF is independent of separation distance, focusing in ultra-short channels results in not only faster separation but also more concentrated bands potentially allowing detection of low-concentration species. PMID:15499934

Han, Jongyoon; Singh, Anup K

2004-09-17

151

Targeted quantitative analysis of synaptic proteins in Alzheimer's disease brain.  

PubMed

Brain tissue from Alzheimer's disease (AD) patients shows significant loss of synapses in selected regions. Synaptic degeneration is the best predictor for loss of cognitive functions ante mortem. The molecular mechanisms underlying this degeneration remain unknown. Our previous two-dimensional gel-electrophoresis proteomics study found that 26 synaptic proteins are differentially expressed in Alzheimer's brain. It is difficult to quantify global protein expression using this technique because (a) several proteins can migrate together and (b) isoforms of the same protein can migrate to different places. The present study estimated global synaptic protein levels by label-free multiple reaction monitoring. Multiple reaction monitoring is a powerful and sensitive mass spectrometry technique that specifically targets multiple protein of interests. The severely AD-affected hippocampus was compared with motor cortex, a relatively spared region. We targeted ten proteins in autopsy brain based on the earlier study. Analytes separated by high performance liquid-chromatography were monitored on a hybrid triple quadrupole linear ion trap mass spectrometer in multiple reaction monitoring mode. With the use of an internal standard protein, linear and highly reproducible (CV<9%) label-free assays were achieved. Data were contrasted with the gel-based study to highlight differences and similarities. Significantly higher expression levels of peroxiredoxin-1 (may provide antioxidant protection) and dihydropyrimidinase-related protein-1 (associated with cytoskeletal remodeling) were found in AD hippocampus. Significantly lower levels of peroxiredoxin-1 and the energy-related enzymes creatine kinase B and fructose-bisphosphate aldolase C were found in non-AD hippocampus. Our previously reported difference in synaptotagmin expression is probably isoform-specific. These findings suggest potential roles of key proteins in synaptic loss in AD, and/or a protective mechanism in non-AD brain tissue. PMID:24893329

Chang, Rachel Yoon Kyung; Etheridge, Naomi; Dodd, Peter R; Nouwens, Amanda S

2014-09-01

152

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.

2012-01-01

153

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.

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

2012-01-01

154

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.

1981-03-01

155

Assessing association between protein truncating variants and quantitative traits  

PubMed Central

Motivation: In sequencing studies of common diseases and quantitative traits, power to test rare and low frequency variants individually is weak. To improve power, a common approach is to combine statistical evidence from several genetic variants in a region. Major challenges are how to do the combining and which statistical framework to use. General approaches for testing association between rare variants and quantitative traits include aggregating genotypes and trait values, referred to as ‘collapsing’, or using a score-based variance component test. However, little attention has been paid to alternative models tailored for protein truncating variants. Recent studies have highlighted the important role that protein truncating variants, commonly referred to as ‘loss of function’ variants, may have on disease susceptibility and quantitative levels of biomarkers. We propose a Bayesian modelling framework for the analysis of protein truncating variants and quantitative traits. Results: Our simulation results show that our models have an advantage over the commonly used methods. We apply our models to sequence and exome-array data and discover strong evidence of association between low plasma triglyceride levels and protein truncating variants at APOC3 (Apolipoprotein C3). Availability: Software is available from http://www.well.ox.ac.uk/~rivas/mamba Contact: donnelly@well.ox.ac.uk

Rivas, Manuel A.; Pirinen, Matti; Neville, Matthew J.; Gaulton, Kyle J.; Moutsianas, Loukas; Lindgren, Cecilia M.; Karpe, Fredrik; McCarthy, Mark I.; Donnelly, Peter

2013-01-01

156

Pressure-Assisted Capillary Electrophoresis Coupling with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging for Quantitative Analysis of Complex Peptide Mixtures  

PubMed Central

Herein we report a pressure-assisted capillary electrophoresis-mass spectrometric imaging (PACE-MSI) platform for peptide analysis. This new platform has addressed the sample diffusion and peak splitting problems that appeared in our previous groove design, and it enables homogenous deposition of the CE trace for high-throughput MALDI imaging. In the coupling of CE to MSI, individual peaks (m/z) can be visualized as discrete colored image regions and extracted from the MS imaging data, thus eliminating issues with peak overlapping and reducing reliance on an ultra-high mass resolution mass spectrometer. Through a PACE separation, 46 tryptic peptides from bovine serum albumin and 150 putative neuropeptides from the pericardial organs of a model organism blue crab Callinectes sapidus were detected from the MALDI MS imaging traces, enabling four to six-fold increase of peptide coverage as compared with direct MALDI MS analysis. For the first time, quantitation with high accuracy was obtained using PACE-MSI for both digested tryptic peptides and endogenous neuropeptides from complex biological samples in combination with isotopic formaldehyde labeling. Although MSI is typically employed in tissue imaging, we show in this report that, it offers a unique tool for quantitative analysis of complex trace-level analytes with CE separation. These results demonstrate a great potential of the PACE-MSI platform for enhanced quantitative proteomics and neuropeptidomics.

Zhang, Zichuan; Ye, Hui; Wang, Junhua; Hui, Limei; Li, Lingjun

2012-01-01

157

ShapeFinder: A software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis  

PubMed Central

Analysis of the long-range architecture of RNA is a challenging experimental and computational problem. Local nucleotide flexibility, which directly reports underlying base pairing and tertiary interactions in an RNA, can be comprehensively assessed at single nucleotide resolution using high-throughput selective 2?-hydroxyl acylation analyzed by primer extension (hSHAPE). hSHAPE resolves structure-sensitive chemical modification information by high-resolution capillary electrophoresis and typically yields quantitative nucleotide flexibility information for 300–650 nucleotides (nt) per experiment. The electropherograms generated in hSHAPE experiments provide a wealth of structural information; however, significant algorithmic analysis steps are required to generate quantitative and interpretable data. We have developed a set of software tools called ShapeFinder to make possible rapid analysis of raw sequencer data from hSHAPE, and most other classes of nucleic acid reactivity experiments. The algorithms in ShapeFinder (1) convert measured fluorescence intensity to quantitative cDNA fragment amounts, (2) correct for signal decay over read lengths extending to 600 nts or more, (3) align reactivity data to the known RNA sequence, and (4) quantify per nucleotide reactivities using whole-channel Gaussian integration. The algorithms and user interface tools implemented in ShapeFinder create new opportunities for tackling ambitious problems involving high-throughput analysis of structure–function relationships in large RNAs.

Vasa, Suzy M.; Guex, Nicolas; Wilkinson, Kevin A.; Weeks, Kevin M.; Giddings, Morgan C.

2008-01-01

158

ShapeFinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis.  

PubMed

Analysis of the long-range architecture of RNA is a challenging experimental and computational problem. Local nucleotide flexibility, which directly reports underlying base pairing and tertiary interactions in an RNA, can be comprehensively assessed at single nucleotide resolution using high-throughput selective 2'-hydroxyl acylation analyzed by primer extension (hSHAPE). hSHAPE resolves structure-sensitive chemical modification information by high-resolution capillary electrophoresis and typically yields quantitative nucleotide flexibility information for 300-650 nucleotides (nt) per experiment. The electropherograms generated in hSHAPE experiments provide a wealth of structural information; however, significant algorithmic analysis steps are required to generate quantitative and interpretable data. We have developed a set of software tools called ShapeFinder to make possible rapid analysis of raw sequencer data from hSHAPE, and most other classes of nucleic acid reactivity experiments. The algorithms in ShapeFinder (1) convert measured fluorescence intensity to quantitative cDNA fragment amounts, (2) correct for signal decay over read lengths extending to 600 nts or more, (3) align reactivity data to the known RNA sequence, and (4) quantify per nucleotide reactivities using whole-channel Gaussian integration. The algorithms and user interface tools implemented in ShapeFinder create new opportunities for tackling ambitious problems involving high-throughput analysis of structure-function relationships in large RNAs. PMID:18772246

Vasa, Suzy M; Guex, Nicolas; Wilkinson, Kevin A; Weeks, Kevin M; Giddings, Morgan C

2008-10-01

159

Detection of tetracysteine-tagged proteins using a biarsenical fluorescein derivative through dry microplate array gel electrophoresis.  

PubMed

The design of an extended-run 96-well sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system and the development of protein detection technology based upon fluorescein derivatives that bind to peptide epitope tags, allows the creation of a truly high-throughput analysis of protein expression, where less than 20 min are needed to separate proteins and analyze results. We demonstrate the overall capabilities of such a method combination in a complex cell lysate background, while comparing the specific results obtained using a biarsenical fluorescein-derivative and tetracysteine epitope-tagged proteins with total protein staining using a fluorescent gel stain and with Western blotting where an anti-oligohistidine (His) tag antibody has been employed. When applied on purified target proteins without extraneous protein background, the demonstrated sensitivity of the assay on the extended-run 96-array precast SDS-PAGE system allows detection of quantities of tagged protein as low as 1 pmol per band. PMID:15300761

Feldman, Galia; Bogoev, Roumen; Shevirov, Julia; Sartiel, Adam; Margalit, Ilana

2004-08-01

160

Use of two-dimensional gel electrophoresis in predictive toxicology: identification of potential early protein biomarkers in chemically induced hepatocarcinogenesis.  

PubMed

Our current approach focused on the identification of potential early protein biomarker signatures which are indicative of the carcinogenic processes in rats exposed to 20 mg/kg of the liver carcinogen N-nitrosomorpholine (NNM). Treated liver was investigated at different timepoints. Therefore, proteins were separated by two-dimensional gel electrophoresis as a first step prior to identification of differentially expressed proteins by mass spectrometry. Proteomic analysis of liver samples after one day of exposure revealed significant upregulation of proteins involved in response to cellular stress induced by NNM (superoxide dismutase, heat shock protein 60, peroxiredoxin). Eighteen weeks after withdrawal of NNM, we were able to identify cancer-related proteins in rat liver bearing malignant, transformed cells (caspase-8 precursor, vimentin, Rho GDP dissociation inhibitor). Some of these proteins were already deregulated after three weeks of exposure indicating their potential usefulness as early predictive biomarkers for liver carcinogenicity (annexin A5, fructose-1,6-bisphosphatase). As regulatory toxicology approaches usually include the investigation of carcinogenicity in two-years studies in rodents, especially the detection of early protein biomarker signatures which precede the appearance of neoplasia, demonstrates the high potential of proteomics approaches to substantially reduce the time and costs of carcinogenicity testing. PMID:15816005

Fella, Kerstin; Glückmann, Matthias; Hellmann, Jürgen; Karas, Michael; Kramer, Peter-Jürgen; Kröger, Michaela

2005-05-01

161

Proteomic analysis of plasma proteins in diabetic rats by 2D electrophoresis and MALDI-TOF-MS.  

PubMed

Despite tremendous advances in our understanding of the molecular basis of diabetes mellitus, substantial gaps still remain in our understanding of disease pathogenesis and in the development of effective strategies for early diagnosis and treatment. The proteomic approach has offered many opportunities and challenges in identifying new marker proteins and therapeutic targets, i.e., using 2D-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. The differential protein expressions were analyzed in alloxan-induced diabetic rats treated with Cynodon dactylon leaf extract. The plant extract was administered for 15 days that resulted in a significant increase in plasma insulin and C-peptide levels. We have also identified four differentially expressed proteins from rat plasma. These four diabetes-associated proteins were broadly classified into three groups as per their function: (1) lipid metabolism-associated protein (Apo A-IV), (2) antioxidant activity-related proteins [preprohaptoglobin and heat shock proteins B8 (HspB8)], and (3) muscle function-related protein (TPM3). Apo A-IV, HspB8, and preprohaptoglobin may play a key role in the recovery of diabetes mellitus and also prevent the diabetes-associated complications such as prevention of oxidative stress due to free radical and free hemoglobin. These results show the value of proteomic approach in identifying the potential markers that may eventually serve as diagnostic markers or therapeutic targets. PMID:22258647

Karthik, D; Ilavenil, S; Kaleeswaran, B; Sunil, S; Ravikumar, S

2012-03-01

162

Gold nanoparticles amplified ultrasensitive quantification of human urinary protein by capillary electrophoresis with on-line inductively coupled plasma mass spectroscopic detection.  

PubMed

Quantitative analysis of proteins play pivotal roles in basic discovery research and clinical applications, and the analytical challenge is to provide sufficient sensitivity to determine the proteins at endogenous levels. Here, we report a strategy for ultrasensitive quantification of human urinary protein by capillary electrophoresis with on-line inductively coupled plasma mass spectroscopic detection (CE-ICPMS) in conjunction with gold nanoparticles (AuNPs) amplification. The albumin in the sample solution was incubated with excess AuNPs to form the AuNP-albumin adduct. The excess AuNPs and the AuNP-albumin adduct were then effectively separated by CE for on-line ICPMS detection. As a result of AuNPs-tagging, more than 2000 gold atoms on average were attached to each albumin molecule to successfully achieve a significant amplification of ICPMS signal with extremely low limit of detection (0.5 pM for 280 nL of sample injection, corresponding to 0.1 amol) and a wide linear response over 4 orders of magnitude. The relative standard deviations of the migration time, peak area, and peak height for seven replicate injections of a mixture of 0.4 pM AuNPs and 9.0 pM albumin ranged from 1.8% to 4.4%. The developed method was successfully applied for detecting albumin in human urine samples with quantitative recoveries in the range of 93.0-99.7%. The methodology demonstrated here has potential for simultaneous determination of low-abundance multiple biomarkers of interest via multiple nanomaterials tags because of high-resolution CE separation and ultrasensitive ICPMS detection. PMID:20450228

Liu, Jing-Min; Li, Yan; Jiang, Yan; Yan, Xiu-Ping

2010-07-01

163

Quantitating metabolites in protein precipitated serum using NMR spectroscopy.  

PubMed

Quantitative NMR-based metabolite profiling is challenged by the deleterious effects of abundant proteins in the intact blood plasma/serum, which underscores the need for alternative approaches. Protein removal by ultrafiltration using low molecular weight cutoff filters thus represents an important step. However, protein precipitation, an alternative and simple approach for protein removal, lacks detailed quantitative assessment for use in NMR based metabolomics. In this study, we have comprehensively evaluated the performance of protein precipitation using methanol, acetonitrile, perchloric acid, and trichloroacetic acid and ultrafiltration approaches using 1D and 2D NMR, based on the identification and absolute quantitation of 44 human blood metabolites, including a few identified for the first time in the NMR spectra of human serum. We also investigated the use of a "smart isotope tag," (15)N-cholamine for further resolution enhancement, which resulted in the detection of a number of additional metabolites. (1)H NMR of both protein precipitated and ultrafiltered serum detected all 44 metabolites with comparable reproducibility (average CV, 3.7% for precipitation; 3.6% for filtration). However, nearly half of the quantified metabolites in ultrafiltered serum exhibited 10-74% lower concentrations; specifically, tryptophan, benzoate, and 2-oxoisocaproate showed much lower concentrations compared to protein precipitated serum. These results indicate that protein precipitation using methanol offers a reliable approach for routine NMR-based metabolomics of human blood serum/plasma and should be considered as an alternative to ultrafiltration. Importantly, protein precipitation, which is commonly used by mass spectrometry (MS), promises avenues for direct comparison and correlation of metabolite data obtained from the two analytical platforms to exploit their combined strength in the metabolomics of blood. PMID:24796490

Nagana Gowda, G A; Raftery, Daniel

2014-06-01

164

Electrophoresis experiments for space  

NASA Astrophysics Data System (ADS)

It has long been hoped that space could alleviate the problems of large-scale, high-capacity electrophoresis. Support media and reduced chamber dimensions of capillary electrophoresis have established the physical boundaries for Earth-based systems. Ideally, electrophoresis conducted in a virtual weightless environment in an unrestricted ``free'' fluid should have great potential. The electrophoresis and isoelectric focusing experiments done in the reduced gravity over the past twenty-five years have demonstrated the absence of thermal convection and sedimentation as well as the presence of electrohydrodynamics that requires careful control. One commercial venture produced gram amounts of an electrophoretically purified protein during seven Space Shuttle flights but the market disappeared in the six years between experiment conception and performance on the Space Shuttle. Our accumulated experience in microgravity plus theoretical models predict improvements that should be possible with electrophoresis if past problems are considered and both invention of new technologies and innovation of procedures on the Space Station are encouraged. .

Snyder, Robert S.; Rhodes, Percy H.

2000-01-01

165

An optimized procedure for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of hydrophobic peptides from an integral membrane protein.  

PubMed

A procedure for successful analysis of the hydrophobic tryptic peptides of the Neurospora crassa plasma membrane H+-ATPase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. The features of this procedure that are essential for the best results include (i) treatment of the hydrophobic peptide samples with neat trifluoroacetic acid, (ii) dissolution and disaggregation of the hydrophobic peptide samples with SDS at 0 degrees C, (iii) SDS-PAGE of the hydrophobic peptide samples in gels containing a 200:1 ratio of acrylamide to bisacrylamide and a 5-20% convex acrylamide gradient, and (iv) silver-staining of the gels after electrophoresis. This method results in the reproducible resolution and visualization of the H+-ATPase hydrophobic tryptic peptides, which range in size from ca. 5 to 21 kDa, as well as other peptides and proteins ranging in size from ca. 2.5 to 150 kDa. The methods described should also prove useful in other studies where resolution and visualization of hydrophobic peptides of integral membrane proteins are required. PMID:2525882

Hennessey, J P; Scarborough, G A

1989-02-01

166

Two-dimensional electrophoresis protein profiling and identification in rat bronchoalveolar lavage fluid following allergen and endotoxin challenge.  

PubMed

The protein content of bronchoalveolar lavage fluid (BALF) from actively sensitised Brown Norway (BN) rats challenged with allergen (ovalbumin, OA) and from naïve Brown Norway rats challenged with endotoxin (lipopolysaccharide, LPS) was analyzed and compared to healthy controls treated with vehicle only. BALF proteins were analyzed by one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis and identified by peptide mass fingerprinting matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or nanoliquid chromatography-tandem MS (nanoLC-MS/MS) after in-gel trypsin digestion of selected 2-D gel spots. Our study shows that the BALF protein profile is significantly different in animals after allergen (OA) or endotoxin (LPS) challenge as compared to controls, concerning the content of proteins derived from plasma or produced locally in the lung. In both challenges the following proteins presented patterns which differed qualitatively compared to control: T-kininogen I and II, alpha-1-antitrypsin, calgranulin A, fetuin A and B, and haptoglobin. Other proteins were diminished in both challenges, such as Clara cell 10 kDa secretory protein (CC10) and pulmonary surfactant associated protein B (SP-B); c-reactive protein increased in the OA-challenge and decreased in the LPS-challenge, and pulmonary surfactant associated protein A (SP-A) was decreased in the OA-challenge and was not significantly changed in the LPS-challenge. The identified proteins could be important not only for the diagnosis but have also interesting implications for medical treatment of lung inflammatory conditions. Furthermore, even if based on a limited number of animals, our results are of interest for the identification of lung protein markers and a better understanding of the mechanisms involved in the pathogenesis of lung diseases. PMID:15221771

Signor, Luca; Tigani, Bruno; Beckmann, Nicolau; Falchetto, Rocco; Stoeckli, Markus

2004-07-01

167

Fast top-down intact protein characterization with capillary zone electrophoresis-electrospray ionization tandem mass spectrometry  

PubMed Central

Capillary zone electrophoresis (CZE)-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was applied for rapid top-down intact protein characterization. A mixture containing four model proteins (cytochrome c, myoglobin, bovine serum albumin (BSA) and beta casein) was used as the sample. The CZE-ESI-MS system was first evaluated with the mixture. The four model proteins and five impurities were baseline separated within 12 min. The limits of detection (s/n = 3) of the four model proteins ranged from 20 amole (cytochrome c) to 800 amole (BSA). The relative standard deviations of migration time and intensity for the four model proteins were less than 3% and 30%, respectively, in quintuplicate runs. CZE-ESI-MS/MS was then applied for top-down characterization of the mixture. Three of the model proteins (all except BSA) and an impurity (bovine transthyretin) were confidently identified by database searching of the acquired tandem spectra from protein fragmentation. Modifications including phosphorylation, N-terminal acetylation, and heme group binding were identified.

Sun, Liangliang; Knierman, Michael D.; Zhu, Guijie; Dovichi, Norman J.

2013-01-01

168

Proteomic analysis of surface proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry  

PubMed Central

Background Trichinella spiralis is a zoonotic tissue-dwelling parasitic nematode that infects humans and other mammals. Its surface proteins are recognized as antigenic in many infected hosts, being directly exposed to the host’s immune system and are the main target antigens that induce the immune responses. The larval surface proteins may also interact with intestinal epithelial cells and may play an important role in the invasion and development process of T. spiralis. The purpose of this study was to analyze and characterize the surface proteins of T. spiralis muscle larvae by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Methods The surface proteins of T. spiralis muscle larvae were stripped from the cuticle of live larvae by the cetyltrimethylammonium bromide (CTAB) and sodium deoxycholate. The surface protein stripping was examined by an immunofluorescent test (IFT). The surface proteins were analyzed by SDS-PAGE and Western blotting, and then identified by 2-DE and MALDI-TOF/TOF mass spectrometry analysis. Results The IFT results showed that the surface proteins-stripped larvae were not recognized by sera of mice immunized with surface antigens. Western blotting showed 7 of 12 protein bands of the surface proteins were recognized by mouse infection sera at 18 dpi and at 42 dpi. The 2-DE results showed that a total of approximately 33 proteins spots were detected with molecular weights varying from 10 to 66 kDa and isoelectric point (pI) from 4 to 7. Twenty-seven of 33 protein spots were identified and characterized to correlate with 15 different proteins. Out of the 14 proteins identified as T. spiralis proteins, 5 proteins (partial P49 antigen, deoxyribonuclease II family protein, two serine proteases, and serine proteinase) had catalytic and hydrolase activity. All of these 5 proteins were also associated with metabolic processes and 2 of the five proteins were associated with cellular processes. Conclusions In this study, T. spiralis muscle larval surface proteins have been identified, which will provide useful information to elucidate the host-parasite interaction, identify the invasion-related proteins, early diagnostic antigens and the targets for a vaccine.

2013-01-01

169

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

Microsoft Academic Search

Background  Wheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the\\u000a abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers\\u000a with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use\\u000a MS\\/MS to distinguish the

Frances M Dupont; William H Vensel; Charlene K Tanaka; William J Hurkman; Susan B Altenbach

2011-01-01

170

Quantitation of talinolol and other beta-blockers by capillary electrophoresis for in vitro drug absorption studies.  

PubMed

A capillary zone electrophoresis method is described for the enantioseparation of talinolol using heptakis(2,3-diacetyl-6-sulfo)-beta-cyclodextrin (HDAS-beta-CD) as a chiral selector. After liquid-liquid extraction of talinolol from physiological solution, electrokinetic injection was employed to improve the sensitivity. The use of a coated capillary was necessary to achieve stable and reproducible enantioseparations. A baseline separation of the talinolol enantiomers was achieved in less than 10 min using 100 mM phosphate solution as background electrolyte and pH 3.5, at the presence of 3.0 mM HDAS-beta-CD and at 20 degrees C. In addition, this analytical condition proved to be useful for the enantioseparation of a number of other beta-blocking agents such as alprenolol, atenolol, bisoprolol, celiprolol, metipranolol, oxprenolol, and sotalol. For determining talinolol, the method could be validated in terms of precision, accuracy and linearity, and was found to be suitable in determination of talinolol enantiomers in highly diluted samples obtained from in vitro experiments. PMID:12900875

Awadallah, Bilal; Schmidt, Peter C; Holzgrabe, Ulrike; Wahl, Martin A

2003-08-01

171

HP-Lattice QSAR for dynein proteins: experimental proteomics (2D-electrophoresis, mass spectrometry) and theoretic study of a Leishmania infantum sequence.  

PubMed

The toxicity and inefficacy of actual organic drugs against Leishmaniosis justify research projects to find new molecular targets in Leishmania species including Leishmania infantum (L. infantum) and Leishmaniamajor (L. major), both important pathogens. In this sense, quantitative structure-activity relationship (QSAR) methods, which are very useful in Bioorganic and Medicinal Chemistry to discover small-sized drugs, may help to identify not only new drugs but also new drug targets, if we apply them to proteins. Dyneins are important proteins of these parasites governing fundamental processes such as cilia and flagella motion, nuclear migration, organization of the mitotic splinde, and chromosome separation during mitosis. However, despite the interest for them as potential drug targets, so far there has been no report whatsoever on dyneins with QSAR techniques. To the best of our knowledge, we report here the first QSAR for dynein proteins. We used as input the Spectral Moments of a Markov matrix associated to the HP-Lattice Network of the protein sequence. The data contain 411 protein sequences of different species selected by ClustalX to develop a QSAR that correctly discriminates on average between 92.75% and 92.51% of dyneins and other proteins in four different train and cross-validation datasets. We also report a combined experimental and theoretic study of a new dynein sequence in order to illustrate the utility of the model to search for potential drug targets with a practical example. First, we carried out a 2D-electrophoresis analysis of L. infantum biological samples. Next, we excised from 2D-E gels one spot of interest belonging to an unknown protein or protein fragment in the region M<20,200 and pI<4. We used MASCOT search engine to find proteins in the L. major data base with the highest similarity score to the MS of the protein isolated from L. infantum. We used the QSAR model to predict the new sequence as dynein with probability of 99.99% without relying upon alignment. In order to confirm the previous function annotation we predicted the sequences as dynein with BLAST and the omniBLAST tools (96% alignment similarity to dyneins of other species). Using this combined strategy, we have successfully identified L. infantum protein containing dynein heavy chain, and illustrated the potential use of the QSAR model as a complement to alignment tools. PMID:18662882

Dea-Ayuela, María Auxiliadora; Pérez-Castillo, Yunierkis; Meneses-Marcel, Alfredo; Ubeira, Florencio M; Bolas-Fernández, Francisco; Chou, Kuo-Chen; González-Díaz, Humberto

2008-08-15

172

Characterization by affinity electrophoresis of an alpha-1,6-glucan-binding protein from Streptococcus sobrinus.  

PubMed

Glucan-binding protein 1 (GBP1), the most abundant glucan-binding protein isolated from culture supernatants of Streptococcus sobrinus 6715-49, has been purified by affinity chromatography on Sephadex G-50 followed by gel permeation chromatography with Bio-Gel P-10. The specificity and affinity of GBP1 for glucans were assessed by affinity electrophoresis. GBP1 did not detectably bind to glucans lacking linear arrays of alpha-1,6 linkages. The association constant for the linear alpha-1,6-glucan Dextran T2000 was 3 x 10(7) M-1. Providing small isomaltosaccharide ligands to compete with this dextran indicated that the binding site maximally accommodated isomaltosaccharides with a degree of polymerization of 8. When glucans produced by purified S. sobrinus glucosyltransferases were tested, GBP1 displayed the highest affinity for the glucan from the soluble-product, primer-independent glucosyltransferase. PMID:2445685

Landale, E C; McCabe, M M

1987-12-01

173

Multilayer polymer microchip capillary array electrophoresis devices with integrated on-chip labeling for high-throughput protein analysis  

PubMed Central

It is desirable to have inexpensive, high-throughput systems that integrate multiple sample analysis processes and procedures, for applications in biology, chemical analysis, drug discovery, and disease screening. In this paper, we demonstrate multilayer polymer microfluidic devices with integrated on-chip labeling and parallel electrophoretic separation of up to 8 samples. Microchannels were distributed in two different layers and connected through interlayer through-holes in the middle layer. A single set of electrophoresis reservoirs and one fluorescent label reservoir address parallel analysis units for up to 8 samples. Individual proteins and a mixture of cancer biomarkers have been successfully labeled on-chip and separated in parallel with this system. A detection limit of 600 ng/mL was obtained for heat shock protein 90. Our integrated on-chip labeling microdevices show great potential for low-cost, simplified, rapid and high-throughput analysis.

Yu, Ming; Wang, Qingsong; Patterson, James E.; Woolley, Adam T.

2011-01-01

174

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.

2011-01-01

175

Identification of a S100 calcium-binding protein expressed in HL60 cells treated with all- trans retinoic acid by two-dimensional electrophoresis and mass spectrometry  

Microsoft Academic Search

To characterize the alteration of protein expression during tumor cell differentiation induced by all-trans retinoic acid (ATRA) and to understand downstream signaling and molecular mechanism of ATRA action, we compared the protein expression profiles in HL-60 cells with ATRA treatment using two-dimensional electrophoresis (2-DE). Although many changes in protein expression were found in 2-DE maps, here we identified two protein

Yuzhang Wu; Zhirong Mou; Jintao Li; Wei Zhou; Bing Wei; Linyun Zou

2004-01-01

176

[Progress in combination of gel electrophoresis and laser ablation inductively coupled plasma mass spectrometry for trace elements determination in proteins].  

PubMed

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has become a very efficient and sensitive trace, ultratrace, and surface analytical technique for the in situ study of the concentration and distribution of the elements in life sciences with high spatial resolution. It is being used more and more frequently in biological, medical materials and protein research, which will lead to a better understanding of physiology and pathology process in cells and tissues. The present review mainly introduces the strategies of combination of gel electrophoresis (GE) with LA-ICP-MS for the quantification of trace elements in proteins, including the proteins separation, elements detection and calibration methods. The paper emphasizes the basic conditions of the proteins separation, focusing on the stability of proteins during GE and the treatment methods of staining and drying of the gel to enable successful detection of the elements by LA-ICP-MS. In addition, the application of GE-LA-ICP-MS in phosphoproteins, selenoproteins and metal-binding proteins is introduced in detail. The prospects and challenge for this technique are discussed as well for further study. PMID:22497164

Wang, Ying; Guo, Yan-li; Yuan, Hong-lin; Wei, Yong-feng; Yan, Hong-tao; Chen, Hui-hui

2012-01-01

177

Secreted proteins of human monocytes. Analysis by two-dimensional gel electrophoresis and effect of lipopolysaccharide.  

PubMed

A monocyte-rich preparation from the adherent cell fraction of human peripheral blood leukocytes was incubated for 1-8 h with [35S]methionine or [3H]leucine in the presence and absence of bacterial lipopolysaccharide (LPS). The macromolecules released into the supernatant were analysed by two-dimensional gel electrophoresis and radioautography. A complex labelling pattern involving at least 20 easily demonstrable and apparently distinct products with a broad range of molecular masses and isoelectric points was observed. LPS or LPS plus actinomycin in combination markedly stimulated the labelling and release of at least twelve different macromolecules ranging in apparent Mr from 12,000 to 46,000. Studies with monocytes that had been additionally purified by centrifugal elutriation and with the monocyte-like human cell line U-937 indicated that monocytes rather than contaminating cells were the source of these products. The majority of the secreted products were unique and did not cross-react with antibodies to interleukin 1 or tumour necrosis factor. The high resolving capacity of two-dimensional gel electrophoresis may be useful to define further the diverse biological activities and potential monokines released from monocytes at various stages of their differentiation and activation. PMID:3257692

Panuska, J R; Fukui, K; Parker, C W

1988-01-15

178

Secreted proteins of human monocytes. Analysis by two-dimensional gel electrophoresis and effect of lipopolysaccharide.  

PubMed Central

A monocyte-rich preparation from the adherent cell fraction of human peripheral blood leukocytes was incubated for 1-8 h with [35S]methionine or [3H]leucine in the presence and absence of bacterial lipopolysaccharide (LPS). The macromolecules released into the supernatant were analysed by two-dimensional gel electrophoresis and radioautography. A complex labelling pattern involving at least 20 easily demonstrable and apparently distinct products with a broad range of molecular masses and isoelectric points was observed. LPS or LPS plus actinomycin in combination markedly stimulated the labelling and release of at least twelve different macromolecules ranging in apparent Mr from 12,000 to 46,000. Studies with monocytes that had been additionally purified by centrifugal elutriation and with the monocyte-like human cell line U-937 indicated that monocytes rather than contaminating cells were the source of these products. The majority of the secreted products were unique and did not cross-react with antibodies to interleukin 1 or tumour necrosis factor. The high resolving capacity of two-dimensional gel electrophoresis may be useful to define further the diverse biological activities and potential monokines released from monocytes at various stages of their differentiation and activation. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 6. Fig. 7.

Panuska, J R; Fukui, K; Parker, C W

1988-01-01

179

Binary Oscillatory Crossflow Electrophoresis  

NASA Technical Reports Server (NTRS)

We present preliminary results of our implementation of a novel electrophoresis separation technique: Binary Oscillatory Cross flow Electrophoresis (BOCE). The technique utilizes the interaction of two driving forces, an oscillatory electric field and an oscillatory shear flow, to create an active binary filter for the separation of charged species. Analytical and numerical studies have indicated that this technique is capable of separating proteins with electrophoretic mobilities differing by less than 10%. With an experimental device containing a separation chamber 20 cm long, 5 cm wide, and 1 mm thick, an order of magnitude increase in throughput over commercially available electrophoresis devices is theoretically possible.

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

1996-01-01

180

Protein Extraction for Two-Dimensional Gel Electrophoresis of Proteomic Profiling in Turfgrass  

Microsoft Academic Search

Protein extraction for two-dimensional gel elec- trophoresis (2-DE) from plant samples is chal- lenging due to low protein content and high level of contaminants. Proteomic research in turfgrass is limited by the lack of effi cient protein extrac- tion methods. To establish an effective protocol of protein extraction suitable for 2-DE analysis in turfgrasses, four protein extraction meth- ods (chloroform\\/acetone,

Chenping Xu; Yan Xu; Bingru Huang

2008-01-01

181

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

PubMed

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

2014-01-01

182

Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris.  

PubMed

Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates of P. mirabilis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs. PMID:1400963

Kappos, T; John, M A; Hussain, Z; Valvano, M A

1992-10-01

183

Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris.  

PubMed Central

Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates of P. mirabilis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs. Images

Kappos, T; John, M A; Hussain, Z; Valvano, M A

1992-01-01

184

Differentially regulated proteins in Prevotella intermedia after oxidative stress analyzed by 2D electrophoresis and mass spectrometry.  

PubMed

Prevotella intermedia is a rod-shaped, Gram-negative anaerobic bacterium found in human indigenous microbiota that plays an important role in opportunistic infections. The successful colonization depends on the ability of anaerobes to respond to oxidative stress (OS) in oxygenated tissues as well as to resist oxidative events from the host immune system until anaerobic conditions are present at the infection site. As knowledge of the mechanisms of protection against OS in Prevotella is limited, studies are needed to clarify aspects of molecular biology, physiology and ecology of this bacterium. The aim of this study was to access the proteins differentially regulated in P. intermedia after exposure to molecular oxygen by using two-dimensional gel electrophoresis (2DE) associated with the approach of MALDI-TOF/TOF Tandem Mass Spectrometry. The identity of the protein was evaluated by database search for homologous genomic sequences of P. intermedia strain 17 (TIGR). Twenty five out of 72 proteins found were identified as up-regulated (17) or down-regulated (9). These proteins were related to a variety of metabolic process, some of which could be associated to antioxidant and redox regulatory roles. Our data indicate that OS may stimulate an adaptive response in P. intermedia whose effect on its biology may be evidenced by the increase in aerotolerance and changes in protein abundance in the oxygen adapted cells. PMID:22193554

Santos, Simone G; Diniz, Cláudio G; Silva, Vânia L; Lima, Francisca L; Andrade, Hélida M; Chapeaurouge, Donat A; Perales, Jonas; Serufo, José Carlos; Carvalho, Maria Auxiliadora R; Farias, Luiz M

2012-02-01

185

Identification of Besnoitia besnoiti proteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis.  

PubMed

Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio ± 1.5, P<0.05 in t-test). Furthermore, 25 differentially expressed spots were selected and analysed by MALDI-TOF/MS. As a result, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research. PMID:23594379

Fernández-García, Aurora; Alvarez-García, Gema; Marugán-Hernández, Virginia; García-Lunar, Paula; Aguado-Martínez, Adriana; Risco-Castillo, Verónica; Ortega-Mora, Luis M

2013-07-01

186

Interactions of tumour-targeting nanoparticles with proteins: potential of using capillary electrophoresis as a direct probe.  

PubMed

Metal-based nanoscale particles possess unique optoelectronic or magnetic properties that make them highly promising as imaging agents in cancer therapy research. The fate of nanoparticles in vivo and particularly, the delivery to tumours are closely related to their interactions with plasma proteins. Furthermore, proteins can be used to modify the nanoparticle surface in order to facilitate active targeting to tumours. Therefore, there is an ongoing need for new and more capable analytical methodologies to characterize the protein-nanoparticle binding. Due to the small-sample volume requirement, high degree of resolution and, most importantly, mild, species-friendly separation conditions, capillary electrophoresis (CE) is gaining increasing popularity in the analysis of protein-nanoparticle interaction. This perspective article highlights the potential of CE in studying reactions associated with protein-mediated transformations of nanoparticles, with the focus on quantum dots, gold and iron oxide nanoparticles. Different ways by which CE can be applied to such monitoring are summarized and critically assessed using a representative coverage of recent publications. PMID:23011516

Aleksenko, Svetlana S; Shmykov, Alexei Y; Oszwa?dowski, S?awomir; Timerbaev, Andrei R

2012-11-01

187

SDS-polyacrylamide gel electrophoresis of buffalo bulls seminal plasma proteins and their relation with semen freezability.  

PubMed

The objective of this study was to evaluate the protein profiles of seminal plasma in buffalo bulls and to examine their correlation with semen characteristics. Semen of 10 buffalo bulls were collected by a bovine artificial vagina. Semen characteristics (motility, morphology, viability and concentration) were recorded. A part of the semen sample (1 ml) was diluted by tris-egg yolk-glycerol extender, packed in French straws and was frozen in liquid nitrogen. The straws were later thawed and semen characteristics were compared with those of the fresh semen. Seminal plasma was harvested by centrifugation; treated with cold ethanol and then, underwent SDS-polyacrylamide gel electrophoresis (PAGE). Twenty five protein bands were identified on the gel, of which those of <35.5 kDa were prominent (72% of the bands). Of these protein fractions, 24.5 kDa was significantly correlated with sperm progressive motility in fresh and viability in frozen-thawed semen while 45 kDa bands were correlated with abnormal morphology in frozen-thawed semen; 55 kDa protein fractions were correlated with sperm viability of fresh semen. Progressive motility, viability and abnormal sperm morphology of frozen-thawed semen were highly correlated with these parameters in the fresh semen. In conclusion, seminal plasma protein fractions in buffalo bulls are similar to those reported in other animal species and have some correlations with semen characteristics before and after freezing. PMID:17433580

Asadpour, R; Alavi-Shoushtari, S M; Rezaii, S Asri; Ansari, M H Kh

2007-12-01

188

Quartz crystal microbalances for quantitative biosensing and characterizing protein multilayers  

Microsoft Academic Search

The use of quartz crystal microbalances (QCMs) for quantitative biosensing and characterization of protein multilayers is demonstrated in three case studies. Monolayers of QCM-based affinity biosensors were investigated first. Layers of a thiol-containing synthetic peptide constituting an epitope of the foot-and-mouse-disease virus were formed on gold electrodes via self-assembly. The binding of specific antibodies to epitope-modified gold electrodes was detected

Jan Rickert; Andreas Brecht; Wolfgang Göpel

1997-01-01

189

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.

1987-05-01

190

Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms  

NASA Technical Reports Server (NTRS)

Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

1999-01-01

191

Global subcellular characterization of protein degradation using quantitative proteomics.  

PubMed

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

2013-03-01

192

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

PubMed

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 increased or decreased more than 2 fold. Five spots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recognised by specific antibodies. The glutathione S-transferase (GSTs) subunits Ybl, Yc and Yf were identified by the simultaneous analysis of both glutathione-binding cytosolic proteins and the corresponding standards. As estimated by internal optical density (IOD) of spots, the changes caused by protein depletion in GAPDH and GST subunit contents were similar to those obtained by other methods. By means of mass spectrometric analysis of tryptic peptides generated from spots and/or comparison of two-dimensional gel electrophoretic patterns, carbonic anhydrase III (CAIII), Cu, Zn superoxide dismutase (CuZnSOD) and a cytochrome P450 cytosolic protein (cyt P450) were identified. These three proteins, as well as GSTs, are related with intracellular detoxification and free radical scavenging systems. Their contents were regulated by dietary protein restriction in a manner indicative of diminished liver defence against oxidising agents. PMID:11451382

Sanllorenti, P M; Rosenfeld, J; Ronchi, V P; Ferrara, P; Conde, R D

2001-04-01

193

Identification of differentially expressed proteins in sulfadiazine resistant and sensitive strains of Toxoplasma gondii using difference-gel electrophoresis (DIGE).  

PubMed

Treatment options for toxoplasmosis in humans are generally limited to the use of sulfonamide and/or pyrimethamine-based compounds. However, there is increasing evidence for clinical therapy failures in patients suggesting the existence of drug resistance in these classes of drug. In vitro resistance to sulfadiazine has been detected in three strains of Toxoplasma gondii isolated from clinical cases. In order to begin to understand the mechanisms of resistance, we undertook a difference-gel electrophoresis (DIGE) approach combined with mass spectrometry to identify proteins that are differentially expressed in sulfadiazine-resistance strains of the parasite. Naturally resistant strains TgA 103001 (Type I), TgH 32006 (Type II) and TgH 32045 (Type II variant) were compared to sensitive strains RH (Type I) and ME-49 (Type II) using DIGE and the modulated proteins analyzed using LC-MS/MS. In total, 68 differentially expressed protein spots were analyzed by mass spectrometer and 31 unique proteins, including four hypothetical proteins, were identified. Among the differentially expressed proteins, 44% were over-expressed in resistant strains and 56% were over-expressed in sensitive strains. The virulence-associated rhoptry protein, ROP2A, was found in greater abundance in both naturally resistant Type II strains TgH 32006 and TgH 32045 compared to the sensitive strain ME-49. Enolase 2 and IMC1 were found to be in greater abundance in sensitive strains RH and ME-49, and MIC2 was found to be more abundant in the sensitive strain ME-49. Proteins regulation of ROP2, MIC2, ENO2, IMC1 and GRA7 were confirmed by Western blot analysis. In addition, gene expression patterns of ROP2, MIC2, ENO2 and IMC1 were analyzed with qRT-PCR. This study provides the first proteomics insights into sulfadiazine resistance in T. gondii resistant strains isolated from clinical cases. PMID:24533291

Doliwa, Christelle; Xia, Dong; Escotte-Binet, Sandie; Newsham, Emma L; Sanya J, Sanderson; Aubert, Dominique; Randle, Nadine; Wastling, Jonathan M; Villena, Isabelle

2013-12-01

194

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.

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

2013-01-01

195

Identification of a recombinant synaptobrevin-thioredoxin fusion protein by capillary zone electrophoresis using laser-induced fluorescence detection.  

PubMed

Capillary zone electrophoresis (CZE) was utilized to identify a synaptobrevin-thioredoxin fusion protein (TSB-51). TSB-51 is a substrate for cleavage by botulinum toxin B at the Q(76)-F(77) site. TSB-51 was derivatized with a fluorophore, CBQCA [3-(4-carboxy-benzoyl)-2-quinoline-carboxaldehyde], for 4 h at room temperature. Optimal conditions for CZE separation of the TSB-51-CBQCA complex were determined: buffer (sodium borate), pH (9.0), applied voltage (25 kV), temperature (25 degrees C) and forward polarity. SDS-PAGE showed that TSB-51 had a molecular mass of approximately 19 kDa. The protein was transferred to PVDF membrane and sequenced by the Edman degradation method verifying the first twelve amino acids as SDKIIHLTDDSF. TSB-51 was also collected during CZE separation and subsequently sequenced yielding the first three amino acids as SDK. This CZE-LIF method coupled with the CBQCA derivatization, fraction collection and Edman sequencing allowed for identification of the recombinant protein, a fast separation run time and utilization of small volumes of peptide (1.5 ng protein/23.6 nl injection). This method will be used for monitoring the endopeptidase activity of botulinum toxin B on TSB-51. PMID:9271130

Asermely, K E; Broomfield, C A; Nowakowski, J; Courtney, B C; Adler, M

1997-07-18

196

Optimization of the capillary zone electrophoresis loading limit and resolution of proteins, using triethylamine, ammonium formate and acidic pH  

Microsoft Academic Search

Capillary zone electrophoresis (CZE) of five model proteins (lysozyme, myoglobin, ribonuclease A, ?-lactalbumin, and trypsinogen), using ammonium formate as the electrophoretic buffer and triethylamine (TEA) as a buffer additive at pH 2.5, was used for protein separation. The electrophoretic behavior of these proteins was examined with respect to various concentrations (10–40 mM) of TEA and of ammonium formate. Based on

Huey G. Lee; Dominic M. Desiderio

1997-01-01

197

Application of two-dimensional electrophoresis and mass spectrometry to screen endometriosis?related proteins.  

PubMed

The aim of the present study was to detect the differences in the protein expression between Uyghur females with or without endometriosis (EM). The two dimensional (2D) gel images of proteins extracted from the serum of Uyghur and Han females with EM and the controls were analyzed using Phoretix 2D software, and the differentially expressed proteins were identified primarily by database query. Having compared the reproducible 2D gel images of proteins from the serum of Uihgur and Han females with and without EM, 13 differentially expressed proteins were obtained from Uyghur females and eight differentially expressed proteins were obtained from Han females. The present study demonstrated ethnic differences in gene and protein expression between Uyghur and Han females with EM. PMID:24737119

Tuerxun, Hanikezi; Zhang, Yanmei; Ji, Fei; Aili, Aixingzi; Yang, Xinhua; Ding, Yan

2014-07-01

198

Optimized sample preparation for two-dimensional gel electrophoresis of soluble proteins from chicken bursa of Fabricius  

PubMed Central

Background Two-dimensional gel electrophoresis (2-DE) is a powerful method to study protein expression and function in living organisms and diseases. This technique, however, has not been applied to avian bursa of Fabricius (BF), a central immune organ. Here, optimized 2-DE sample preparation methodologies were constructed for the chicken BF tissue. Using the optimized protocol, we performed further 2-DE analysis on a soluble protein extract from the BF of chickens infected with virulent avibirnavirus. To demonstrate the quality of the extracted proteins, several differentially expressed protein spots selected were cut from 2-DE gels and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results An extraction buffer containing 7 M urea, 2 M thiourea, 2% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol (DTT), 0.2% Bio-Lyte 3/10, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 U/ml Deoxyribonuclease I (DNase I), and 0.25 mg/ml Ribonuclease A (RNase A), combined with sonication and vortex, yielded the best 2-DE data. Relative to non-frozen immobilized pH gradient (IPG) strips, frozen IPG strips did not result in significant changes in the 2-DE patterns after isoelectric focusing (IEF). When the optimized protocol was used to analyze the spleen and thymus, as well as avibirnavirus-infected bursa, high quality 2-DE protein expression profiles were obtained. 2-DE maps of BF of chickens infected with virulent avibirnavirus were visibly different and many differentially expressed proteins were found. Conclusion These results showed that method C, in concert extraction buffer IV, was the most favorable for preparing samples for IEF and subsequent protein separation and yielded the best quality 2-DE patterns. The optimized protocol is a useful sample preparation method for comparative proteomics analysis of chicken BF tissues.

Wu, Yongping; Zhou, Jiyong; Zhang, Xin; Zheng, Xiaojuan; Jiang, Xuetao; Shi, Lixue; Yin, Wei; Wang, Junhua

2009-01-01

199

Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants.  

PubMed Central

The proteins of the major human snRNPs U1, U2, U4/U6 and U5 were characterised by two-dimensional electrophoresis, with isoelectric focussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second. With the exception of protein F, which exhibits an acidic pl value (pl = 3.3), the snRNP proteins are basic. Post-translational modification was found among the proteins associated specifically with the U1 and U2 particles. The most complex modification pattern was observed for the U1-specific 70K protein. This was found in at least 13 isoelectric variants, with pl values ranging from 6.7 to 8.7; these variants differed also in molecular weight. All of the 70K variants are phosphorylated in the cell. Thin-layer analysis of their tryptic phosphopeptides revealed that the 70K variants have four major phosphopeptides in common, in addition to which at least four additional serine residues are phosphorylated to different extents. The comparative phosphopeptide analysis shows that differential phosphorylation alone is not sufficient to explain the occurrence of the many isoelectric variants of 70K, so that the final charge of the 70K variants is determined both by phosphorylation and by other, as yet unidentified posttranslational modifications. By two-dimensional separation of snRNP proteins obtained from mouse Ehrlich ascites tumour cells, it was shown that the pattern of pl values of the mouse proteins was almost identical with the corresponding pattern for human proteins. Even the complex modification patterns of the 70K protein are identical in mouse and man, indicating that the presence in the cell of so many variants of this protein may have functional importance. The major difference between murine and human snRNP proteins is the absence of protein B' from mouse snRNPs. This suggests that the homologous protein B may be able to carry out the task of protein B'. Images

Woppmann, A; Patschinsky, T; Bringmann, P; Godt, F; Luhrmann, R

1990-01-01

200

A visual detection of protein content based on titration of moving reaction boundary electrophoresis.  

PubMed

A visual electrophoretic titration method was firstly developed from the concept of moving reaction boundary (MRB) for protein content analysis. In the developed method, when the voltage was applied, the hydroxide ions in the cathodic vessel moved towards the anode, and neutralized the carboxyl groups of protein immobilized via highly cross-linked polyacrylamide gel (PAG), generating a MRB between the alkali and the immobilized protein. The boundary moving velocity (V(MRB)) was as a function of protein content, and an acid-base indicator was used to denote the boundary displacement. As a proof of concept, standard model proteins and biological samples were chosen for the experiments to study the feasibility of the developed method. The experiments revealed that good linear calibration functions between V(MRB) and protein content (correlation coefficients R>0.98). The experiments further demonstrated the following merits of developed method: (1) weak influence of non-protein nitrogen additives (e.g., melamine) adulterated in protein samples, (2) good agreement with the classic Kjeldahl method (R=0.9945), (3) fast measuring speed in total protein analysis of large samples from the same source, and (4) low limit of detection (0.02-0.15 mg mL(-1) for protein content), good precision (R.S.D. of intra-day less than 1.7% and inter-day less than 2.7%), and high recoveries (105-107%). PMID:23567122

Wang, Hou-Yu; Guo, Cheng-Ye; Guo, Chen-Gang; Fan, Liu-Yin; Zhang, Lei; Cao, Cheng-Xi

2013-04-24

201

Applying microfluidic techniques in quantitative studies of protein aggregation  

NASA Astrophysics Data System (ADS)

Protein aggregation and fibrillation is involved in a number of devastating diseases, of which we have a limited understanding at present. Microfluidic techniques can be used in developing quantitative assays to study individual aspects of protein aggregation. Under certain conditions bovine insulin aggregates to give spherulites; spherical structures with fibrils growing and branching out from a central core. Drawing a parallel to actin polymerisation of the cell's cytoskeleton, fibril growth generates force. The force generated by polymerisation at fibril ends during spherulite growth can be measured in a microfluidic environment (TPJ Knowles et al, PNAS, 2009). By measuring the bending of four polydimethylsiloxane walls by a growing spherulite positioned in the centre, the force generated by polymerisation at fibril termini can be calculated. By growing the spherulites with a constant flow of monomer, the maximum force able to be generated by fibril growth, the stall force, can be calculated. This gives insight into the energy landscape of protein aggregation.

Herling, Therese

2012-02-01

202

Quantitative and qualitative differences in protein expression between papillary thyroid carcinoma and normal thyroid tissue  

Microsoft Academic Search

In order to better understand basic mechanisms of tumor development and identify potential new biomarkers, we have performed difference gel electrophoresis (DIGE) and peptide mass fingerprinting on pooled protein extracts from patients with papillary thyroid carcinoma (PTC) compared with matched normal thyroid tissue. Image analysis of DIGE gels comparing PTC and matched normal thyroid tissue protein indicated that 25% of

Lewis M. Brown; Steve M. Helmke; Stephen W. Hunsucker; Romana T. Netea-Maier; Simon A. Chiang; David E. Heinz; Kenneth R. Shroyer; Mark W. Duncan; Bryan R. Haugen

2006-01-01

203

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.

1999-02-12

204

Rapid capillary coating by epoxy-poly-(dimethylacrylamide): performance in capillary zone electrophoresis of protein and polystyrene carboxylate.  

PubMed

A fast and simple method for the internal coating of capillaries in capillary zone electrophoresis (CZE) is that with epoxy-poly(dimethylacrylamide) (EPDMA). Duration of coating by that method is 30 min, compared with that of 24 h when using uncross-linked polyacrylamide (PA) under otherwise identical conditions. Under the conditions used for the CZE of proteins (pH 9.0, 2% polyethylene glycol), the capillary coating with EPDMA is stable for at least 50 consecutive runs as judged by the constancy of low electroosmotic flow, equalling the stability of coating achieved by PA. Protein mobilities and protein peak asymmetry (suggestive of reversible interaction with the capillary wall) are also found to be the same in EPDMA and PA coated capillaries. Differences between EPDMA and PA coating also exist: The former is unstable upon lowering the ionic strength of the buffer to 0.003, upon the addition of sodium dodecyl sulfate (SDS) to the buffer and in application to the hydrophobic analyte, polystyrene carboxylate. PMID:11296919

Chiari, M; Cretich, M; Stastna, M; Radko, S P; Chrambach, A

2001-01-01

205

Isolation of bifidobacteria from breast milk and assessment of the bifidobacterial population by PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR.  

PubMed

The objective of this work was to elucidate if breast milk contains bifidobacteria and whether they can be transmitted to the infant gut through breastfeeding. Twenty-three women and their respective infants provided samples of breast milk and feces, respectively, at days 4 to 7 after birth. Gram-positive and catalase-negative isolates from specific media with typical bifidobacterial shapes were identified to the genus level by F6PPK (fructose-6-phosphate phosphoketolase) assays and to the species level by 16S rRNA gene sequencing. Bifidobacterial communities in breast milk were assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and their levels were estimated by quantitative real-time PCR (qRTi-PCR). Bifidobacteria were present in 8 milk samples and 21 fecal samples. Bifidobacterium breve, B. adolescentis, and B. bifidum were isolated from milk samples, while infant feces also contained B. longum and B. pseudocatenulatum. PCR-DGGE revealed the presence of one to four dominant bifidobacterial bands in 22 milk samples. Sequences with similarities above 98% were identified as Bifidobacterium breve, B. adolescentis, B. longum, B. bifidum, and B. dentium. Bifidobacterial DNA was detected by qRTi-PCR in the same 22 milk samples at a range between 40 and 10,000 16S rRNA gene copies per ml. In conclusion, human milk seems to be a source of living bifidobacteria for the infant gut. PMID:19088308

Martín, Rocío; Jiménez, Esther; Heilig, Hans; Fernández, Leonides; Marín, María L; Zoetendal, Erwin G; Rodríguez, Juan M

2009-02-01

206

Combination of Competitive Quantitative PCR and Constant-Denaturant Capillary Electrophoresis for High-Resolution Detection and Enumeration of Microbial Cells  

PubMed Central

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 104 cells ml?1. The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells ml?1 by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples.

Lim, Eelin L.; Tomita, Aoy V.; Thilly, William G.; Polz, Martin F.

2001-01-01

207

Quantitative analysis of 17 amino acids in the connective tissue of patients with pelvic organ prolapse using capillary electrophoresis-tandem mass spectrometry.  

PubMed

The simultaneous determination of 17 amino acids in connective tissue using capillary electrophoresis is described in this study. Separation was carried out on a fused silica capillary column (80 cm x 50 mm i.d.) with 1M formic acid as the running electrolyte. The detection was conducted on a mass spectrometer by selective reaction monitoring (SRM) mode via an electrospray ionization source. Tissue samples were prepared by reduction and acid hydrolysis to extract amino acids; over 84.3% recovery was seen for all compounds. The method allowed for sensitive, reproducible, and reliable quantification, and all 17 amino acids were separated using this method. Good linearity over the investigated concentration ranges was observed, with values of R higher than 0.993 for all the analytes. Precision and accuracy examined at three concentration levels ranged from 0.2% to 19.5% and 84.1% to 120.0%, respectively. Matrix effects were also tested and ranged from -9.1% to 15.4%. The validated method was applied to the quantitation of 17 amino acids in pelvic connective tissue of pelvic organ prolapsed patients. Methionine, glutamine, and histidine were significantly higher in the experimental patients compared to the controls. This suggests that changes in the amino acid concentrations within the connective tissue could be a factor in the genesis of pelvic organ prolapse. Therefore, this method is potentially applicable for amino acid analysis in tissue, providing a more complete understanding of pelvic organ prolapse. PMID:18339589

Shama, Naz; Bai, Sang Wook; Chung, Bong Chul; Jung, Byung Hwa

2008-04-01

208

Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers  

SciTech Connect

The authors have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA. After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controller scanner. The medium lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards. The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single stand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single stand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis. The method has been used to determined pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ. It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single stand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amendable to radioactive labeling.

Freeman, S.E.; Blackett, A.D.; Monteleone, D.C.; Setlow, R.B.; Sutherland, B.M.; Sutherland, J.C.

1986-10-01

209

Protein analysis in the rat auditory brainstem by two-dimensional gel electrophoresis and mass spectrometry  

Microsoft Academic Search

A catalogue of the protein repertoire of processing centres in the central auditory system would greatly foster our knowledge on the anatomical and functional properties of this sensory system. Towards this goal, we report on the first mapping study of the protein content in the superior olivary complex (SOC) and the inferior colliculus (IC) of the rat auditory brainstem. The

Hans Gerd Nothwang; Michael Becker; Kornelia Ociepka; Eckhard Friauf

2003-01-01

210

Characterization of Soluble Amaranth and Soybean Proteins Based on Fluorescence, Hydrophobicity, Electrophoresis, Amino Acid Analysis, Circular Dichroism, and Differential Scanning Calorimetry Measurements  

Microsoft Academic Search

Intrinsic fluorescence (IF), surface hydrophobicity (So), electrophoresis, amino acid analysis, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study folded and unfolded soluble proteins from Amaranthus hypochondriacus (A. h.) and soybean (S). Globulin (Glo) and albumin subfractions (Alb-1 and Alb-2) were extracted from A. h. and S and denatured with urea. Electrophoretic and functional properties indicated a

Shela Gorinstein; Efren Delgado-Licon; Elke Pawelzik; Herry Heriyati Permady; Moshe Weisz; Simon Trakhtenberg

2001-01-01

211

A novel microfluidic chip electrophoresis strategy for simultaneous, label-free, multi-protein detection based on a graphene energy transfer biosensor.  

PubMed

A new type of high-throughput and parallel optical sensing platform with a single-color probe based on microfluidic chip electrophoresis combined with aptamer-carboxyfluorescein/graphene oxide energy transfer is reported here. Label-free protein multi-targets were detected, even in challenging complex samples without any pre-treatment. PMID:24755615

Lin, Fengming; Zhao, Xiaochao; Wang, Jianshe; Yu, Shiyong; Deng, Yulin; Geng, Lina; Li, HuanJun

2014-06-01

212

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

PubMed

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

2011-11-25

213

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.

2007-04-19

214

Gel Electrophoresis  

NSDL National Science Digital Library

In the early days of DNA manipulation, DNA fragments were laboriously separated by gravity. In the 1970s, the powerful tool of DNA gel electrophoresis was developed. This process uses electricity to separate DNA fragments by size as they migrate through a gel matrix. This animation from Cold Spring Harbor Laboratory's Dolan DNA Learning Center presents Gel Electrophoresis through a series of illustrations of the processes involved.

2012-01-20

215

A study of the antigenicity of Rickettsia helvetica proteins using two-dimensional gel electrophoresis.  

PubMed

Rickettsia helvetica is an obligate intracellular Gram-negative microorganism found in Ixodes ricinus ticks. When R. helvetica was first discovered in 1979, little was known about its physiology and it fell into oblivion until it recently was suspected of being pathogenic to humans. However, all efforts to isolate R. helvetica from patients have been unsuccessful, although serological responses against R. helvetica can be demonstrated. The aim of our study was to investigate the protein profile of R. helvetica and study the antigenicity of its proteins using two-dimensional (2D) immunoblot in order to characterize the immunological response against R. helvetica infection. Our results show that in addition to the known PS120 and OmpB antigenic R. helvetica proteins, three other antigens exist: a 60 kDa GroEL protein, a 10 kDa GroES protein and a hitherto unknown 35 kDa hypothetical protein that has similarities with ORF-RC0799 of Rickettsia conorii. Furthermore, the lipopolysaccharide showed strong antigenicity. In this study, we present the first proteome map and the first 2D immunoblot profile of R. helvetica and finally we present the 35 kDa R. helvetica as an additional antigen to the previously known rickettsial antigens. PMID:19338513

Hajem, Nedaa; Weintraub, Andrej; Nimtz, Manfred; Römling, Ute; Påhlson, Carl

2009-04-01

216

Multi-dimension microchip-capillary electrophoresis device for determination of functional proteins in infant milk formula.  

PubMed

To improve resolution of important minor proteins and eliminate time-consuming precipitation of major protein with associated analyte co-precipitation risk, a multi-dimension strategy is adopted in the 2D microchip-CE device to isolate major proteins on-chip, enrich minor proteins in capillary before their separation in CE for UV quantitation. A standard fluorescent protein mixture containing FITC-BSA, myoglobin and cytochrome as specific pI markers has prepared to demonstrate capability of the device to fractionate minor proteins by IEF. The results using a standard protein mixture with profile resembling infant milk formula show a complete isolation of high abundance proteins by a 2-min 1D IEF run. The subsequent t-ITP/CZE run by on-chip high voltage switching delivers a high stacking ratio, realizing 60 folds enrichment of isolated protein fractions. All five important functional proteins (LF, IgG, ?-LA, ?-LgA and ?-LgB) known to fortify infant milk formula are isolated and determined using two consecutive t-ITP-CZE runs within a 18-min total assay time, a significant saving compared to several hours conventional pretreatment. For a 100g infant milk formula sample, working ranges of 20-8000mg, repeatability 3.8-5.3% and detection limits 2.3-10mg have been achieved to meet government regulations. Method reliability is established by 100% recoveries and agreeable results within expected ranges and labeled values. The capability of the device for field operation, rapid assay with quick results, label-free universal detection, simple operation by aqueous dissolution before injection, and the demanding matching in 2D separation based on isolated fractions at specified pI ranges, closely matched migration time and baseline-resolved peak shape makes the device a general tool to detect unknown proteins and determine known minor proteins in protein-rich samples with interfering constituents. PMID:23870546

Wu, Ruige; Wang, Zhiping; Zhao, Wenfeng; Yeung, William Shu-Biu; Fung, Ying Sing

2013-08-23

217

Acid and Base-Induced Proteins during Aerobic and Anaerobic Growth of Escherichia coli Revealed by Two-Dimensional Gel Electrophoresis  

Microsoft Academic Search

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 pro- teins, 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

DARCY BLANKENHORN; JUDITH PHILLIPS; JOAN L. SLONCZEWSKI

1999-01-01

218

High throughput and rapid screening of marine protein hydrolysates enriched in peptides with angiotensin-I-converting enzyme inhibitory activity by capillary electrophoresis  

Microsoft Academic Search

Twelve kinds of marine protein materials, including fish, shrimp, seashell, algae and seafood wastes were selected for the hydrolysis using four different proteases. The IC50 values for angiotensin-converting enzyme (ACE) inhibitory activity of 48 hydrolysates were rapidly determined by capillary electrophoresis (CE). The values ranged from 0.17 to 501.7mg\\/ml, and were affected by both the marine protein resources and the

Hai-Lun He; Xiu-Lan Chen; Hao Wu; Cai-Yun Sun; Yu-Zhong Zhang; Bai-Cheng Zhou

2007-01-01

219

Low-flow sheathless capillary electrophoresis-mass spectrometry for sensitive glycoform profiling of intact pharmaceutical proteins.  

PubMed

Capillary electrophoresis coupled to time-of-flight mass spectrometry (CE-TOF-MS) via a porous tip sheathless electrospray ionization (ESI) interface was studied for the characterization of pharmaceutical glycoproteins. To achieve optimal glycoform separation, background electrolytes of low pH were used in conjunction with a capillary with a neutral coating exhibiting near-zero electroosmotic flow. Crucial interfacing parameters, like ESI voltage and ESI tip-to-end plate distance, were optimized for very low flow rates (?5 nL/min) in order to attain maximum sensitivity and stable performance. Under optimal conditions, the sheathless CE-MS interface provided significantly increased ionization efficiencies for intact proteins and decreased ionization suppression leading to detection limits in the picomolar-range. Analysis of a sample of recombinant human interferon-? allowed the assignment of at least 18 glycoforms, plus a variety of deamidation, succinimide, and oxidation products, representing a considerable improvement over sheath-liquid CE-MS. The sheathless CE-MS system also proved highly suitable for the glycoprofiling of recombinant human erythropoietin, revealing 74 glycoforms in a 60-min run. In addition, oxidation and acetylation products were detected, overall resulting in assignment of more than 250 different isoforms. Semiquantitative glycoprofiles could be derived for both pharmaceutical proteins, with estimated glycoform concentrations analyzed ranging from 0.35 to 950 nM. These profiles may be very useful for quality control of biopharmaceuticals and their biosimilars. PMID:23323765

Haselberg, Rob; de Jong, Gerhardus J; Somsen, Govert W

2013-02-19

220

Two-dimensional polyacrylamide gel electrophoresis of extracellular soybean pathogenesis-related proteins using PhastSystem.  

PubMed

Acidic and basic pathogenesis-related proteins (PR-Ps) were extracted from the intercellular fluid (IF) of soybean leaves, locally infected with tobacco necrosis virus and showing necrotic local lesions. Proteins were detected by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using PhastSystem and precast commercially available gels. Extracts from healthy leaves were run as controls. PR-Ps were first run under native PAGE conditions or isoelectric focusing (IEF), the gels stained with Coomassie Blue, then run under sodium dodecyl sulfate (SDS)-denaturing conditions and finally stained with silver. Ten major acidic PR-Ps were separated; their Mr's were close to those found by conventional PAGE. Their isoelectric points ranged from 3.5 to 5.0. Ten basic PR-Ps were separated and their Mr's estimated. None of these acidic or basic soybean PR-Ps was a glycoprotein. PAGE with PhastSystem and precast gels gives reliable results, comparable with those from conventional 2D-PAGE, with simpler experimental procedures. By electrophoresing Coomassie-stained gels with SDS in the second dimension, we were able to control the first-dimensional separation and to avoid laborious protocols generally adopted with unstained gels. PMID:2318193

Roggero, P; Pennazio, S

1990-01-01

221

Functional Maps of Protein Complexes from Quantitative Genetic Interaction Data  

PubMed Central

Recently, a number of advanced screening technologies have allowed for the comprehensive quantification of aggravating and alleviating genetic interactions among gene pairs. In parallel, TAP-MS studies (tandem affinity purification followed by mass spectroscopy) have been successful at identifying physical protein interactions that can indicate proteins participating in the same molecular complex. Here, we propose a method for the joint learning of protein complexes and their functional relationships by integration of quantitative genetic interactions and TAP-MS data. Using 3 independent benchmark datasets, we demonstrate that this method is >50% more accurate at identifying functionally related protein pairs than previous approaches. Application to genes involved in yeast chromosome organization identifies a functional map of 91 multimeric complexes, a number of which are novel or have been substantially expanded by addition of new subunits. Interestingly, we find that complexes that are enriched for aggravating genetic interactions (i.e., synthetic lethality) are more likely to contain essential genes, linking each of these interactions to an underlying mechanism. These results demonstrate the importance of both large-scale genetic and physical interaction data in mapping pathway architecture and function.

Bandyopadhyay, Sourav; Kelley, Ryan; Krogan, Nevan J.; Ideker, Trey

2008-01-01

222

Determination of Transaminase Activity of Serum Protein Fractions separated by Paper Electrophoresis  

Microsoft Academic Search

THE electrophoretic mobility of glutamic-oxalacetic acid transaminase in blood serum has been found to be between those for alpha2- and beta-globulins1. In our experiments2 the glutamic-oxalacetic acid transaminase was associated mainly with alpha2-globulins. This communication deals with this transaminase and glutamic-pyruvic acid transaminase and their relations to serum protein fractions.

Miroslav Sevela

1958-01-01

223

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.

1983-01-01

224

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.  

PubMed

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

2013-10-21

225

Application of the copolymers containing sulfobetaine methacrylate in protein separation by capillary electrophoresis.  

PubMed

This study describes the formation of highly efficient antiprotein adsorption random copolymer coating of poly(N,N-dimethylacrylamide-co-sulfobetaine methacrylate) (poly(DMA-co-SBMA)) on the fused-silica capillary inner wall. Firstly, the poly(DMA-co-SBMA)s with different feed ratio (SBMA/DMA) were synthesized via the reversible addition fragmentation chain transfer polymerization. And then, X-ray photoelectron spectroscopy (XPS) and water contact angle (CA) were used to investigate the composition and hydrophilicity of poly(DMA-co-SBMA) coating formed on the glass slide surfaces. CA measurements revealed that the poly(DMA-co-SBMA) coating became more hydrophilic with the increment of feed ratio (SBMA/DMA), and at the same time, the XPS results showed that the coating ability was also increased with the increment of feed ratio. Followed, the copolymer was applied to coat the fused-silica capillary inner wall, and the coated capillary was used to separate the mixture of proteins (lysozyme, cytochrome c, ribonuclease A, and ?-chymotrypsinogen A) in a pH range from 3.0 to 5.0. Under the optimum conditions, an excellent separation of basic proteins with peak efficiencies ranging from 551,000 to 1509,000 N/m had been accomplished within 10 min. Furthermore, the effect of coating composition on protein separation was also investigated through the comparison of separation efficiency achieved by using bare, PSBMA- and poly(DMA-co-SBMA)-coated capillary, respectively. PMID:23909655

Cao, Fuhu; Tan, Lin; Xiang, Lina; Liu, Songtao; Wang, Yanmei

2013-01-01

226

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.

1985-01-01

227

Quantitative structure--plasma protein binding relationships of acidic drugs.  

PubMed

One of the most important factors, affecting significantly the overall pharmacokinetic and pharmacodynamic profile of a drug, is its binding to plasma protein (PPB). In the present study, we focus on a set of 132 diverse acidic drugs binding to plasma proteins to different extent and develop quantitative structure-plasma protein binding relationships (QSPPBR) to predict their unbound fraction in plasma (f(u)) using 178 molecular descriptors. QSPPBR models were derived after variable selection by genetic algorithm followed by stepwise regression and tested by cross- and external validation. The final model has r(2) value of 0.771, q(2) value of 0.737, and four outliers. It predicts 57% of the f(u) values with less than twofold error. According to the molecular descriptors selected as the most predictive for PPB, the lipophilicity of the drugs, the presence of aromatic rings, cyano groups, and H-bond donor-acceptor pairs increase the PPB, whereas the presence of tertiary carbon atoms, four-member rings, and iodine atoms decrease PPB. These descriptors were summarized into a short seven-item checklist of criteria responsible for PPB. The checklist could be used as a guide for evaluation of PPB of acidic drug candidates, similarly to the Lipinski's rule of five used for evaluation of oral permeability of drugs. PMID:22961754

Zhivkova, Zvetanka; Doytchinova, Irini

2012-12-01

228

Quaternized cellulose-supported gold nanoparticles as capillary coatings to enhance protein separation by capillary electrophoresis.  

PubMed

Gold nanoparticles (Au NPs) were synthesized and stabilized by using water-soluble quaternized cellulose (QC) as support matrix through a straightforward and environmentally friendly aqueous-phase approach. The structure and morphology of QC-supported Au NPs (QC-Au NPs) were investigated systematically by UV-visible, FT-IR, x-ray diffraction and TEM measurement. The Au NPs with mean diameter of about 7nm were shown to efficiently redisperse in water due to the strong interaction between QC and Au NPs, and the solutions were quite stable after storage for nearly 4 months at room temperature. QC-Au NPs were subsequently used as novel physically adsorbed coatings for protein separation by CE. The separation performance was significantly improved in the capillary coated by QC-Au NPs compared with that of the uncoated capillary or QC coated capillary. A small quantity of Au NPs (Au content of 4.6%) was adequate for the obvious improvement of coating ability. The theoretical plate number of lysozyme in QC-Au1 NPs coated capillary was 2.9 times as much as that in QC coated capillary. We have demonstrated the separation of six model proteins with RSD of migration time less than 2.79% and RSD of peak area less than 4.81%. Furthermore, QC-Au NPs was applied to the analysis of closely related proteins and biological samples. With simplicity, high resolution and reproducibility, the proposed method shows potential for applications in proteomics and clinical diagnosis. PMID:24745845

You, Jun; Zhao, Lingguo; Wang, Gongwei; Zhou, Haitao; Zhou, Jinping; Zhang, Lina

2014-05-23

229

Hybrid phospholipid bilayer coatings for separations of cationic proteins in capillary zone electrophoresis.  

PubMed

Protein separations in CZE suffer from nonspecific adsorption of analytes to the capillary surface. Semipermanent phospholipid bilayers have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self-assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3-cyanopropyldimethylchlorosilane (CPDCS) or n-octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m(-2) , respectively, compared to 17 ± 1 mJ m(-2) for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine to CPDCS- or ODCS-modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3-1.9 × 10(-4) cm(2) V(-1) s(-1) ) compared to CPDCS- and ODCS-modified or bare capillaries (3.6 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , 4.8 ± 0.4 × 10(-4) cm(2) V(-1) s(-1) , and 6.0 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , respectively), with increased stability compared to phospholipid bilayer coatings. HPB-coated capillaries yielded reproducible protein migration times (RSD ? 3.6%, n ? 6) with separation efficiencies as high as 200 000 plates/m. PMID:24459085

Gallagher, Elyssia S; Adem, Seid M; Bright, Leonard K; Calderon, Isen A C; Mansfield, Elisabeth; Aspinwall, Craig A

2014-04-01

230

Discrimination between peak spreading in capillary zone electrophoresis of proteins due to interaction with the capillary wall and due to protein microheterogeneity.  

PubMed

Our study attempts to find an approach to distinguishing between the contribution to peak spreading in capillary zone electrophoresis (CZE) due to protein microheterogeneity and that due to interaction with the capillary wall, by analyzing correlations between observed peak spreading and peak asymmetry. The peak asymmetry was measured as ln[(tm-t1)/(t2-tm)] where tm, t1, and t2 are migration times at the mode of the peak and at the intersection of the peak width at half-height with the ascending and descending limbs, respectively. Two isoforms of recombinant green fluorescent protein (GFP-1 and GFP-2, 27 kDa molecular mass), glucose-6-phosphate dehydrogenase (GPD, 104 kDa), and the naturally fluorescent protein R-phycoerythrin (PHYCO, 240 kDa) were subjected to CZE in polyacrylamide-coated fused-silica capillaries of 50 and 100 microns diameters under varying conditions of protein concentration, field strength, and the initial zone length. Under conditions such that contributions to peak spreading from axial diffusion, thermal effects, and electrophoretic dispersion are negligible, the analysis of the interrelations between peak width and peak asymmetry was found to allow a conclusion as to the cause of peak spreading in CZE of protein. It appears that the peak width of GFP-2 originates mostly in protein microheterogeneity while that of GFP-1 is due to protein-capillary wall interactions. For PHYCO, both microheterogeneity and protein-capillary wall interactions contribute to peak spreading. GPD exhibits relatively little microheterogeneity or interaction with capillary walls. Thus, its peak width appears to be mostly affected by an extracolumn source of spreading such as the initial zone length. PMID:11197181

Stastna, M; Radko, S P; Chrambach, A

2001-01-01

231

Optimal protein extraction methods from diverse sample types for protein profiling by using Two-Dimensional Electrophoresis (2DE).  

PubMed

There is a great diversity of protein samples types and origins, therefore the optimal procedure for each sample type must be determined empirically. In order to obtain a reproducible and complete sample presentation which view as many proteins as possible on the desired 2DE gel, it is critical to perform additional sample preparation steps to improve the quality of the final results, yet without selectively losing the proteins. To address this, we developed a general method that is suitable for diverse sample types based on phenolchloroform extraction method (represented by TRI reagent). This method was found to yield good results when used to analyze human breast cancer cell line (MCF-7), Vibrio cholerae, Cryptocaryon irritans cyst and liver abscess fat tissue. These types represent cell line, bacteria, parasite cyst and pus respectively. For each type of samples, several attempts were made to methodically compare protein isolation methods using TRI-reagent Kit, EasyBlue Kit, PRO-PREP™ Protein Extraction Solution and lysis buffer. The most useful protocol allows the extraction and separation of a wide diversity of protein samples that is reproducible among repeated experiments. Our results demonstrated that the modified TRI-reagent Kit had the highest protein yield as well as the greatest number of total proteins spots count for all type of samples. Distinctive differences in spot patterns were also observed in the 2DE gel of different extraction methods used for each type of sample. PMID:22433892

Tan, A A; Azman, S N; Abdul Rani, N R; Kua, B C; Sasidharan, S; Kiew, L V; Othman, N; Noordin, R; Chen, Y

2011-12-01

232

Optimal protein extraction methods from diverse sample types for protein profiling by using Two-Dimensional Electrophoresis (2DE)  

Microsoft Academic Search

There is a great diversity of protein samples types and origins, therefore the optimal procedure for each sample type must be determined empirically. In order to obtain a reproducible and complete sample presentation which view as many proteins as possible on the desired 2DE gel, it is critical to perform additional sample preparation steps to improve the quality of the

Y. Chen

2011-01-01

233

Reference values for hematology, plasma biochemical analysis, plasma protein electrophoresis, and Aspergillus serology in elegant-crested tinamou (Eudromia elegans).  

PubMed

Elegant-crested tinamou (Eudromia elegans) are members of the family Tinamidae. A review of postmortem records at the Saint Louis Zoo suggests that this species is highly susceptible to aspergillosis. Antemortem detection of Aspergillus infections can be aided by evaluating results of hematologic, plasma biochemical, protein electrophoretic, and Aspergillus antigen and antibody serologic testing. To establish species-specific reference intervals for these parameters in elegant-crested tinamou, blood samples were collected and analyzed from 19 healthy captive adult and subadult elegant-crested tinamou. The predominant circulating leukocyte in elegant-crested tinamou is the lymphocyte. Prealbumin electrophoresis fractions were detected in 4 of 19 birds (21%), and globulin fractions were detected in all birds. All birds had measurable titers for Aspergillus antibody and galactomannan antigen, including 3 birds with antibody levels considered to be positive. Galactomannan levels were all below the positive threshold of 1.0. Results of hematologic and plasma biochemical analyses performed on blood samples obtained from terminally ill elegant-crested tinamou with confirmed aspergillosis were compared with results from healthy individuals. Significant differences were found in the hematocrit, total white blood cell count, heterophil and monocyte counts, and albumin and globulin levels. PMID:23772450

Black, Peter A; Macek, Michael; Tieber, Anne; Weber, Martha

2013-03-01

234

Affinity capillary electrophoresis: important application areas and some recent developments.  

PubMed

Affinity capillary electrophoresis (ACE) is a broad term referring to the separation by capillary electrophoresis of substances that participate in specific or non-specific affinity interactions during electrophoresis. The interacting molecules can be found free in solution or can be immobilized to a solid support. Every ACE mode has advantages and disadvantages. Each can be used for a wide variety of applications. This paper focuses on applications that include purification and concentration of analytes present in diluted solutions or complex matrices, quantitation of analytes based on calibration curves, and estimation of binding constants from direct and derived binding curves based on quantitation of analytes or on analyte migration shifts. A more recent chemicoaffinity strategy in capillary electrophoresis/capillary electrochromatography (CE/CEC) termed molecular imprinting ('plastic antibodies') is discussed as well. Although most ACE studies are aimed at characterizing small-molecular mass analytes such as drugs, hormones, and peptides, some efforts have been pursued to characterize larger biopolymers including proteins, such as immunoglobulins. Examples of affinity interactions that have been studied are antigen-antibody, hapten-antibody, lectin-sugar, drug-protein, and enzyme-substrate complexes using ultraviolet, laser-induced fluorescence, and mass spectrometer detectors. This paper also addresses the critical issue of background electrolyte selection and quantitation of analytes. Specific examples of bioaffinity applications are presented, and the future of ACE in the biomedical field is discussed. PMID:9792496

Heegaard, N H; Nilsson, S; Guzman, N A

1998-09-11

235

Aptamers in Affinity Separations:Capillary Electrophoresis  

NASA Astrophysics Data System (ADS)

Assays employing aptamers in capillary electrophoresis (CE), including competitive and noncompetitive assays, fluorescence polarization (FP) assays, nonequilibrium capillary electrophoresis of equilibrium mixtures, and affinity-polymerase chain reaction-CE assays, are summarized. These assays can be used to estimate dissociation rate and equilibrium binding constants, determine binding stoichiometries, study molecular interactions, and quantitatively determine specific analytes (e.g., proteins) in complex media. They can potentially be completed in under 60 s, detect zeptomol (10-24) amounts of analyte, be utilized in complex media with little or no cross reaction, and target a number of different analytes of biological, environmental, and clinical importance. This chapter briefly overviews the process of aptamer selection using CE and discusses the various CE-based bioanalytical methods that have been used to study biomolecular interactions.

Guthrie, Jeffrey W.; Shao, Yuanhua; Le, X. Chris

236

Optimization of Quantitative Proteomics Using 2-Dimensional Difference Gel Electrophoresis to Characterize Molecular Mechanisms of Chemical Warfare Nerve Agent Exposure in the Rat Brain.  

National Technical Information Service (NTIS)

This report describes the optimization and use of 2-dimensional difference gel electrophoresis (2-D DIGE) to investigate the proteomic changes induced by chemical warfare nerve agents (CWNAs) in whole cell homogenates and isolated mitochondria from rat br...

H. M. Hoard-Fruchey R. K. Kan

2010-01-01

237

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

2007-01-01

238

Quantitative Analysis of Metabolic Profile and Tamm-Horsfall Protein in Pediatric Stone Patients  

NASA Astrophysics Data System (ADS)

According to some studies, the secretion of urinary glycoprotein Tamm-Horsfall protein (uromodulin) plays a significant part in the suppression of calcium nephrolith formation. The aim of the present study was to detect correlation of Tamm-Horsfall protein with soluble Ca2+ in the urine of urolithic patients and compare it with a control group, in order to estimate the degree of the disease progress, as well as the prognosis for the disease. The research included 29 urolithic patients, aged 8, 0+/-0, 2 y. The content of soluble Ca2+ in fresh urine was estimated by the method of capillary zone electrophoresis. The level of Tamm-Horsfall protein in urine was measured from cryoprecipitate using SDS polyacrylamide electrophoresis and semiquantitative analysis. Urine from the urolithic patients was consistently higher in Ca2+ and in Tamm-Horsfall protein concentrations than urine from controls, at all periods of the day.

Kogan, M. I.; Matishov, D. G.; Shin, E. F.; Sizonov, V. V.

2008-09-01

239

Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose.  

PubMed Central

We have developed a general two-step method for obtaining peptide fragments for sequence analysis from picomole quantities of proteins separated by gel electrophoresis. After separation by one- or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out, and each protein is digested in situ by proteolytic enzymes such as trypsin or staphylococcal V-8 protease. The resulting peptide fragments are separated by narrow-bore reverse-phase HPLC, collected, and sequenced in a gas-phase sequenator. Excellent peptide recoveries and the absence of extraneous contaminants in the separation of the peptide fragment mixture allow the generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and/or used to search sequence data bases for related proteins. This method has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from one- and two-dimensional polyacrylamide gels. Images

Aebersold, R H; Leavitt, J; Saavedra, R A; Hood, L E; Kent, S B

1987-01-01

240

Quantitative analysis of pheromone-binding protein specificity.  

PubMed

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, using ?-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila odorant-binding protein that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in Escherichia?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 Laughlin, Ha, Jones and Smith model of pheromone reception are discussed. PMID:23121132

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

2013-02-01

241

Electrophoresis in space.  

PubMed

Programs for free flow electrophoresis in microgravity over the past 25 years are reviewed. Several studies accomplished during 20 spaceflight missions have demonstrated that sample throughput is significantly higher in microgravity than on the ground. Some studies have shown that resolution is also increased. However, many cell separation trials have fallen victim to difficulties associated with experimenting in the microgravity environment such as microbial contamination, air bubbles in electrophoresis chambers, and inadequate facilities for maintaining cells before and after separation. Recent studies suggest that the charge density of cells at their surface may also be modified in microgravity. If this result is confirmed, a further cellular mechanism of "sensing" the low gravity environment will have been found. Several free fluid electrophoresis devices are now available. Most have been tried at least once in microgravity. Newer units not yet tested in spaceflight have been designed to accommodate problems associated with space processing. The USCEPS device and the Japanese FFEU device are specifically designed for sterile operations, whereas the Octopus device is designed to reduce electroosmotic and electrohydrodynamic effects, which become dominant and detrimental in microgravity. Some of these devices will also separate proteins by zone electrophoresis, isotachophoresis, or isoelectric focusing in a single unit. Separation experiments with standard test particles are useful and necessary for testing and optimizing new space hardware. A cohesive free fluid electrophoresis program in the future will obviously require (1) flight opportunities and funding, (2) identification of suitable cellular and macromolecular candidate samples, and (3) provision of a proper interface of electrophoresis processing equipment with biotechnological facilities--equipment like bioreactors and protein crystal growth chambers. The authors feel that such capabilities will lead to the production of commercially useful quantities of target products and to an accumulation of new knowledge relating to the complexities of electrostatic phenomena at the cell surface. PMID:10660776

Bauer, J; Hymer, W C; Morrison, D R; Kobayashi, H; Seaman, G V; Weber, G

1999-01-01

242

Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis  

PubMed Central

A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates, and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 nM and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn2+ and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min?1 mg?1) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min?1 mg?1). Zn2+, 1,2-naphthoquinone and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 ?M, and 100 nM, respectively. Dephosphorylation of the reporter following loading into living single cells occurred at rates of at least 2 pmol min?1 mg?1. When single cells were exposed to 1,2-naphthoquinone (50 ?M), Zn2+ (100 ?M), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens.

Phillips, Ryan M.; Bair, Eric; Lawrence, David S.; Sims, Christopher E.; Allbritton, Nancy L.

2013-01-01

243

Quantitative methods to analyze subnuclear protein organization in cell populations with varying degrees of protein expression  

PubMed Central

The control of gene transcription is dependent on DNA-binding and coregulatory proteins that assemble in distinct regions of the cell nucleus. We use multispectral wide-field microscopy of cells expressing transcriptional coregulators labeled with fluorescent proteins (FP) to study the subnuclear localization and function of these factors in living cells. In coexpression studies, the glucocorticoid receptor interacting protein (GRIP) coactivator protein and the silencing mediator of retinoid and thyroid (SMRT) corepressor protein form spherical subnuclear focal bodies that are spatially distinct, suggesting that specific protein interactions concentrate these divergent proteins in separate subnuclear regions. However, the variability of these subnuclear bodies between cells within the population makes analysis based on “representative images” difficult, if not impossible. To address this issue, we develop a protocol for unbiased selection of cells from the population, followed by the automated quantification of the subnuclear organization of the labeled proteins. Statistical methods identify a significant linear correlation between the FP-coregulator expression level and subnuclear focal body formation for both FP-GRIP and FP-SMRT. Importantly, we confirm that these changes in subnuclear organization could be statistically normalized for differences in coregulator expression level. This integrated quantitative image analysis method will allow the rigorous comparison of different experimental cell populations that express variable levels of FP fusion proteins.

Voss, Ty C.; Demarco, Ignacio A.; Booker, Cynthia F.; Day, Richard N.

2005-01-01

244

Beyond hairballs: the use of quantitative mass spectrometry data to understand protein-protein interactions  

PubMed Central

The past 10 years have witnessed a dramatic proliferation in the availability of protein interaction data. However, for interaction mapping based on affinity purification coupled with mass spectrometry (AP-MS), there is a wealth of information present in the datasets that often goes unrecorded in public repositories, and as such remains largely unexplored. Further, how this type of data is represented and used by bioinformaticians has not been well established. Here, we point out some common mistakes in how AP-MS data are handled, and describe how protein complex organization and interaction dynamics can be inferred using quantitative AP-MS approaches.

Gingras, Anne-Claude; Raught, Brian

2012-01-01

245

whittled M. Winnik Robert M. DeKroon Joseph S. Y. Jeong Mahaiula Mocane Jenifer B. Robinette Christina Osro Nedyalka N. Dicheva Eric hamlet Oscar Alzada. Analysis Proteins die Mold Mass Spectrometry.Difference Gel Electrophoresis (die)  

EPA Pesticide Factsheets

Did you mean: whittled M. Winnik Robert M. DeKroon Joseph S. Y. Jeong Mahaiula Mocane Jenifer B. Robinette Christina Osro Nedyalka N. Dicheva Eric hamlet Oscar Alzada. Analysis Proteins die Mold Mass Spectrometry.Difference Gel Electrophoresis (die) ?

246

The application of amine-terminated silicon quantum dots on the imaging of human serum proteins after polyacrylamide gel electrophoresis (PAGE).  

PubMed

Novel amine-terminated silicon (Si) quantum dots (QDs) were synthesized and applied for the detection of human serum proteins on gels directly after polyacrylamide gel electrophoresis (PAGE). The diameter of these stable amine-terminated Si?QDs was in the range of 0.5-2.0 nm. In this study, the fluorescent imaging conditions, such as the buffer solution, pH value, buffer concentration and quantity of Si?QDs, were optimized and the possible mechanisms of Si?QDs-protein interaction were analyzed. The mode of Si?QDs and human serum albumin association was found to occur by hydrogen bond interactions; this was probably attributed to the interaction between the amino group of amine-terminated Si?QDs and the carboxyl group of proteins. Meanwhile, human serum proteins separated by native 1D and native 2D electrophoresis were detected by Si QD-based fluorescent imaging. Some proteins, such as isoform 1 of ?-1-antitrypsin, complement C3 (Fragment) and hemopexin, which were identified by mass spectrometry (MS), were easily detected by using Si?QDs, but not with CBB-R250 staining. The Si?QDs-based fluorescent imaging technique with high resolution is a sensitive and dependable method for direct detection of human serum proteins, and has enormous potential in clinical diagnosis. PMID:22249969

Liu, Pingping; Na, Na; Huang, Lingyun; He, Dacheng; Huang, Changgang; Ouyang, Jin

2012-01-27

247

Protein Microarrays for Quantitative Detection of PAI-1 in Serum  

PubMed Central

Objective Plasminogen activator inhibitor-1 (PAI-1), one crucial component of the plasminogen activator system, is a major player in the pathogenesis of many vascular diseases as well as in cancer. High levels of PAI-1 in breast cancer tissue are associated with poor prognosis. The aim of this study is to evaluate rigorously the potential of serum PAI-1 concentration functioning as a general screening test in diagnostic or prognostic assays. Methods A protein-microarray-based sandwich fluorescence immunoassay (FIA) was developed to detect PAI-1 in serum. Several conditions of this microarray-based FIA were optimized to establish an efficacious method. Serum specimens of 84 healthy women and 285 women with breast cancer were analyzed using the optimized FIA microarray. Results The median serum PAI-1 level of breast cancer patients was higher than that of healthy women (109.7 ng/ml vs. 63.4 ng/ml). Analysis of covariance revealed that PAI-1 levels of the two groups were significantly different (P<0.001) when controlling for an age effect on PAI-1 levels. However, PAI-1 values in TNM stage I?IV patients respectively were not significantly different from each other. Conclusion This microarray-based sandwich FIA holds potential for quantitative analysis of tumor markers such as PAI-1.

Ma, Xu

2012-01-01

248

A quantitative slot blot assay for host cell protein impurities in recombinant proteins expressed in E. coli  

Microsoft Academic Search

Residual host cell protein impurities in recombinant proteins intended for human use must be accurately quantified to help establish their safety. We describe a novel means of host cell protein quantitation, in which a slot blot system was employed together with scanning laser densitometry to allow picogram level sensitivity in detection of residual host cell proteins in unpurified fermentation products

Daming Zhu; Allan J. Saul; Aaron P. Miles

2005-01-01

249

Electrophoresis experiments in microgravity  

NASA Technical Reports Server (NTRS)

The use of the microgravity environment to separate and purify biological cells and proteins has been a major activity since the beginning of the NASA Microgravity Science and Applications program. Purified populations of cells are needed for research, transplantation and analysis of specific cell constituents. Protein purification is a necessary step in research areas such as genetic engineering where the new protein has to be separated from the variety of other proteins synthesized from the microorganism. Sufficient data are available from the results of past electrophoresis experiments in space to show that these experiments were designed with incomplete knowledge of the fluid dynamics of the process including electrohydrodynamics. However, electrophoresis is still an important separation tool in the laboratory and thermal convection does limit its performance. Thus, there is a justification for electrophoresis but the emphasis of future space experiments must be directed toward basic research with model experiments to understand the microgravity environment and fluid analysis to test the basic principles of the process.

Snyder, Robert S.; Rhodes, Percy H.

1991-01-01

250

Gene expression patterns in the black blowfly (Phormia regina) as revealed by two-dimensional electrophoresis of proteins. I. Developmental stage-specific and sex-specific differences  

SciTech Connect

The black blowfly, Phormia regina, has been implicated in human myiasis and as a contact vector of viral and bacterial diseases present in carrion to which female flies are attracted for egg deposition. Inbred strains of P. regina are an excellent model system for studying gene expression in the developmental stages of such holometabolous dipteran parasites. However, information regarding gene and protein expression patterns in regina is limited. The authors used ISO-DALT high-resolution, two-dimensional electrophoresis with solver staining to establish fundamental protein maps for examination of the stage-specific gene expression patterns in the 615 most abundant proteins of the eggs, first- and third-instar larvae, pupae, and male and female adults. They also used a differential extraction technique to identify the major cuticular proteins of the adults. The results show 48 clearly identifiable stage-specific and sex-specific proteins.

Harrison, H.H. (The University of Chicago Pritzker School of Medicine, Il (United States)); Joslyn, D.J. (Rutgers University at Camden, NJ (United States))

1991-12-01

251

Two-dimensional electrophoresis of acidic proteins isolated from ozone-stressed Norway spruce needles (Picea abies L. Karst): separation method and image processing.  

PubMed

The effect of ozone stress (< 10,200 or 400 micrograms m-3) on the protein pattern of Picea abies L. Karst needles was studied by fumigating potted grafts from mature trees. Clonal trees of Norway spruce were fumigated in open-top chambers for one growing season. Proteins, soluble and membrane bound, were extracted from the needles in a four-step procedure: (i) grinding in liquid nitrogen and then in a pH 5.0 buffer with thiourea added to inhibit phenol oxidase and polyvinylpolypyrrolidone (PVP) added to bind phenolic compounds; (ii) precipitation of proteins with acetone at -20 degrees C; (iii) solubilization in a modified lysis buffer; and (iv) dialysis against the modified lysis buffer. Proteins were separated by two-dimensional electrophoresis according to O'Farrell's method. The changes in the silver-stained protein pattern were evaluated by using image processing methods. Electrophoretograms from stressed and nonstressed trees are presented. PMID:7498181

Davidsen, N B

1995-07-01

252

Quantitative double-label radiography of two-dimensional protein gels using color negative film and computer analysis.  

PubMed

We have devised a method of data collection and computer analysis which allows utilization of the resolving power of two-dimensional gel electrophoresis of proteins, in conjunction with the versatility of using two different radionuclides simultaneously. Cultures of Escherichia coli growing with exponential growth rate constants (mu) of 0.32 and 1.43 were labeled with [3H]leucine and [14C]leucine, respectively; these samples were mixed, and cell protein was separated on a two-dimensional gel. Spacial and quantitative data for both radionuclides were recorded on color negative film by radiographic exposure. Data for 14C alone were then collected photographically from the red-light-sensitive layer of the film using a red filter, while data for 3H and spillover of 14C were collected photographically from the blue-light-sensitive layer using a blue filter. These two data sets were analyzed by CINT, a computer program for analysis of two-dimensional gels, and quantitative data for 3H were calculated after determination of spillover of 14C in a manner analogous to quantification of 3H and 14C by liquid scintillation counting. Quantitative data from over 1000 protein spots representing from 0.002% to 10% of the total 3H or 14C, respectively, are available in a matter of hours. We have used this method to analyze the effect of growth rate and medium composition on the relative levels of individual proteins in a pathogenic strain of E. coli which contains group 111 O-antigen. As expected, the relative levels of aminoacyl-tRNA synthetases, protein chain elongation factors, ribosomal proteins, and the alpha-subunit of RNA polymerase are all increased with increased growth rate; the magnitude of these changes agreed with previous data derived using other strains of E. coli. Alterations in the levels of other proteins identified on the two-dimensional gels could be interpreted in terms of changes in medium composition. When compared to manual data collection by excising radiolabeled proteins and quantifying 3H and 14C in a liquid scintillation counter following combustion to H2O and CO2, respectively, this new method of data collection and computer analysis increases the resolution of data collection and decreases the time involved from days to hours. PMID:6341053

Goldman, R C; Trus, B L; Leive, L

1983-04-01

253

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

PubMed Central

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.

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

2010-01-01

254

Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae  

PubMed Central

Background Two-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics. Currently, most 2DE experiments are performed using immobilized pH gradient (IPG) in the first dimension; however, some laboratories still use carrier ampholytes-based isoelectric focusing technique. The aim of this study was to directly compare IPG-based and non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE techniques by using the same samples and identical second dimension procedures. We have used commercially available Invitrogen ZOOM IPGRunner and WITAvision systems for IPG and NEPHGE, respectively. The effectiveness of IPG-based and NEPHGE-based 2DE methods was compared by analysing differential protein expression during cytosolic unfolded protein response (UPR-Cyto) in Saccharomyces cerevisiae. Results Protein loss during 2DE procedure was higher in IPG-based method, especially for basic (pI?>?7) proteins. Overall reproducibility of spots was slightly better in NEPHGE-based method; however, there was a marked difference when evaluating basic and acidic protein spots. Using Coomassie staining, about half of detected basic protein spots were not reproducible by IPG-based 2DE, whereas NEPHGE-based method showed excellent reproducibility in the basic gel zone. The reproducibility of acidic proteins was similar in both methods. Absolute and relative volume variability of separate protein spots was comparable in both 2DE techniques. Regarding proteomic analysis of UPR-Cyto, the results exemplified parameters of general comparison of the methods. New highly basic protein Sis1p, overexpressed during UPR-Cyto stress, was identified by NEPHGE-based 2DE method, whereas IPG-based method showed unreliable results in the basic pI range and did not provide any new information on basic UPR-Cyto proteins. In the acidic range, the main UPR-Cyto proteins were detected and quantified by both methods. The drawback of NEPHGE-based 2DE method is its failure to detect some highly acidic proteins. The advantage of NEPHGE is higher protein capacity with good reproducibility and quality of spots at high protein load. Conclusions Comparison of broad range (pH 3–10) gradient-based 2DE methods suggests that NEPHGE-based method is preferable over IPG (Invitrogen) 2DE method for the analysis of basic proteins. Nevertheless, the narrow range (pH 4–7) IPG technique is a method of choice for the analysis of acidic proteins.

2013-01-01

255

PPINGUIN: Peptide Profiling Guided Identification of Proteins improves quantitation of iTRAQ ratios  

PubMed Central

Background Recent development of novel technologies paved the way for quantitative proteomics. One of the most important among them is iTRAQ, employing isobaric tags for relative or absolute quantitation. Despite large progress in technology development, still many challenges remain for derivation and interpretation of quantitative results. One of these challenges is the consistent assignment of peptides to proteins. Results We have developed Peptide Profiling Guided Identification of Proteins (PPINGUIN), a statistical analysis workflow for iTRAQ data addressing the problem of ambiguous peptide quantitations. Motivated by the assumption that peptides uniquely derived from the same protein are correlated, our method employs clustering as a very early step in data processing prior to protein inference. Our method increases experimental reproducibility and decreases variability of quantitations of peptides assigned to the same protein. Giving further support to our method, application to a type 2 diabetes dataset identifies a list of protein candidates that is in very good agreement with previously performed transcriptomics meta analysis. Making use of quantitative properties of signal patterns identified, PPINGUIN can reveal new isoform candidates. Conclusions Regarding the increasing importance of quantitative proteomics we think that this method will be useful in practical applications like model fitting or functional enrichment analysis. We recommend to use this method if quantitation is a major objective of research.

2012-01-01

256

Sorbitol dehydrogenase overexpression and other aspects of dysregulated protein expression in human precancerous colorectal neoplasms: a quantitative proteomics study.  

PubMed

Colorectal adenomas are cancer precursor lesions of the large bowel. A multitude of genomic and epigenomic changes have been documented in these preinvasive lesions, but their impact on the protein effectors of biological function has not been comprehensively explored. Using shotgun quantitative MS, we exhaustively investigated the proteome of 30 colorectal adenomas and paired samples of normal mucosa. Total protein extracts were prepared from these tissues (prospectively collected during colonoscopy) and from normal (HCEC) and cancerous (SW480, SW620, Caco2, HT29, CX1) colon epithelial cell lines. Peptides were labeled with isobaric tags (iTRAQ 8-plex), separated via OFFGEL electrophoresis, and analyzed by means of LC-MS/MS. Nonredundant protein families (4325 in tissues, 2017 in cell lines) were identified and quantified. Principal component analysis of the results clearly distinguished adenomas from normal mucosal samples and cancer cell lines from HCEC cells. Two hundred and twelve proteins displayed significant adenoma-related expression changes (q-value < 0.02, mean fold change versus normal mucosa ±1.4), which correlated (r = 0.74) with similar changes previously identified by our group at the transcriptome level. Fifty-one (?25%) proteins displayed directionally similar expression changes in colorectal cancer cells (versus HCEC cells) and were therefore attributed to the epithelial component of adenomas. Although benign, adenomas already exhibited cancer-associated proteomic changes: 69 (91%) of the 76 protein up-regulations identified in these lesions have already been reported in cancers. One of the most striking changes involved sorbitol dehydrogenase, a key enzyme in the polyol pathway. Validation studies revealed dramatically increased sorbitol dehydrogenase concentrations and activity in adenomas and cancer cell lines, along with important changes in the expression of other enzymes in the same (AKR1B1) and related (KHK) pathways. Dysregulated polyol metabolism might represent a novel facet of metabolome remodeling associated with tumorigenesis. PMID:24567419

Uzozie, Anuli; Nanni, Paolo; Staiano, Teresa; Grossmann, Jonas; Barkow-Oesterreicher, Simon; Shay, Jerry W; Tiwari, Amit; Buffoli, Federico; Laczko, Endre; Marra, Giancarlo

2014-05-01

257

Efficient analysis of egg yolk proteins and their thermal sensitivity using sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing and nonreducing conditions.  

PubMed

The multiple functional properties of egg yolk are mostly influenced by its complex protein composition. The high lipid content of egg yolk as well as the low solubility of delipidated egg yolk lipoproteins make analysis by conventional chromatographic or electrophoretic techniques a difficult task. This work describes a method to profile egg yolk proteins after delipidation with acetone using sodium dodecyl sulfate polyacrylamide gel electrophoresis on precast 8-18% T polyacrylamide gradient gels. Twenty bands were obtained for the whole egg yolk profile with molecular weights ranging between 5 and 221 kDa. The bands were identified based on their molecular weight and by comparison with isolated egg yolk subfractions. The dissociation behavior under reducing and nonreducing conditions provided additionally helpful information for identification and characterization of the yolk proteins. The method presented is very well suited for assaying the thermal sensitivity of whole yolk and its components and thus for the characterization of heat treatment processes. PMID:16302743

Guilmineau, Fabien; Krause, Ingolf; Kulozik, Ulrich

2005-11-30

258

Relative quantitation of proteins fractionated by the ProteomeLab™ PF 2D system using isobaric tags for relative and absolute quantitation (iTRAQ)  

Microsoft Academic Search

We describe an optimised protocol for application of isobaric tags for relative and absolute quantitation (iTRAQ) and tandem\\u000a mass spectrometry to obtain relative quantitative data from peptides derived from tryptic digestions of proteins fractionated\\u000a by using the 2D liquid-phase ProteomeLab™ PF 2D technique. This methodology is suitable for the quantitation of proteins from\\u000a a pool of co-eluting proteins which are

Helena Skalnikova; Pavel Rehulka; Josef Chmelik; Jirina Martinkova; Michaela Zilvarova; Suresh Jivan Gadher; Hana Kovarova

2007-01-01

259

Fluorescence detection for gel and capillary electrophoresis  

SciTech Connect

First, an indirect fluorescence detection system for the separation of proteins via gel electrophoresis. Quantities as low as 50 nanograms of bovine serum albumin and soybean trypsin inhibitor are separated and detected visually without the need for staining of the analytes. This is very similar to levels of protein commonly separated with gel electrophoresis.

Hogan, B.

1992-07-21

260

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.

2013-01-01

261

Two-dimensional gel electrophoresis pattern (pH 6-11) and identification of water-soluble barley seed and malt proteins by mass spectrometry.  

PubMed

A protocol was established for two-dimensional gel electrophoresis (2-DE) of barley seed and malt proteins in the pH range of 6-11. Proteins extracted from flour in a low-salt buffer were focused after cup-loading onto IPG strips. Successful separation in the second dimension was achieved using gradient gels in a horizontal SDS-PAGE system. Silver staining of gels visualized around 380 (seed) and 500 (malt) spots. Thirty-seven different proteins from seeds were identified in 60 spots, among these 46 were visualized also in the malt 2-D pattern. Proteins were identified by peptide mass fingerprinting and by tandem MS sequencing after in-gel digestion by trypsin. In addition, the N-terminal sequence of 10 different proteins from 11 spots was determined after electroblotting to a polyvinylidene difluoride (PVDF) membrane. Five identified proteins (in 9 spots) are involved in glycolysis, 12 in defence against pathogens (21 spots), 4 in storage, folding, and synthesis of proteins, and in nitrogen metabolism (5 spots), 6 in carbohydrate metabolism (11 spots), and 4 in stress and detoxification (9 spots). Six proteins (7 spots) were not grouped in these categories, and 3 were not ascribed a function. The presented 2-D patterns and identifications will be used to describe proteome differences between cultivars and changes during malting. PMID:14997495

Bak-Jensen, Kristian Sass; Laugesen, Sabrina; Roepstorff, Peter; Svensson, Birte

2004-03-01

262

A strategy of plasma protein quantitation by selective reaction monitoring of an intact protein.  

PubMed

Immunoassays are used extensively in the quantitative analysis of proteins in plasma, urine, and other biological matrixes to support preclinical and clinical studies. Although immunoassays are both sensitive and rapid, difficulties during development of these assays are compounded by the need to have a specific antibody or antigen to the protein of interest. Furthermore, calibration curves of immunoassays are inherently nonlinear, and the technique often detects many structurally related components in addition to the analyte of interest. We have developed a novel strategy of analyzing protein concentrations in plasma by utilizing 96-well solid-phase extraction and LC-MS/MS detection of the intact protein. This strategy has been successfully applied in method development and assay validation of quantitatively analyzing protein rK5 concentrations in monkey plasma samples. Additional techniques such as precolumn regeneration and column heating were also incorporated into the assay. Total run time for each sample was approximately 15 min. An LLOQ of 99.2 ng/mL from a sample volume of 50 microL, corresponding to only 380 fmol (3.97 ng) of the rK5 analyte being injected onto the analytical column (assuming 100% extraction recovery), was obtained. The validated linear dynamic range was between 99.2 and 52 920.0 ng/mL, with a correlation coefficient (r(2)) ranging from 0.9972 and 0.9994. The intraassay CV for this assay was between 0.6 and 3.8%, and the interassay CV was between 1.7 and 3.2%. Interassay mean accuracies were between 101.5 and 104.7%. The assay has proven rugged and specific and has been employed to generate data in support of preclinical studies. This strategy for rK5 assay could be used for the development of bioanalytical assays to provide preclinical and clinical support for other protein drug candidates and, furthermore, for the validation of biomarkers discovered from proteomic research. PMID:14670064

Ji, Qin C; Rodila, Ramona; Gage, Eric M; El-Shourbagy, Tawakol A

2003-12-15

263

Quantitative and Qualitative Differences in Protein Expression Between Papillary Thyroid Carcinoma and Normal Thyroid Tissue†  

PubMed Central

In order to better understand basic mechanisms of tumor development and identify potential new biomarkers, we have performed difference gel electrophoresis (DIGE) and peptide mass fingerprinting on pooled protein extracts from patients with papillary thyroid carcinoma (PTC) compared with matched normal thyroid tissue. Image analysis of DIGE gels comparing PTC and matched normal thyroid tissue protein indicated that 25% of the protein spots were differentially expressed at a 2.5-fold cutoff and 35% at two-fold. Comparison between two different pools of protein from normal thyroid tissues revealed differential protein expression of only 4% at 2.5-fold and 6% at two-fold cutoff. One hundred ninety-two protein spots were identified by MALDI-TOFMS, representing 90 distinct proteins. Excluding albumin, globins and thyroglobulin, imaging software determined 31 proteins to be differentially expressed at the two-fold (or greater) level. Individual gel comparisons (PTC vs. matched normal) from five patients established that 15/31 (48%) of these proteins exhibited statistically significant differential expression. Previously identified molecular markers in this group of proteins include cathepsin B, cytokeratin 19, and galectin-3. Novel differentially expressed proteins include S100A6, moesin, HSP70 (BiP), peroxiredoxin 2, protein phosphatase 2, selenium binding protein 1, vitamin D binding protein, and proteins involved in mitochondrial function. The use of two-dimensional gel electrophoresis (2DGE) revealed a significantly altered protein mass and/or pI in 10%–15% of proteins, suggesting alternatively spliced forms and other posttranslational modification of proteins revealed by this approach. We confirmed S100A6 as a potentially useful biomarker using immunohistochemical analysis (85% sensitivity and 69% specificity for distinguishing benign from malignant thyroid neoplasms). In summary, proteomic analysis of PTC using DIGE and mass spectrometry has confirmed several known biomarkers, uncovered novel potential biomarkers, and provided insights into global pathophysiologic changes in PTC. Many of the differences observed would not have been detected by genomic or other proteomic approaches

Brown, Lewis M.; Helmke, Steve M.; Hunsucker, Stephen W.; Netea-Maier, Romana T.; Chiang, Simon A.; Heinz, David E.; Shroyer, Kenneth R.; Duncan, Mark W.; Haugen, Bryan R.

2007-01-01

264

Label-Free Protein Quantitation Using Weighted Spectral Counting  

PubMed Central

Mass spectrometry (MS)-based shotgun proteomics allows protein identifications even in complex biological samples. Protein abundances can then be estimated from the counts of MS/MS spectra attributable to each protein, provided that one corrects for differential MS-detectability of the contributing peptides. We describe the use of a method, APEX, which calculates Absolute Protein EXpression levels based on learned correction factors, MS/MS spectral counts, and each protein's probability of correct identification. The APEX-based calculations consist of three parts: (1) Using training data, peptide sequences and their sequence properties, a model is built that can be used to estimate MS-detectability (Oi) for any given protein. (2) Absolute abundances of proteins measured in an MS/MS experiment are calculated with information from spectral counts, identification probabilities and the learned Oi -values. (3) Simple statistics allow for significance analysis of differential expression in two distinct biological samples, i.e., measuring relative protein abundances. APEX-based protein abundances span more than four orders of magnitude and are applicable to mixtures of hundreds to thousands of proteins from any type of organism.

Vogel, Christine; Marcotte, Edward M.

2012-01-01

265

Characterization of human placental alkaline phosphatase by activity and protein assays, capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.  

PubMed

Placental alkaline phosphatase (PLAP) that had been isolated from human placenta was further purified using subsequent ion-exchange chromatography (IEC), affinity chromatography (AC) and centrifugal membrane concentration (CMC). During the process, the PLAP samples from the different stages of purification were characterized regarding purity and activity. This was accomplished by combining Lowry analysis, enzymatic activity assay, capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE) and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS). The sample obtained after IEC had a rather low specific activity (6.8 U/mg) and appeared to contain several major contaminants, among which was human serum albumin (HSA). AC followed by CMC yielded PLAP with a specific activity of 128 U/mg. The purity and identity of the protein was indicated by MALDI-TOF-MS yielding a spectrum with one major peak at m/z 58,101. Interestingly, CZE of the pure PLAP revealed a cluster of peaks, which probably reflects the presence of various glycoforms and/or oligomers. The same analytical approach was used to characterize commercially available PLAP. This sample showed a moderate specific activity (15 U/mg) and appeared to be highly impure containing various other proteins. PMID:11393719

Eriksson, H J; Somsen, G W; Hinrichs, W L; Frijlink, H W; de Jong, G J

2001-05-01

266

Characterization of the osteogenic stromal cell line MN7: identification of secreted MN7 proteins using two-dimensional polyacrylamide gel electrophoresis, western blotting, and microsequencing.  

PubMed

Proteins secreted by the osteogenic stromal cell line MN7 were analyzed using two-dimensional polyacrylamide gel electrophoresis (PAGE), western blotting, immunodetection, and microsequencing. Trichloroacetic acid-precipitated proteins from the conditioned medium of MN7 cell cultures, harvested at different times of growth, were dissolved in denaturing and reducing sample buffer and separated in the first dimension according to isoelectric point and in the second dimension according to molecular weight. Protein patterns were visualized using silver staining. Among the 350 separated protein spots, we identified type I collagen, bone sialoprotein, osteonectin, and cathepsin B by western blotting and immunodetection using polyclonal antibodies. Osteocalcin could not be detected in the conditioned medium of MN7 cells. Furthermore, 15 MN7-specific protein spots were localized after comparison with two-dimensional PAGE patterns from the conditioned medium of the nonosteogenic stromal cell lines MM1 and MV1. Microsequencing of the internal peptides of five selected spots revealed three known proteins, namely the carboxyl-terminal propeptide of the alpha 2 chain of collagen type I, cathepsin L, and the tissue inhibitor of metalloproteinases-2, an 18 kilodalton peptide fragment from osteopontin that has not previously been described, and a novel glycosylated 85 kD protein with an average isoelectric point of 5.7. All identified proteins did not vary in presence between the different time points analyzed by two-dimensional PAGE. The use of two-dimensional PAGE to investigate the secreted proteins of MN7 cells will enable us to establish a complete protein data base of extracellular osteoblast-specific proteins. Furthermore, two-dimensional PAGE in combination with other techniques is a fast and accurate method for the identification of novel proteins that could function as markers in osteoblast differentiation and/or bone formation. PMID:8079665

Mathieu, E; Meheus, L; Raymackers, J; Merregaert, J

1994-06-01

267

Plasma biomarker discovery using 3D protein profiling coupled with label-free quantitation.  

PubMed

In-depth quantitative profiling of human plasma samples for biomarker discovery remains quite challenging. One promising alternative to chemical derivatization with stable isotope labels for quantitative comparisons is direct, label-free, quantitative comparison of raw LC-MS data. But, in order to achieve high-sensitivity detection of low-abundance proteins, plasma proteins must be extensively pre-fractionated, and results from LC-MS runs of all fractions must be integrated efficiently in order to avoid misidentification of variations in fractionation from sample to sample as "apparent" biomarkers. This protocol describes a powerful 3D protein profiling method for comprehensive analysis of human serum or plasma proteomes, which combines abundant protein depletion and high-sensitivity GeLC-MS/MS with label-free quantitation of candidate biomarkers. PMID:21468938

Beer, Lynn A; Tang, Hsin-Yao; Barnhart, Kurt T; Speicher, David W

2011-01-01

268

iTRAQ-based quantitative analysis of protein mixtures with large fold change and dynamic range.  

PubMed

Quantitation of changes in protein abundance is key to understanding the alterations that biological systems undergo and to discovering novel biomarkers. Currently, HPLC-MS/MS can be used to quantify changes in protein expression levels [Ong, S. E. and Mann, M., Nat. Chem. Biol. 2005, 1, 252-262]. Nevertheless, quantitative analysis of protein mixtures by HPLC-MS/MS is still hampered by the wide range of protein expression levels, the high dynamic range of protein concentrations and the lack of reliable quantitation algorithms [D'Ascenzo, M., et al. Brief. Funct. Genomic. Proteomic. 2008, 7, 127-135; Lin, W. T., et al., J. Proteome Res. 2006, 5, 2328-2338; Matthiesen, R., et al. J. Proteome Res. 2005, 4, 2338-2347; Yu, C. Y., et al. Nucleic Acids Res. 2007, 35, W707-W712]. In this context, we describe two different samples (4-protmix and 8-protmix) suitable for relative protein quantitation using iTRAQ. Using the 4-protmix, relative protein changes of up to 24-fold were measured. The 8-protmix allowed the quantitation of the relative protein changes in a mixture of proteins within the range of two orders of magnitude in concentration and ten-fold differences in relative abundance. We propose that the two samples are suited to test the iTRAQ quantitative proteomic workflow. We analyzed the iTRAQ samples with a LTQ Orbitrap using "higher energy collision-induced dissociation" fragmentation [Olsen, J. V., et al., Nat. Methods 2007, 4, 709-712] and quantified with Proteome Discoverer v.1.1 (Thermo Fisher Scientific). We believe that the presented protein mixtures will be useful to assess the performance of the iTRAQ-based quantitation proteomic strategy in any laboratory. PMID:20029838

Casado-Vela, Juan; Martínez-Esteso, María José; Rodriguez, Eva; Borrás, Eva; Elortza, Felix; Bru-Martínez, Roque

2010-01-01

269

Quantitative targeted proteomics for membrane transporter proteins: method and application.  

PubMed

Although global proteomics has shown promise for discovery of many new proteins, biomarkers, protein modifications, and polymorphisms, targeted proteomics is emerging in the proteomics research field as a complement to untargeted shotgun proteomics, particularly when a determined set of low-abundance functional proteins need to be measured. The function and expression of proteins related to drug absorption, distribution, metabolism, and excretion (ADME) such as cytochrome P450 enzymes and membrane transporters are of great interest in biopharmaceutical research. Since the variation in ADME-related protein expression is known to be a major complicating factor encountered during in vitro-in vivo and in vivo-in vivo extrapolations (IVIVE), the accurate quantification of the ADME proteins in complex biological systems becomes a fundamental element in establishing IVIVE for pharmacokinetic predictions. In this review, we provide an overview of relevant methodologies followed by a summary of recent applications encompassing mass spectrometry-based targeted quantifications of membrane transporters. PMID:24830943

Qiu, Xi; Zhang, Hui; Lai, Yurong

2014-07-01

270

Two-dimensional difference gel electrophoresis.  

PubMed

Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as "spots" with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With the appropriate imaging system, difference gel electrophoresis (DIGE) is capable of reliably detecting as little as 0.2 fmol of protein, and protein differences down to ±15%, over a ?20,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2-3 days to complete. PMID:22585495

Minden, Jonathan S

2012-01-01

271

Quantitative cytospectrophotometrical determination of the total protein thiols with “Mercurochrom”  

Microsoft Academic Search

Mercurochrom (2,7-dibromo-4-(hydroxymercuri)-fluoresceine-disodium salt) reacts histochemically not only with protein-SH-groups, but is also bound unspecifically to cellular proteins. The amount of the unspecifical staining approximately equals the specific SH-staining. Two methods are described to remove the unspecifically bound Mercurochrom, without influencing the specific reaction with the protein thiols. The first applies 0,1 m thioglycolate pH 4.0 (MT4-method), the other a special

G. Nöhammer; G. Desoye; G. Khoschsorur

1981-01-01

272

Identification of dengue RNA binding proteins using RNA chromatography and quantitative mass spectrometry.  

PubMed

A major challenge in dengue virus (DENV) research has been to understand the interaction of the viral RNA with host cell proteins during infection. Until recently, there were no comprehensive studies identifying host RNA binding proteins that interact with DENV RNA (Ward et al. RNA Biol 8 (6):1173-1186, 2011). Here, we describe a method for identifying proteins that associate with DENV RNA using RNA chromatography and quantitative mass spectrometry. The method utilizes a tobramycin RNA aptamer incorporated into an RNA containing the dengue 5' and 3' untranslated regions (UTRs) in order to reversibly bind RNA to a tobramycin matrix. The RNA-tobramycin matrix is incubated with SILAC-labeled cell lysates, and bound proteins are eluted using an excess of tobramycin. The eluate is analyzed using quantitative mass spectrometry, which allows direct and quantitative comparison of proteins bound to DENV UTRs and a control RNA-tobramycin matrix. This technique has the advantage of allowing one to distinguish between specific and nonspecific binding proteins based on the ratio of protein preferentially bound to the DENV UTRs versus the control RNA. This methodology can also be used for validation of quantitative mass spectrometry results using conventional Western blotting for specific proteins. Furthermore, though it was specifically developed to identify DENV RNA binding proteins, the RNA chromatography method described here can be applied to a broad range of viral and cellular RNAs for identification of interacting proteins. PMID:24696342

Ward, Alex M; Gunaratne, J; Garcia-Blanco, Mariano A

2014-01-01

273

Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size  

SciTech Connect

In biofluids (e.g., blood plasma) nanoparticles are readily embedded in layers of proteins that can affect their biological activity and biocompatibility. Herein, we report a study on the interactions between human plasma proteins and nanoparticles with a controlled systematic variation of properties using stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS) based quantitative proteomics. Novel protocol has been developed to simplify the isolation of nanoparticle bound proteins and improve the reproducibility. Plasma proteins associated with polystyrene nanoparticles with three different surface chemistries and two sizes as well as for four different exposure times (for a total of 24 different samples) were identified and quantified by LC-MS analysis. Quantitative comparison of relative protein abundances were achieved by spiking an 18 O-labeled 'universal reference' into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantitation across the sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive pattern that classifies the nanoparticles based on their surface properties and size. In addition, data on the temporal study indicated that the stable protein 'corona' that was isolated for the quantitative analysis appeared to be formed in less than 5 minutes. The comprehensive results obtained herein using quantitative proteomics have potential implications towards predicting nanoparticle biocompatibility.

Zhang, Haizhen; Burnum, Kristin E.; Luna, Maria L.; Petritis, Brianne O.; Kim, Jong Seo; Qian, Weijun; Moore, Ronald J.; Heredia-Langner, Alejandro; Webb-Robertson, Bobbie-Jo M.; Thrall, Brian D.; Camp, David G.; Smith, Richard D.; Pounds, Joel G.; Liu, Tao

2011-12-01

274

A novel specific heparin-binding activity of bovine folate-binding protein characterized by capillary electrophoresis.  

PubMed

Folate-binding proteins (FBPs) are ubiquitous, soluble and membrane-bound high-affinity receptors for folate, an essential nutrient involved in nucleic and amino acid metabolism. In the course of optimizing CE separation conditions for FBP purified from cow's milk we discovered a novel specific heparin-binding activity of FBP by affinity CE. Heparin is a highly sulfated glycosaminoglycan and thus prone to induce anodic migration shifts of complexing analytes. Prior complexation of FBP with folate abolished heparin binding, and thus folate competes with heparin for binding to FBP. It was estimated that heparin bound several orders of magnitude less strongly than folate with an average dissociation constant in the 1-10 microM range. In contrast to the mobility shifts induced by heparin, free and folate-bound FBP were not separated by CE. However, binding of folate induced a distinct increase in FBP-peak symmetry, and using heparin as an affinity displacer, the free FBP in equilibrium with folate-FBP complexes could readily be separated from the complexes. While the folate-FBP interaction was too strong to be characterized quantitatively because of inadequate detection limits of a UV-based detection system, it was possible to estimate the folate-FBP binding stoichiometry using this approach. The heparin interaction fractionated FBP into distinct subfractions, and the CE approach thus promises to be useful for unraveling the complex oligomerization behavior of FBP isoforms as well as for evaluating the FBP affinity for various species and analogs of glycosaminoglycans and folate. PMID:16470783

Heegaard, Niels H H; Hansen, Steen I; Holm, Jan

2006-03-01

275

Quantitative measurement of protein digestion in simulated gastric fluid  

Microsoft Academic Search

The digestibility of novel proteins in simulated gastric fluid is considered to be an indicator of reduced risk of allergenic potential in food, and estimates of digestibility for transgenic proteins expressed in crops are required for making a human-health risk assessment by regulatory authorities. The estimation of first-order rate constants for digestion under conditions of low substrate concentration was explored

Rod A. Herman; Valerie A. Korjagin; Barry W. Schafer

2005-01-01

276

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

PubMed

The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase. PMID:20801349

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

2010-09-15

277

Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis.  

PubMed

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

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

2013-10-01

278

Quantitative Proteomics Identify Novel miR-155 Target Proteins  

PubMed Central

Background MicroRNAs are 22 nucleotides long non-coding RNAs and exert their function either by transcriptional or translational inhibition. Although many microRNA profiles in different tissues and disease states have already been discovered, only little is known about their target proteins. The microRNA miR-155 is deregulated in many diseases, including cancer, where it might function as an oncoMir. Methodology/Principal Findings We employed a proteomics technique called “stable isotope labelling by amino acids in cell culture” (SILAC) allowing relative quantification to reliably identify target proteins of miR-155. Using SILAC, we identified 46 putative miR-155 target proteins, some of which were previously reported. With luciferase reporter assays, CKAP5 was confirmed as a new target of miR-155. Functional annotation of miR-155 target proteins pointed to a role in cell cycle regulation. Conclusions/Significance To the best of our knowledge we have investigated for the first time miR-155 target proteins in the HEK293T cell line in large scale. In addition, by comparing our results to previously identified miR-155 target proteins in other cell lines, we provided further evidence for the cell line specificity of microRNAs.

Lossner, Christopher; Meier, Jan; Warnken, Uwe; Rogers, Michael A.; Lichter, Peter; Pscherer, Armin; Schnolzer, Martina

2011-01-01

279

Quantitative proteomic analysis of cold-responsive proteins in rice.  

PubMed

Rice is susceptible to cold stress and with a future of climatic instability we will be unable to produce enough rice to satisfy increasing demand. A thorough understanding of the molecular responses to thermal stress is imperative for engineering cultivars, which have greater resistance to low temperature stress. In this study we investigated the proteomic response of rice seedlings to 48, 72 and 96?h of cold stress at 12-14°C. The use of both label-free and iTRAQ approaches in the analysis of global protein expression enabled us to assess the complementarity of the two techniques for use in plant proteomics. The approaches yielded a similar biological response to cold stress despite a disparity in proteins identified. The label-free approach identified 236 cold-responsive proteins compared to 85 in iTRAQ results, with only 24 proteins in common. Functional analysis revealed differential expression of proteins involved in transport, photosynthesis, generation of precursor metabolites and energy; and, more specifically, histones and vitamin B biosynthetic proteins were observed to be affected by cold stress. PMID:21433000

Neilson, Karlie A; Mariani, Michael; Haynes, Paul A

2011-05-01

280

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.

2010-01-01

281

Bloodstream Infection in Neutropenic Cancer Patients Related to Short-Term Nontunnelled Catheters Determined by Quantitative Blood Cultures, Differential Time to Positivity, and Molecular Epidemiological Typing with Pulsed-Field Gel Electrophoresis  

PubMed Central

To determine the rate of catheter-related bloodstream infection (CRBSI) among cases of primary bloodstream infection (BSI) in febrile neutropenic cancer patients with short-term nontunnelled catheters, quantitative paired blood cultures (Isolator) from the central venous catheter (CVC) and peripheral vein were obtained between November 1999 and January 2001. Bactec blood culture bottles were obtained to determine the differential time to positivity (DTP). CRBSI was defined as a quantitative blood culture ratio of >5:1 (CVC versus peripheral) with proven identity of isolates from positive peripheral and CVC blood cultures as confirmed by pulsed-field gel electrophoresis. Forty-nine episodes of primary BSI were detected among 235 cancer patients with febrile neutropenia. Of these, 18 episodes (37%) were CRBSI and 31 (63%) were BSI with an unknown portal of entry. Coagulase-negative staphylococci were present in nine cases of CRBSI (50%). The identity of isolates from peripheral and CVC blood cultures was confirmed in all cases. Earlier positivity (>2 h) of CVC-drawn versus peripheral blood cultures was observed in 18 of 22 CRBSI-associated blood cultures (sensitivity, 82%; specificity, 88%; positive predictive value, 75%; negative predictive value, 92%). In summary, CRBSI accounted for 37% of cases of primary BSI in this population of neutropenic cancer patients. DTP compares favourably with quantitative blood cultures for the diagnosis of CRBSI and may be particularly useful for patients in whom catheter salvage is highly desirable.

Seifert, Harald; Cornely, Oliver; Seggewiss, Kerstin; Decker, Mathias; Stefanik, Danuta; Wisplinghoff, Hilmar; Fatkenheuer, Gerd

2003-01-01

282

Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2)  

PubMed Central

Background Genotyping of Plasmodium falciparum based on PCR amplification of the polymorphic genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) is well established in the field of malaria research to determine the number and types of concurrent clones in an infection. Genotyping is regarded essential in anti-malarial drug trials to define treatment outcome, by distinguishing recrudescent parasites from new infections. Because of the limitations in specificity and resolution of gel electrophoresis used for fragment analysis in most genotyping assays it became necessary to improve the methodology. An alternative technique for fragment analysis is capillary electrophoresis (CE) performed using automated DNA sequencers. Here, one of the most widely-used protocols for genotyping of P. falciparum msp1 and msp2 has been adapted to the CE technique. The protocol and optimization process as well as the potentials and limitations of the technique in molecular epidemiology studies and anti-malarial drug trials are reported. Methods The original genotyping assay was adapted by fluorescent labeling of the msp1 and msp2 allelic type specific primers in the nested PCR and analysis of the final PCR products in a DNA sequencer. A substantial optimization of the fluorescent assay was performed. The CE method was validated using known mixtures of laboratory lines and field samples from Ghana and Tanzania, and compared to the original PCR assay with gel electrophoresis. Results The CE-based method showed high precision and reproducibility in determining fragment size (< 1 bp). More genotypes were detected in mixtures of laboratory lines and blood samples from malaria infected children, compared to gel electrophoresis. The capacity to distinguish recrudescent parasites from new infections in an anti-malarial drug trial was similar by both methods, resulting in the same outcome classification, however with more precise determination by CE. Conclusion The improved resolution and reproducibility of CE in fragment sizing allows for comparison of alleles between separate runs and determination of allele frequencies in a population. The more detailed characterization of individual msp1 and msp2 genotypes may contribute to improved assessments in anti-malarial drug trials and to a further understanding of the molecular epidemiology of these polymorphic P. falciparum antigens.

Liljander, Anne; Wiklund, Lisa; Falk, Nicole; Kweku, Margaret; Martensson, Andreas; Felger, Ingrid; Farnert, Anna

2009-01-01

283

Identification of drug-binding sites on human serum albumin using affinity capillary electrophoresis and chemically modified proteins as buffer additives.  

PubMed

A technique based on affinity capillary electrophoresis (ACE) and chemically modified proteins was used to screen the binding sites of various drugs on human serum albumin (HSA). This involved using HSA as a buffer additive, following the site-selective modification of this protein at two residues (tryptophan 214 or tyrosine 411) located in its major binding regions. The migration times of four compounds (warfarin, ibuprofen, suprofen and flurbiprofen) were measured in the presence of normal or modified HSA. These times were then compared and the mobility shifts observed with the modified proteins were used to identify the binding regions of each injected solute on HSA. Items considered in optimizing this assay included the concentration of protein placed into the running buffer, the reagents used to modify HSA, and the use of dextran as a secondary additive to adjust protein mobility. The results of this method showed good agreement with those of previous reports. The advantages and disadvantages of this approach are examined, as well as its possible extension to other solutes. PMID:11920883

Kim, Hee Seung; Austin, John; Hage, David S

2002-03-01

284

Detection of differential levels of proteins in the urine of patients with endometrial cancer: analysis using two-dimensional gel electrophoresis and o-glycan binding lectin.  

PubMed

Cancers can cause some proteins to be aberrantly excreted or released in the urine, which can be used as biomarkers. To screen for potential biomarkers for endometrial cancer (ECa), the urinary proteins from patients who were newly diagnosed with early stage ECa and untreated controls were separated using two-dimensional gel electrophoresis (2-DE) and followed by image analysis. The altered levels of zinc alpha-2 glycoprotein, alpha 1-acid glycoprotein, and CD59 were detected in the patients compared to the controls. In addition, the urine of the ECa patients was also found to contain relatively lower levels of a fragment of nebulin when the 2-DE separated urinary proteins were probed using champedak galactose binding (CGB) lectin. The different levels of the nebulin fragment were further validated by subjecting the urinary protein samples to CGB lectin affinity chromatography and analysis of the bound fractions by LC-MS/MS. Our data is suggestive of the potential use of the differentially expressed urinary proteins as biomarkers for ECa although this requires further extensive validation on clinically representative populations. PMID:22949810

Mu, Alan Kang-Wai; Lim, Boon-Kiong; Hashim, Onn Haji; Shuib, Adawiyah Suriza

2012-01-01

285

QUANTITATIVE DOT-IMMUNOBINDING ASSAY FOR PROTEINS USING NITROCELLULOSE MEMBRANE FILTERS  

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...

286

Quantitative structure activity relationship modelling of peptides and proteins as a tool in food science  

Microsoft Academic Search

Peptides and proteins contribute to physical properties, biological activities and sensory characteristics of foods. Studies on the isolation and characterisation of peptides and proteins allow compilation of data sets on their structures and properties\\/activities. Quantitative structure activity relationship (QSAR) modelling provides methodology to find mathematical expressions for such relationships which may then be useful for estimating activities of any related

Are Hugo Pripp; Tomas Isaksson; Leszek Stepaniak; Terje Sørhaug; Ylva Ardö

2005-01-01

287

Application of a nitrocellulose immunoassay for quantitation of proteins secreted in cultured media  

Microsoft Academic Search

A macro-dot immunoassay was developed to quantitate proteins (antigens) secreted in the culture media of primary rat hepatocytes. Dilutions of protein standards and undiluted spent culture media were applied to numbered sheets of nitrocellulose (NC) paper by vacuum filtration (in volumes up to 1 ml) through a specially designed macrofiltration apparatus constructed of plexiglas. Sequential incubation of the NC with

F. M. LaDuca; C. V. Dang; W. R. Bell

1986-01-01

288

Quantitative variation in cystic fibrosis-associated proteins in cystic fibrosis patients, carriers, and controls  

Microsoft Academic Search

Serum samples from patients with cystic fibrosis (CF), obligate heterozygotes, and normal controls have been examined by isoelectric focusing (IEF). Our results suggest that cystic fibrosis protein (CFP) is a normal serum protein exhibiting quantitative variation primarily dependent on possession of the CF allele. It is concluded that detection of CFP by IEF is an inappropriate screening test for the

A. Jamieson; Elizabeth Mackinlay; D. A. Aitken; A. Cooke; M. A. Ferguson-Smith

1985-01-01

289

Quantitative Analysis of Protein-Lipid Interactions Using Tryptophan Fluorescence  

NSDL National Science Digital Library

The fluorescent properties of the amino acid tryptophan make it a useful tool for fluorometric assays. Because tryptophan fluorescence is remarkably sensitive to the polarity of the environment, it can be used to determine the affinity of tryptophan-containing peptides for phospholipid vesicles of varying compositions. Here, we describe a method for using tryptophan fluorescence to determine the binding affinities of peptides derived from the proteins Raf-1 and KSR-1 to small unilamellar vesicles containing phosphatidic acid. The method can be extrapolated to measure the binding of other tryptophan-containing peptides or proteins to lipid vesicles.

Catherine A. Kraft (University of Pittsburgh School of Medicine;The Department of Pharmacology and Chemical Biology REV); Jose Luis Garrido (University of Pittsburgh School of Medicine;The Department of Pharmacology and Chemical Biology REV); Luis Leiva-Vega (University of Pittsburgh School of Medicine;The Department of Pharmacology and Chemical Biology REV); Guillermo Romero (University of Pittsburgh School of Medicine;The Department of Pharmacology and Chemical Biology REV)

2009-12-01

290

Primitive Model Electrophoresis.  

PubMed

The electrophoresis of a spherical macroion is calculated with the new primitive model electrophoresis (PME) theory, which incorporates the ionic size. The results are compared with the classical theory of Wiersema, O'Brien, and White. The PME mobility, as a function of the macroion's surface charge (sigma) or zeta potential (zeta), is found to depend on the ionic valence, radius and concentration, and macroion hydrodynamical radius (A); i.e., it is not universal with kappaA, as predicted by the classical theory. The mobility is very nonlinear as a function of zeta. This behavior is related to the nonlinear dependence of zeta, as a function of sigma, when ionic size is included in the theory. In the classical theory zeta is a monotonic function of sigma. Important quantitative and/or qualitative differences between PME and the classical theory are found. However, in the limit of zero ionic diameter, and/or low salt concentration, and low macroion charge, the new theory reduces to the classical theory. The agreement of the PME with experimental data is very good. In particular, the prediction of reversed mobility is corroborated by recent experimental data. Copyright 2001 Academic Press. PMID:11426992

Lozada-Cassou, Marcelo; González-Tovar, Enrique

2001-07-15

291

Quantitative Binding Assays using Green Fluorescent Protein as a Label.  

National Technical Information Service (NTIS)

A heterogeneous binding assay for an analyte in a fluid sample is developed, which uses a green fluorescent protein (GFP) label. A ligand-GFP conjugate has a specific binding affinity for an anti-ligand immobilized on a support. The anti-ligand also has a...

S. Daunert J. C. Lewis E. C. Hernandez

2005-01-01

292

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

2007-01-01

293

A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: A follow up  

Microsoft Academic Search

With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid

B. E. Maserti; C. M. Della Croce; F. Luro; R. Morillon; M. Cini; L. Caltavuturo

2007-01-01

294

High throughput and rapid screening of marine protein hydrolysates enriched in peptides with angiotensin-I-converting enzyme inhibitory activity by capillary electrophoresis.  

PubMed

Twelve kinds of marine protein materials, including fish, shrimp, seashell, algae and seafood wastes were selected for the hydrolysis using four different proteases. The IC(50) values for angiotensin-converting enzyme (ACE) inhibitory activity of 48 hydrolysates were rapidly determined by capillary electrophoresis (CE). The values ranged from 0.17 to 501.7mg/ml, and were affected by both the marine protein resources and the selected proteases. Hydrolysates of the lowest IC(50) values were from shrimp (Acetes chinensis), shark meat, mackerel bone, Polysiphonia urceolata and Spirulina platensis, indicating these five kinds of marine food proteins contained beneficial materials for the production of ACE inhibitory peptides by proteolysis. The hydrolysates obtained using proteases Protamex and SM98011 had lower IC(50) values, showing these two proteases were superior to others. The CE method achieved the same sensitivity as the high performance liquid chromatography (HPLC) method. However, the CE method was faster and, as a result, more economical. Therefore, CE had potential for rapid screening of marine protein hydrolysates enriched in ACE inhibitory peptides. PMID:17317156

He, Hai-Lun; Chen, Xiu-Lan; Wu, Hao; Sun, Cai-Yun; Zhang, Yu-Zhong; Zhou, Bai-Cheng

2007-12-01

295

The use of 2D-electrophoresis and de novo sequencing to characterize inter- and intra-cultivar protein polymorphisms in an allopolyploid crop.  

PubMed

Polyploidy and allopolyploidy have played an important role in the evolution of many plants and crops. Several techniques exist to characterize allopolyploid varieties. Analyzing the consequences of genomic reorganization at the gDNA level is a prerequisite but a better insight into the consequences for the phenotype is also primordial. As such, protein polymorphism analysis is important in understanding plant and crop biodiversity and is a driving force behind crop improvement. Our strategy to analyze protein isoforms and to detect possible gene silencing or deletion in bananas was based on protein analysis. Bananas are a good representative of a complex allopolyploid and important crop. We combined two-dimensional electrophoresis (2DE) and 2D DIGE with de novo MS/MS sequence determination to characterize a range of triploid varieties. Via Principal Component Analysis (PCA) and hierarchical clustering we were able to blindly classify the different varieties according to their presumed genome constitution. We report for the first time the application of an automated approach for the derivatization of peptides for facilitated MS/MS de novo sequence determination. We conclude that the proteome does not always correspond to the presumed genome formulae and that proteomics is a powerful tool to characterize varieties. The observations at the protein level provide good indications for a more complex genome structure and genomic rearrangement in some banana varieties. PMID:21109271

Carpentier, Sebastien Christian; Panis, Bart; Renaut, Jenny; Samyn, Bart; Vertommen, Annelies; Vanhove, Anne-Catherine; Swennen, Rony; Sergeant, Kjell

2011-07-01

296

Identification of multiresistant Staphylococcus epidermidis in neonates of a secondary care hospital using pulsed field gel electrophoresis and quantitative antibiogram typing.  

PubMed Central

AIMS: To determine the diversity of types of Staphylococcus epidermidis in a neonatal care unit of a secondary care hospital in the Netherlands. METHODS: In a prospective study, specimens from nose, ear, axilla, umbilicus, and groin were taken from patients twice a week during a period of up to two weeks. All isolates were typed by both pulsed field gel electrophoresis (PFGE) and antibiogram analysis. RESULTS: Fifty three S epidermidis isolates from 15 of 24 patients were obtained in one to four surveys. Fourteen isolates from six patients had a common PFGE pattern and were of one multiresistant antibiogram type. The remaining 39 isolates were allocated to 24 sporadic PFGE types and were more susceptible to antibiotics. Colonisation with the multiresistant strain correlated with a long period of stay and with the use of specific antibiotics. The multiresistant isolates were related closely to isolates of S epidermidis found in a recent study in a teaching hospital in the vicinity of the secondary care hospital. CONCLUSION: Repeated sampling and the use of two typing methods allowed the identification of two closely related multiresistant S epidermidis strains in two hospitals in the same area. Images

Sloos, J H; Horrevorts, A M; Van Boven, C P; Dijkshoorn, L

1998-01-01

297

A new quantitative method for circulating DNA level in human serum by capillary zone electrophoresis with laser-induced fluorescence detection.  

PubMed

We present a method for the quantification of circulating DNA in serum by capillary zone electrophoresis (CZE) with laser-induced fluorescence detection (LIF). The serum was digested by proteinase to release free DNA. SYBR Gold was utilized as DNA intercalating dye, fluorescein as internal standard (ISTD). CZE-LIF was applied for the separation and quantification of total circulating DNA. Good linearity (R = 0.9992) in the low range of DNA concentrations (0.5-40 ng/mL) and a detection limit of 0.5 ng/mL for DNA (S/N = 3) were obtained. Our data demonstrated that CZE-LIF system has a good linearity with excellent sensitivity and satisfactory reproducibility in the quantification of circulating DNA in serum. This method was successfully used for the quantification of circulating DNA levels in serum. We observed that the circulating DNA levels in certain cancer patients were significantly higher than that in healthy individuals. Compared to current methods, our protocol does not need the extraction of DNA from serum. Our preliminary results have illustrated that CZE-LIF system is simple, rapid, and sensitive, and it is well suitable for large-scale quantification of circulating DNA levels in clinical diagnosis. PMID:15213980

Zhang, Peng; Ren, Jicun; Shen, Zuojun

2004-06-01

298

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.

2005-01-01

299

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

PubMed

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

2009-04-24

300

Long-Term Biological Variation of Serum Protein Electrophoresis M-Spike, Urine M-Spike, and Monoclonal Serum Free Light Chain Quantification: Implications for Monitoring Monoclonal Gammopathies  

PubMed Central

BACKGROUND We analyzed serial data in patients with clinically stable monoclonal gammopathy to determine the total variation of serum M-spikes [measured with serum protein electrophoresis (SPEP)], urine M-spikes [measured with urine protein electrophoresis (UPEP)], and monoclonal serum free light chain (FLC) concentrations measured with immunoassay. METHODS Patients to be studied were identified by (a) no treatment during the study interval, (b) no change in diagnosis and <5 g/L change in serum M-spike over the course of observation; (c) performance of all 3 tests (SPEP, UPEP, FLC immunoassay) in at least 3 serial samples that were obtained 9 months to 5 years apart; (d) serum M-spike ?10 g/L, urine M-spike ?200 mg/24 h, or clonal FLC ?100 mg/L. The total CV was calculated for each method. RESULTS Among the cohort of 158 patients, 90 had measurable serum M-spikes, 25 had urine M-spikes, and 52 had measurable serum FLC abnormalities. The CVs were calculated for serial SPEP M-spikes (8.1%), UPEP M-spikes (35.8%), and serum FLC concentrations (28.4%). Combining these CVs and the interassay analytical CVs, we calculated the biological CV for the serum M-spike (7.8%), urine M-spike (35.5%), and serum FLC concentration (27.8%). CONCLUSIONS The variations in urine M-spike and serum FLC measurements during patient monitoring are similar and are larger than those for serum M-spikes. In addition, in this group of stable patients, a measurable serum FLC concentration was available twice as often as a measurable urine M-spike.

Katzmann, Jerry A.; Snyder, Melissa R.; Rajkumar, S. Vincent; Kyle, Robert A.; Therneau, Terry M.; Benson, Joanne T.; Dispenzieri, Angela

2013-01-01

301

Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size  

PubMed Central

Nanoparticle biological activity, biocompatibility and fate can be directly affected by layers of readily adsorbed host proteins in biofluids. Here, we report a study on the interactions between human blood plasma proteins and nanoparticles with a controlled systematic variation of properties using 18O-labeling and LC-MS-based quantitative proteomics. We developed a novel protocol to both simplify isolation of nanoparticle bound proteins and improve reproducibility. LC-MS analysis identified and quantified 88 human plasma proteins associated with polystyrene nanoparticles consisting of three different surface chemistries and two sizes, as well as, for four different exposure times (for a total of 24 different samples). Quantitative comparison of relative protein abundances was achieved by spiking an 18O-labeled “universal” reference into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantification across the entire sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive patterns that classified the nanoparticles based on their surface properties and size. In addition, temporal data indicated that the formation of the stable protein corona was at equilibrium within 5 min. The comprehensive quantitative proteomics results obtained in this study provide rich data for computational modeling and have potential implications towards predicting nanoparticle biocompatibility.

Zhang, Haizhen; Burnum, Kristin E.; Luna, Maria L.; Petritis, Brianne O.; Kim, Jong-Seo; Qian, Wei-Jun; Moore, Ronald J.; Heredia-Langner, Alejandro; Webb-Robertson, Bobbie-Jo M.; Thrall, Brian D.; Camp, David G.; Smith, Richard D.; Pounds, Joel G.; Liu, Tao

2011-01-01

302

Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size.  

PubMed

Nanoparticle biological activity, biocompatibility and fate can be directly affected by layers of readily adsorbed host proteins in biofluids. Here, we report a study on the interactions between human blood plasma proteins and nanoparticles with a controlled systematic variation of properties using (18)O-labeling and LC-MS-based quantitative proteomics. We developed a novel protocol to both simplify isolation of nanoparticle bound proteins and improve reproducibility. LC-MS analysis identified and quantified 88 human plasma proteins associated with polystyrene nanoparticles consisting of three different surface chemistries and two sizes, as well as, for four different exposure times (for a total of 24 different samples). Quantitative comparison of relative protein abundances was achieved by spiking an (18)O-labeled "universal" reference into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantification across the entire sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive patterns that classified the nanoparticles based on their surface properties and size. In addition, temporal data indicated that the formation of the stable protein corona was at equilibrium within 5 min. The comprehensive quantitative proteomics results obtained in this study provide rich data for computational modeling and have potential implications towards predicting nanoparticle biocompatibility. PMID:21956884

Zhang, Haizhen; Burnum, Kristin E; Luna, Maria L; Petritis, Brianne O; Kim, Jong-Seo; Qian, Wei-Jun; Moore, Ronald J; Heredia-Langner, Alejandro; Webb-Robertson, Bobbie-Jo M; Thrall, Brian D; Camp, David G; Smith, Richard D; Pounds, Joel G; Liu, Tao

2011-12-01

303

Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses, and cells (bacteria): Ia. gel antibodies against proteins (transferrins).  

PubMed

Artificial antibodies in the form of gel granules were prepared by the molecular imprinting technique from the monomers acrylamide and N,N'-methylene-bis-acrylamide. Gel granules, freed from the selectively adsorbed protein (the antigen), are neutral and, accordingly, do not migrate in an electrical field. However, upon selective interaction with the antigen at a pH different from its pI, the granules become charged. The selectivity of the gel antibodies was studied by free zone electrophoresis in a tube with inside diameter larger than the size of the granules. Such electrophoretic analyses showed that gel antibodies against iron-free transferrin had a high selectivity for this protein, although some crossreaction took place with iron-saturated transferrin, indicating that these artificial antibodies can easily distinguish the minute differences in the 3-D structure of the transferrins. Analogously, gel antibodies against iron-saturated transferrin were highly selective for this protein with some crossreaction with iron-free transferrin. The mobilities of iron-free and iron-saturated transferrin are very similar, and, therefore, capillary free zone electrophoresis cannot distinguish between these structurally related proteins. However, significant differences in the mobilities of the selective gel granules can be observed depending on their interaction with iron-free or iron-saturated transferrin, i.e., the artificial gel antibodies may become powerful analytical tools. PMID:17305242

Takátsy, Anikó; Kilár, Anikó; Kilár, Ferenc; Hjertén, Stellan

2006-12-01

304

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.

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

2013-01-01

305

Application of a nitrocellulose immunoassay for quantitation of proteins secreted in cultured media  

SciTech Connect

A macro-dot immunoassay was developed to quantitate proteins (antigens) secreted in the culture media of primary rat hepatocytes. Dilutions of protein standards and undiluted spent culture media were applied to numbered sheets of nitrocellulose (NC) paper by vacuum filtration (in volumes up to 1 ml) through a specially designed macrofiltration apparatus constructed of plexiglas. Sequential incubation of the NC with bovine serum albumin blocking buffer, monospecific antibody, and /sup 125/I Protein A enabled quantitation of protein concentration by determination of NC bound radioactivity. Linear and reproducible standard curves were obtained with fibrinogen, albumin, transferrin, and haptoglobin. A high degree of coefficient of correlation between radioactivity (cmp) and protein concentration was found. Intra- and inter-test reproducibility was excellent. By using monospecific antibodies, single proteins (i.e., fibrinogen), as low as 32 ng/ml, could be quantified in heterogeneous protein mixtures and in spent culture media. The assay was sensitive to the difference of fibrinogen secretion under nonstimulatory (serum-free hormonally define medium, SFHD) and stimulatory (SFHD plus hydrocortisone) culture conditions. The procedure and techniques described are applicable to the quantitation of any protein in a suitable buffer.

LaDuca, F.M.; Dang, C.V.; Bell, W.R.

1986-11-01

306

Endogenous Protein "Barcode" for Data Validation and Normalization in Quantitative MS Analysis.  

PubMed

Quantitative proteomic experiments with mass spectrometry detection are typically conducted by using stable isotope labeling and label-free quantitation approaches. Proteins with housekeeping functions and stable expression level such actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase are frequently used as endogenous controls. Recent studies have shown that the expression level of such common housekeeping proteins is, in fact, dependent on various factors such as cell type, cell cycle, or disease status and can change in response to a biochemical stimulation. The interference of such phenomena can, therefore, substantially compromise their use for data validation, alter the interpretation of results, and lead to erroneous conclusions. In this work, we advance the concept of a protein "barcode" for data normalization and validation in quantitative proteomic experiments. The barcode comprises a novel set of proteins that was generated from cell cycle experiments performed with MCF7, an estrogen receptor positive breast cancer cell line, and MCF10A, a nontumorigenic immortalized breast cell line. The protein set was selected from a list of ?3700 proteins identified in different cellular subfractions and cell cycle stages of MCF7/MCF10A cells, based on the stability of spectral count data generated with an LTQ ion trap mass spectrometer. A total of 11 proteins qualified as endogenous standards for the nuclear and 62 for the cytoplasmic barcode, respectively. The validation of the protein sets was performed with a complementary SKBR3/Her2+ cell line. PMID:24856102

Lee, Wooram; Lazar, Iulia M

2014-07-01

307

Gold nanoparticle extraction followed by capillary electrophoresis to determine the total, free, and protein-bound aminothiols in plasma.  

PubMed

This study describes the use of Tween 20-capped gold nanoparticles (Tween 20-AuNPs) for the selective extraction and enrichment of five aminothiols, including glutathione, gamma-glutamylcysteine, cysteine, homocysteine, and cysteineglycine, prior to analysis by capillary electrophoresis with UV detection. Tween 20-AuNPs are capable of extracting aminothiols from a complicated matrix because a Tween 20 capping layer can inhibit effectively the nonspecific adsorption. Moreover, Tween 20-AuNPs had better aminothiol loading compared to Zonyl FSN-100- and Triton X-100-capped AuNPs. The extraction efficiency of aminothiols was highly dependent on the number of Tween 20-AuNPs, the concentration of dithiothreitol, and the type of surfactant (i.e., capping agent). Under optimal extraction conditions, the limits of detection at a signal-to-noise ratio of 3 for five aminothiols were down to 10-65 nM. Total and free aminothiols in plasma were determined by varying the order of disulfide reduction with tris(2-carboxyethyl)phosphine. Efficient separation of total and free aminothiols with baseline resolution was successfully achieved by the addition of cationic polyelectrolyte, poly(diallyldimethylammonium chloride), to the background electrolyte. Because this extraction method provided no matrix effect, the concentrations of total and free aminothiols in plasma can be quantified with an external calibration method. On the basis of the advantages of simplicity, high selectivity, high sensitivity, and good reproducibility, this proposed method may have great potential for disease diagnosis. PMID:20201506

Chang, Chung-Wei; Tseng, Wei-Lung

2010-04-01

308

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

PubMed

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

1999-07-01

309

A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.  

PubMed

Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity. PMID:21520910

Bohler, Sacha; Sergeant, Kjell; Hoffmann, Lucien; Dizengremel, Pierre; Hausman, Jean-Francois; Renaut, Jenny; Jolivet, Yves

2011-07-01

310

High blood pressure effects on the blood to cerebrospinal fluid barrier and cerebrospinal fluid protein composition: a two-dimensional electrophoresis study in spontaneously hypertensive rats.  

PubMed

The aim of the present work is to analyze the cerebrospinal fluid proteomic profile, trying to find possible biomarkers of the effects of hypertension of the blood to CSF barrier disruption in the brain and their participation in the cholesterol and ?-amyloid metabolism and inflammatory processes. Cerebrospinal fluid (CSF) is a system linked to the brain and its composition can be altered not only by encephalic disorder, but also by systemic diseases such as arterial hypertension, which produces alterations in the choroid plexus and cerebrospinal fluid protein composition. 2D gel electrophoresis in cerebrospinal fluid extracted from the cistern magna before sacrifice of hypertensive and control rats was performed. The results showed different proteomic profiles between SHR and WKY, that ?-1-antitrypsin, apolipoprotein A1, albumin, immunoglobulin G, vitamin D binding protein, haptoglobin and ?-1-macroglobulin were found to be up-regulated in SHR, and apolipoprotein E, transthyretin, ?-2-HS-glycoprotein, transferrin, ?-1?-glycoprotein, kininogen and carbonic anhidrase II were down-regulated in SHR. The conclusion made here is that hypertension in SHR produces important variations in cerebrospinal fluid proteins that could be due to a choroid plexus dysfunction and this fact supports the close connection between hypertension and blood to cerebrospinal fluid barrier disruption. PMID:23401751

González-Marrero, Ibrahim; Castañeyra-Ruiz, Leandro; González-Toledo, Juan M; Castañeyra-Ruiz, Agustín; de Paz-Carmona, Hector; Castro, Rafael; Hernandez-Fernaud, Juan R; Castañeyra-Perdomo, Agustín; Carmona-Calero, Emilia M

2013-01-01

311

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.

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

2013-01-01

312

Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.  

PubMed

We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities. PMID:15148295

Nakajima, Kazuki; Kinoshita, Mitsuhiro; Oda, Yasuo; Masuko, Takashi; Kaku, Hanae; Shibuya, Naoto; Kakehi, Kazuaki

2004-09-01

313

Rapid Separation and Quantification of Major Caseins and Whey Proteins of Bovine Milk by Polyacrylamide Gel Electrophoresis  

Microsoft Academic Search

A rapid polyacrylamide gel electro- phoretic method was developed for separating and quantifying major pro- teins in casein and whey protein fractions of bovine milk. For casein separation, best results were achieved by an 8% poly- acrylamide gel containing 4 M urea and a top layer of large pore sample gel; for whey protein the most_ satisfactory separation was with

K. F. Ng-Kwai-Hang; E. M. Kroeker

1984-01-01

314

Macromolecular Assemblage of Aminoacyl-tRNA Synthetases: Quantitative Analysis of Protein-Protein Interactions and Mechanism of Complex Assembly  

Microsoft Academic Search

The structure of the mammalian multi-synthetase complex was investigated in vitro using qualitative and quantitative approaches. This macromolecular assemblage comprises the bifunctional glutamyl-prolyl-tRNA synthetase, the seven monospecific isoleucyl, leucyl, methionyl, glutaminyl, lysyl, arginyl and aspartyl-tRNA synthetases, and the three auxiliary p43, p38 and p18 proteins. The scaffold p38 protein was expressed in Escherichia coli and purified to homogeneity as a

Jean-Charles Robinson; Pierre Kerjan; Marc Mirande

2000-01-01

315

Improved protein arrays for quantitative systems analysis of the dynamics of signaling pathway interactions  

PubMed Central

An improved version of quantitative protein array platform utilizing linear Quantum dot signaling for systematically measuring protein levels and phosphorylation states is presented. The signals are amplified linearly by a confocal laser Quantum dot scanner resulting in ~1000-fold more sensitivity than traditional Western blots, but are not linear by the enzyme-based amplification. Software is developed to facilitate the quantitative readouts of signaling network activities. Kinetics of EGFRvIII mutant signaling was analyzed to quantify cross-talks between EGFR and other signaling pathways.

2011-01-01

316

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.

1979-01-01

317

An Economical Electrophoresis Apparatus  

ERIC Educational Resources Information Center

Describes the production of an electrophoresis apparatus from commonly discarded articles. Outlines paper and gel electrophoresis and its application to the separation of amino acids and intestinal enzymes. (GS)

Andrews, I. M.

1975-01-01

318

Absolute Quantitation of Isoforms of Post-translationally Modified Proteins in Transgenic Organism*  

PubMed Central

Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (Pisf) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent 14N-coded synthetic peptide standards and 15N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (Tisf) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (Raqu). The Pisf was finally determined by integrating the two empirically measured variables using the following equation: Pisf = Tisf · Raqu. The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants.

Li, Yaojun; Shu, Yiwei; Peng, Changchao; Zhu, Lin; Guo, Guangyu; Li, Ning

2012-01-01

319

Introduction to a Protein Interaction System Used for Quantitative Evaluation of Biomolecular Interactions  

PubMed Central

A central goal of molecular biology is the determination of biomolecular function. This comes largely from a knowledge of the non-covalent interactions that biological small and macro-molecules experience. The fundamental mission of the Molecular Interactions Research Group (MIRG) of the ABRF is to show how solution biophysical tools are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies such as biosensors, microcalorimetry or analytical ultracentrifugation, for evalution of such interactions. To this end, the MIRG developed a model system for the quantitative analysis of protein-protein interactions, and validated the system using several orthogonal techniques. In this presentation (MIRG Session 1), the details of the protein-protein interaction system will be described, along with an summary of experimental data obtained in MIRG laboratories. The quantitation of the binding affinity was found to be reproducible in multiple MIRG laboratories and using several techniques such as surface plasmon resonance, isothermal titration calorimetry, and analytical ultracentrifugation. In addition, the proteins were shown to be highly stable and soluble, suggesting that the system was suitable for use in benchmarking. Therefore, in late 2012 the MIRG conducted a benchmarking study to evaluate the capabilities of different laboratories and instrumentation to quantitatively characterize the binding affinity and stiochiometry for the model MIRG system, the results of which will be summarized in the subsequent presentation (MIRG Session 2).

Yamniuk, Aaron

2013-01-01

320

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

PubMed

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 2.1ng/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

2014-10-01

321

Quantitative analysis of surface expression of membrane proteins using cold-adapted proteases.  

PubMed

This unit presents an improved method for quantitative analysis of surface expression of membrane proteins utilizing a cold-adapted trypsin. Preservation of the proteolytic activity of the enzyme at 0° to 4°C allows cleavage of surface-expressed membrane proteins at temperatures at which trafficking of the mammalian plasmalemmal proteins is blocked. This provides an important advantage over established trypsin-cleavage protocols since it can be applied to membrane proteins with a fast turnover rate of the membrane pool and a fast recycling rate. Compared to surface biotinylation, the method is less time consuming. PMID:24510593

Ahmad, Faraz; Kaila, Kai; Blaesse, Peter

2013-01-01

322

A reusable electrochemical proximity assay for highly selective, real-time protein quantitation in biological matrices.  

PubMed

Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)-as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation. PMID:24827871

Hu, Jiaming; Yu, Yajiao; Brooks, Jessica C; Godwin, Leah A; Somasundaram, Subramaniam; Torabinejad, Ferdous; Kim, Joonyul; Shannon, Curtis; Easley, Christopher J

2014-06-11

323

Towards a quantitative understanding of protein hydration and volumetric properties.  

PubMed

Herein, we probe by pressure perturbation calorimetry (PPC) the coefficient of thermal expansion, the volumetric and the hydration properties of variants of a hyperstable variant of staphylococcal nuclease (SNase), Delta+PHS. The temperature-dependent volumetric properties of the folded and unfolded states of the wild-type protein are calculated with previously published data. The present PPC results are used to interpret the volume diagram and expansivity at a molecular level. We conclude that the expansivity of the unfolded state is, to a first approximation, temperature independent, while that of the folded state decreases with increasing temperature. Our data suggest that at low temperature the defining contribution to DeltaV comes mainly from excluded volume differences and DeltaV for unfolding is negative. In contrast, at high temperatures, differential solvation due to the increased exposed surface area of the unfolded state and, in particular, its larger thermal volume linked to the increased conformational dynamics of the unfolded state ensemble takes over and DeltaV for unfolding eventually becomes positive. PMID:18814170

Mitra, Lally; Rouget, Jean-Baptiste; Garcia-Moreno, Bertrand; Royer, Catherine A; Winter, Roland

2008-12-22

324

Capillary electrophoresis with capacitively coupled contactless conductivity detection applied to the quantitation and to the determination of physical-chemical properties of peroxycarboxylates in aqueous medium.  

PubMed

CE with C?D (CE-C?D) was successfully applied to the investigation of performate, peracetate, and perpropionate in aqueous medium. Ionic mobilities, diffusion coefficients, and hydrodynamic radii were obtained for the first time for these species. CE-C?D was also used to estimate the pKa values of the peroxycarboxylic acids. Because the peroxycarboxylates (POCs) undergoes hydrolysis while migrating, a simple calibration curve cannot be used for quantitation. Thus, an indirect calibration approach was used. The new method was used to monitor the formation of peroxycarboxylic acids from hydrogen peroxide and the carboxylic acid as well as to the quantitation of peracetic acid in a commercial sample. The CE-C?D method compares favorably with the conventional titration method because of the possibility of speciation of the POC, the low sample consumption, and the low LOD (14, 8, and 24 ?mol/L for performate, peracetate, and perpropionate, respectively). Although POCs are structural isomers of monoalkyl carbonates, they have greater hydrodynamic radii, which suggests that the positions of the oxygen atoms in the molecules have a direct impact in the charge density and consequently on the hydration atmosphere. PMID:23595363

Vidal, Denis T R; do Lago, Claudimir L

2013-07-01

325

Preparative Sephadex Column Electrophoresis: An Improved Apparatus.  

National Technical Information Service (NTIS)

A preparative column electrophoresis using Sephadex G-25 as supporting medium offers a rapid and inexpensive method of purifying a large quantity of proteins with minimum sample loss. This apparatus has been very effective in separating closely related ho...

E. Kay J. H. C. Shih

1973-01-01

326

Quantitative Glycomics Strategies*  

PubMed Central

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.

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

2013-01-01

327

Quantitative real-time imaging of protein-protein interactions by LSPR detection with micropatterned gold nanoparticles.  

PubMed

Localized surface plasmon resonance (LSPR) offers powerful means for sensitive label-free detection of protein-protein interactions in a highly multiplexed format. We have here established self-assembly and surface modification of plasmonic nanostructures on solid support suitable for quantitative protein-protein interaction analysis by spectroscopic and microscopic LSPR detection. These architectures were obtained by layer-by-layer assembly via electrostatic attraction. Gold nanoparticles (AuNP) were adsorbed on a biocompatible amine-terminated poly(ethylene glycol) (PEG) polymer brush and further functionalized by poly-l-lysine graft PEG (PLL-PEG) copolymers. Stable yet reversible protein immobilization was achieved via tris(nitrilotriacetic acid) groups incorporated into the PLL-PEG coating. Thus, site-specific immobilization of His-tagged proteins via complexed Ni(II) ions was achieved. Functional protein immobilization on the surface was confirmed by real-time detection of LSPR scattering by reflectance spectroscopy. Association and dissociation rate constants obtained for a reversible protein-protein interaction were in good agreement with the data obtained by other surface-sensitive detection techniques. For spatially resolved detection, AuNP were assembled into micropatterns by means of photolithographic uncaging of surface amines. LSPR imaging of reversible protein-protein interactions was possible in a conventional wide field microscope, yielding detection limits of ?30 protein molecules within a diffraction-limited surface area. PMID:24016060

Bhagawati, Maniraj; You, Changjiang; Piehler, Jacob

2013-10-15

328

Multifluorescence confocal microscopy: application for a quantitative analysis of hemostatic proteins in human venous valves.  

PubMed

Confocal laser scanning microscopy is commonly used to visualize and quantify protein expression. Visualization of the expression of multiple proteins in the same region via multifluorescence allows for the analysis of differential protein expression. The defining step of multifluorescence labeling is the selection of primary antibodies from different host species. In addition, species-appropriate secondary antibodies must also be conjugated to different fluorophores so that each protein can be visualized in separate channels. Quantitative analysis of proteins labeled via multifluorescence can be used to compare relative changes in protein expression. Multifluoresecence labeling and analysis of fluorescence intensity within and among human venous specimens, for example, allowed us to determine that the anticoagulant phenotype of the venous valve is defined not by increased anticoagulant expression, but instead by significantly decreased procoagulant protein expression (Blood 114:1276-1279, 2009 and Histochem Cell Biol 135:141-152, 2011). PMID:23026998

Trotman, Winifred E; Taatjes, Douglas J; Bovill, Edwin G

2013-01-01

329

Electrophoresis of Milk Proteins. I. Some Comparisons of Salt-Acid and Salt-Lyophilized Whey Fractions1,2  

Microsoft Academic Search

There are several known methods used for the isolation of whey proteins from milk. ttarland and Ashworth (3) have outlined a salt-acid method which is perhaps one of the simplest and easiest. Their procedure involves the precipita- tion of casein by saturation of skim milk with NaC1 followed by precipitation of the whey proteins upon addition of HC1 to the

William G. Stanley; A. C. Andrews; C. H. Whitnah

1950-01-01

330

Concerted, rapid, quantitative, and site-specific dual labeling of proteins.  

PubMed

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

Sachdeva, Amit; Wang, Kaihang; Elliott, Thomas; Chin, Jason W

2014-06-01

331

Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling  

Microsoft Academic Search

BACKGROUND: The proteomes of mammalian biological fluids, cells and tissues are complex and composed of proteins with a wide dynamic range. The effective way to overcome the complexity of these proteomes is to combine several fractionation steps. OFFGEL fractionation, recently developed by Agilent Technologies, provides the ability to pre-fractionate peptides into discrete liquid fractions and demonstrated high efficiency and repeatability

Jérôme Chenau; Sylvie Michelland; Jonathan Sidibe; Michel Seve

2008-01-01

332

iQuantitator: A tool for protein expression inference using iTRAQ  

Microsoft Academic Search

BACKGROUND: Isobaric Tags for Relative and Absolute Quantitation (iTRAQ™) [Applied Biosystems] have seen increased application in differential protein expression analysis. To facilitate the growing need to analyze iTRAQ data, especially for cases involving multiple iTRAQ experiments, we have developed a modeling approach, statistical methods, and tools for estimating the relative changes in protein expression under various treatments and experimental conditions.

John H Schwacke; Elizabeth G. Hill; Edward L. Krug; Susana Comte-Walters; Kevin L. Schey

2009-01-01

333

The use of a juvenile hormone binding protein for the quantitative assay of juvenile hormone  

Microsoft Academic Search

The suitability of the haemolymph juvenile hormone binding protein (JHBP) of Locusta migratoria for use in a competition assay for juvenile hormone (JH) III has been investigated, and a simple quantitative assay procedure using this protein has been developed. JHBP partially purified from haemolymph of precocene treated adult locusts gives rapid and stable binding of [3H]10R-JH III, and can be

A. V. Glinka; R. P. Braun; J. P. Edwards; G. R. Wyatt

1995-01-01

334

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

Microsoft Academic Search

We developed a novel on-chip activity assay using protein arrays for quantitative and rapid analysis of transglutami-nase\\u000a 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\\u000a diseases and tumor progression. We fabricated the protein arrays by immobilizing N,N?-dimethylcasein (a substrate) on the\\u000a amine surface

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

2009-01-01

335

GENETIC CONTROL OF QUANTITATIVE VARIATION IN SELF-INCOMPATIBILITY PROTEINS DETECTED BY IMMUNODIFFUSION1  

Microsoft Academic Search

Single radial immunodiffusion was used to study the self-incompatibility (S) proteins present in stigmas of two inbred lines of Brassica oleracea: a self- incompatible line and a self-compatible mutant derived from it. The genetic basis of observed quantitative differences in S proteins between the two inbreds was shown to be determined by a single gene with dosage effects. Self-pdli- nation

M. E. NASRALLAH

336

Quantitative analysis of protein-RNA interactions by gel mobility shift  

PubMed Central

The gel mobility shift assay is routinely used to visualize protein-RNA interactions. Its power resides in the ability to resolve free from bound RNA with high resolution in a gel matrix. In this chapter, we review the quantitative application of this approach to elucidate thermodynamic properties of protein-RNA complexes. Assay designs for titration, competition, and stoichiometry experiments are presented for two unrelated model complexes.

Ryder, Sean P.; Recht, Michael I.; Williamson, James R.

2010-01-01

337

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA  

PubMed Central

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection.

Barhoom, Sima; Kaur, Jaskiran; Cooperman, Barry S.; Smorodinsky, Nechama I.; Smilansky, Zeev; Ehrlich, Marcelo; Elroy-Stein, Orna

2011-01-01

338

A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: a follow up.  

PubMed

With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan. PMID:17035105

Maserti, B E; Della Croce, C M; Luro, F; Morillon, R; Cini, M; Caltavuturo, L

2007-04-15

339

Application of PhastSystem to the resolution of bovine milk proteins on urea-polyacrylamide gel electrophoresis.  

PubMed

Optimal conditions were established for alkaline urea-PAGE using modified precast, ultrathin gradient gels on the automated PhastSystem. Profiles of milk proteins showed that the caseins and whey proteins resolved extremely well. Major bands were observed for alpha s1-casein and beta-casein, and alpha s2-casein appeared as a well-resolved doublet. In contrast, kappa-casein separated from other caseins as a faint doublet, and purified kappa-casein appeared as one major and one minor band. Whey proteins (serum albumin, alpha-lactalbumin, beta-lactoglobulin) separated into broad bands resolved from each other and from the caseins. Partially (40%) dephosphorylated whole casein showed multiple bands for alpha s1-casein and beta-casein at different levels of phosphorylation. Separation of genetic phenotypes was observed for beta-lactoglobulin A and B; alpha s1-casein A, B, and C; and beta-casein A, B, and C. Electrophoretic patterns of milk proteins extracted from cheese samples varied among the different types of cheeses. Our modified procedure provides researchers with a rapid technique to separate both caseins and whey proteins on the same urea gel according to their charge to mass ratios. PMID:1597574

Van Hekken, D L; Thompson, M P

1992-05-01

340

Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae  

PubMed Central

The cell wall of yeast consists of an internal skeletal layer and an external layer of glycoproteins covalently linked to the stress-bearing polysaccharides. The cell wall protein (CWP) population consists of over 20 different proteins, and may vary in composition. We present two complementary methods for quantifying CWPs, based on isobaric tagging and tandem MS: (1) absolute quantitation of individual CWPs, allowing estimation of surface densities; and (2) relative quantitation of CWPs, allowing monitoring of the dynamics of the CWP population. For absolute quantitation, we selected a representative group of five proteins (Cwp1p, Crh1p, Scw4p, Gas1p, and Ecm33p), which had 67 × 103, 44 × 103, 38 × 103, 11 × 103 and 6.5 × 103 of wall-bound copies per cell, respectively. As Cwp1p is predominantly incorporated in the birth scar, this corresponds to a protein density of c. 22 × 103 copies ?m?2. For relative quantitation, we compared wild-type cells to gas1? cells, in which the cell wall integrity pathway is constitutively activated. The levels of Crh1p, Crh2p, Ecm33p, Gas5p, Pst1p and Pir3p increased about three- to fivefold, whereas the level of Scw4p was significantly decreased. We propose that our methods are widely applicable to other fungi.

Yin, Qing Yuan; de Groot, Piet W J; de Jong, Luitzen; Klis, Frans M; De Koster, Chris G

2007-01-01

341

Detection and Quantitation of T-2 Mycotoxin Using a Simplified Protein Synthesis Inhibition Assay.  

National Technical Information Service (NTIS)

A simple, rapid and sensitive bioassay is described for the detection and quantitation of T-2 mycotoxin using a protein synthesis assay in cultured cells. Increased sensitivity of the cells of the mycotoxin occurs with time up to approximately 60 min. Tim...

W. L. Thompson R. W. Wannemacher

1983-01-01

342

High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography  

Microsoft Academic Search

It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this informa- tion can be extracted via quantitative proteomic measure- ments. Suitable proteomic techniques need to be sensi- tive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins,

Mass Spectrometry; Hui Zhang; Eugene C. Yi; Xiao-jun Li; Parag Mallick; Karen S. Kelly-Spratt; Christophe D. Masselon; David G. Camp II; Richard D. Smith; Christopher J. Kemp; Ruedi Aebersold

343

Assessing the efficiency of free light chain assay in monitoring patients with multiple myeloma before and after autologous stem cell transplantation along with serum protein electrophoresis and serum protein immunofixation.  

PubMed

Monoclonal gammopathies are a group of disorders, referred to as paraproteinaemias, dysproteinaemias or immunoglobulinopathies, associated with monoclonal proliferation of plasma cells. Monoclonal immunoglobulin secreted by these cells is an indicator of clonal proliferation. The aim of this study is to analyze the efficiency of three methods: serum protein electrophoresis (SPE), serum protein immunofixation (IFE) and FLC (free light chain) assay for the diagnosis and monitoring of the tumor burden in multiple myeloma. In this study we have presented the dynamic evolution of 7 patients with intact immunoglobulin multiple myeloma (IIMM) (2 IgG, kapa; 3 IgG, lambda; 1 IgA, kappa; 1 IgA, lambda) and 2 patients with light chain multiple myeloma before and after autologous peripheral blood stem cell transplantation (PBSCT). All 7 patients fulfilled the four criteria for the diagnosis of IIMM: bone marrow plasma cells exceeding 20%, lytic bone lesions, identification and quantification of M protein by scanning densitometry of electrophoresis gels, IFE (immunofixation protein electrophoresis) confirmed and typed the M protein. All patients had been given cytotoxic chemotherapy (VAD or VELCADE) before autologous (PBSCT). In two of the patients with IIMM both SPE and kappa/lambda ratio fell towards normal range after autologous PBSC and both reported a relapse of the disease after 23 months and 19 months respectively. SPE could not normalize after chemotherapy and transplantation in three patients with IIMM, the kappa/lambda ratio being the only marker used to monitor the tumor kill. In one patient the kappa/lambda ratio could not normalize even after PBSCT still indicating the presence of plasma cell disorder at the time when IFE was still negative. 16 months after PBSCT both SPE and FLC indicated a relapse of the disease. Classical SPE failed to demonstrate the presence of M-protein in light chain multiple myeloma, the diagnosis being established by using IFE and the FLC assay. Because IFE is a qualitative method and its interpretation may be sometimes subjective, FLC was the only method used to follow the disease course. The measurement of kappa/lambda ratio proved to be more sensitive than SPE, IFE and the levels of free light chains kappa or lambda individually indicating whether the treatment is effective or not. PMID:21717807

Dogaru, Monica; Laz?r, Veronica; Coriu, Daniel

2011-01-01

344

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.

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

2014-01-01

345

Towards comprehensive analysis of protein family quantitative stability-flexibility relationships using homology models.  

PubMed

The Distance Constraint Model (DCM) is a computational modeling scheme that uniquely integrates thermodynamic and mechanical descriptions of protein structure. As such, quantitative stability-flexibility relationships (QSFR) that describe the interrelationships of thermodynamics and mechanics can be quickly computed. Using comparative QSFR analyses, we have previously investigated these relationships across a small number of protein orthologs, ranging from two to a dozen [1, 2]. However, our ultimate goal is provide a comprehensive analysis of whole protein families, which requires consideration of many more structures. To that end, we have developed homology modeling and assessment protocols so that we can robustly calculate QSFR properties for proteins without experimentally derived structures. The approach, which is presented here, starts from a large ensemble of potential homology models and uses a clustering algorithm to identify the best models, thus paving the way for a comprehensive QSFR analysis across hundreds of proteins in a protein family. PMID:24061925

Verma, Deeptak; Guo, Jun-Tao; Jacobs, Donald J; Livesay, Dennis R

2014-01-01

346

Monosaccharide and oligosaccharide analysis of proteins transferred to polyvinylidene fluoride membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.  

PubMed

We have developed an intermediate method toward the complete carbohydrate analysis of proteins, which should be universally applicable to all proteins and independent of sample matrix. Using only Coomassie Blue-stained proteins which have been electroblotted onto polyvinylidene fluoride membranes, we report a strategy for: (i) determining unequivocally whether a protein is glycosylated; (ii) obtaining a complete monosaccharide composition; (iii) oligosaccharide mapping which separates most forms according to size, charge and isomerity; and (iv) sequentially releasing and analyzing specific classes of oligosaccharides with endoglycosidases. The method was shown to be applicable to a variety of well characterized soluble glycoproteins and to the membrane-bound protein, the gastric H+, K(+)-ATPase. The monosaccharide composition of the H+,K(+)-ATPase revealed the absence of N-acetylneuraminic or N-glycolylneuraminic acids and a monosaccharide composition which indicated O-linked sugar chains. Oligomannosidic/hybrid and biantennary oligosaccharides were sequentially released and analyzed from one electroblotted band of recombinant tissue plasminogen activator using endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F2, respectively. Sialylated polylactosamine structures were identified and quantified by analyzing high performance liquid chromatography profiles of oligosaccharides first released by peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase and then treated with endo-beta-galactosidase, using a single, stained band of recombinant erythropoietin. This recombinant erythropoietin was found to contain eight times more tetrasialylated oligosaccharides than previously reported (Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076); 47% of released oligosaccharides were identified as polylactosamine structures. PMID:8444888

Weitzhandler, M; Kadlecek, D; Avdalovic, N; Forte, J G; Chow, D; Townsend, R R

1993-03-01

347

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

PubMed

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

2013-09-01

348

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.

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

2013-01-01

349

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.

1995-02-01

350

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

PubMed

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

1989-09-01

351

Simultaneous quantitation of 5- and 7-hydroxyflavone antioxidants and their binding constants with BSA using dual chiral capillary electrophoresis (dCCE) and HPLC with fluorescent detection.  

PubMed

In this article we present two novel uses of the sensitive techniques HPLC fluorescence and dCCE for both the quantitation and binding studies of the 5- and 7-HFs extracted from the plant Alfalfa with Albumin. Ultrasonic extraction method as an extra energy source is used to enhance the extraction efficiency and speed up. The two antioxidants could be easily separated and quantified after a 10.0-min run time. Multiple calibration curves for their analysis exhibited consistent linearity and reproducibility in the range of 0.20-2.00 mg L(-1) for 5-HF (r >0.9979) and 0.01-0.10 mg L(-1) for 7-HF (r >0.9999). Limits of Detection were 0.500 µg L(-1) and 0.025 µg L(-1) for 5-HF and 7-HF respectively. Lower Limits of Quantification were 131.600 µg L(-1) for 5-HF and 6.579 µg L(-1) for 7-HF. Inter-assay imprecision was <10% for both flavones. Mean recovery was 104.76% (range 90%-110%) for 5-HF and 93.18% (range 90%-110%) for 7-HF. Since the intermolecular hydrogen atom transfer in the excited triplet state as well as in the excited singlet state might play an important role in the quenching process of photo-excited molecules in biological systems, the binding constants of these HFs with serum albumin have been also estimated to be 1.910 - 2.019 × 10(5) L mol(-1) and 2.390 - 2.500 × 10(5) L mol(-1) for 5-HF and 7-HF respectively. PMID:24401434

Abo Markeb, Ahmad; Abo El-Maali, Nagwa

2014-02-15

352

Multiplexed quantitation of endogenous proteins in dried blood spots by multiple reaction monitoring-mass spectrometry.  

PubMed

Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personnel. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogs by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than four orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R(2) value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at -20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins. PMID:23221968

Chambers, Andrew G; Percy, Andrew J; Yang, Juncong; Camenzind, Alexander G; Borchers, Christoph H

2013-03-01

353

Using Glycinylation, a Chemical Derivatization Technique, for the Quantitation of Ubiquitinated Proteins  

PubMed Central

The quantitation of lysine post-translational modifications (PTMs) by bottom-up mass spectrometry is convoluted by the need for analogous derivatives and the production of different tryptic peptides from the unmodified and modified versions of a protein. Chemical derivatization of lysines prior to enzymatic digestion circumvents these problems and has proven to be a successful method for lysine PTM quantitation. The most notable example is the use of deuteroacetylation to quantitate lysine acetylation. In this work, levels of lysine ubiquitination were quantitated using a structurally homologous label that is chemically similar to the di-glycine (GlyGly)-tag, which is left at the ubiquitination site upon trypsinolysis. The LC-MS analysis of a chemically equivalent mono-glycine (Gly)-tag that is analogous to the corresponding GlyGly-tag proved that the mono-glycine tag can be used for the quantitation of ubiquitination. A glycinylation protocol was then established for the derivatization of proteins to label unmodified lysine residues with a single glycine tag. Ubiquitin multimers were used to show that after glycinylation and tryptic digestion, the mass spectrometric response from the corresponding analogous tagged peptides could be compared for relative quantitation. For a proof of principle regarding the applicability of this technique to the analysis of ubiquitination in biological samples, the glycinylation technique was used to quantitate the increase in mono-ubiquitinated histone H2B that is observed in yeast which lack the enzyme responsible for deubiquitinating H2B-K123, compared to wild-type yeast.

Fiedler, Katherine L.; Cotter, Robert J.

2013-01-01

354

A sensitive and specific ELISA immunocapture assay for rapid quantitation of influenza A\\/H3N2 neuraminidase protein  

Microsoft Academic Search

Both HA and NA proteins elicit antibodies which have been shown to be capable of altering the course of infection. Nevertheless, while influenza virus vaccine standardization involves hemagglutinin (HA) and neuraminidase (NA) in terms of antigenic characterization, only HA protein quantitation is undertaken. An immunocapture ELISA (EIA) is described for N2 NA quantitation, based on the use of a highly

L Gerentes; N Kessler; M Aymard

1998-01-01

355

Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions  

Microsoft Academic Search

BACKGROUND: We have developed and tested a method for printing protein microarrays and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples,

Brian B Haab; Maitreya J Dunham; Patrick O Brown

2001-01-01

356

Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions  

Microsoft Academic Search

BACKGROUND: We describe a method for printing protein microarrays, and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as

Brain B Haab; Maitreya J Dunham; Patrick O Brown

2000-01-01

357

A quantitative immunopolymerase chain reaction method for detection of vegetative insecticidal protein in genetically modified crops.  

PubMed

Vegetative insecticidal protein (Vip) is being employed for transgenic expression in selected crops such as cotton, brinjal, and corn. For regulatory compliance, there is a need for a sensitive and reliable detection method, which can distinguish between approved and nonapproved genetically modified (GM) events and quantify GM contents as well. A quantitative immunopolymerase chain reaction (IPCR) method has been developed for the detection and quantification of Vip protein in GM crops. The developed assay displayed a detection limit of 1 ng/mL (1 ppb) and linear quantification range between 10 and 1000 ng/mL of Vip-S protein. The sensitivity of the assay was found to be 10 times higher than an analogous enzyme-linked immunosorbent assay for Vip-S protein. The results suggest that IPCR has the potential to become a standard method to quantify GM proteins. PMID:21899299

Kumar, Rajesh

2011-10-12

358

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

PubMed

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

2014-07-17

359

Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS.  

PubMed

Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by µbore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism. PMID:25059147

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

2014-10-01

360

Detection of unwanted protein-bound ligands by capillary zone electrophoresis: the case of hidden ligands that stabilize cholinesterase conformation.  

PubMed

Detection, identification and characterization of compounds present in purified proteins and biopharmaceuticals are of central interest. As well as chemical remedies, proteins of pharmacological interest have to exhibit their nakedness to become therapeutic drugs. Cholinesterases (ChE) are enzymes of major importance for detoxification of poisonous esters. Likewise, ChE are characterized by the high catalytic efficiency of an active site positioned at the bottom of a deep gorge. The gorge can be partially or fully occupied by ligands, i.e., substrates and inhibitors that are currently used in affinity chromatography purification steps. Accordingly, a suitable method allowing to analyse the presence of unwanted ligands and its influence on the functional conformation and stability of these enzymes was essential. We have developed CZE approaches for that purpose. The factors causing discrepancies between data for thermal unfolding of ChE by electrophoretic and by calorimetric methods were investigated. The presence of unwanted hidden ligands bound to purified enzymes was first demonstrated. The incidence of these ligands was discussed. Altogether, our results raised several questions concerning the real conformation of the native state of enzymes. Finally, CZE was proved to be a pertinent tool to validate the conformity of purified enzymes to a status of biopharmaceutical. PMID:11920879

Rochu, Daniel; Renault, Frédérique; Masson, Patrick

2002-03-01

361

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.

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

2009-01-01

362

Comparative investigations of gluten proteins from different wheat speciesI. Qualitative and quantitative composition of gluten protein types  

Microsoft Academic Search

In contrast to the hexaploid common (bread) wheat, little information is available on the qualitative and quantitative compositions\\u000a of gluten proteins from other cultivated wheat species. Therefore, representatives of hexaploid spelt, tetraploid durum wheat\\u000a and emmer, and diploid einkorn were compared with three classes of common wheat (winter wheat, spring wheat, wheat rye hybrid).\\u000a The flours were extracted to yield

Herbert Wieser

2000-01-01

363

Top-down quantitation and characterization of SILAC-labeled proteins.  

PubMed

Stable isotope labeling by amino acids in cell culture (SILAC) has become a popular labeling strategy for peptide quantitation in proteomics experiments. If the SILAC technology could be extended to intact proteins, it would enable direct quantitation of their relative expression levels and of the degree of modification between different samples. Here we show through modeling and experiments that SILAC is suitable for intact protein quantitation and top-down characterization. When SILAC-labeling lysine and/or arginine, peaks of light and heavy SILAC-doublets do not interfere with peaks of different charge states at least between 10 and 200 kDa. Unlike chemical methods, SILAC ensures complete incorporation-all amino acids are labeled. The isotopic enrichment of commercially available SILAC amino acids of nominally 95% to 98% shifts the mass difference between light and heavy state but does not lead to appreciably broadened peaks. We expressed labeled and unlabeled Grb2, a 28 kDa signaling protein, and showed that the two forms can be quantified with an average standard deviation of 6%. We performed on-line top-down sequencing of both forms in a hybrid linear ion trap orbitrap instrument. The quantized mass offset between fragments provided information about the number of labeled residues in the fragments, thereby simplifying protein identification and characterization. PMID:17920290

Waanders, Leonie F; Hanke, Stefan; Mann, Matthias

2007-11-01

364

Methods for Peptide and Protein Quantitation by Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry*  

PubMed Central

Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and ?-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633–641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20–30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications.

Zhang, Haixia; Liu, Qinfeng; Zimmerman, Lisa J.; Ham, Amy-Joan L.; Slebos, Robbert J. C.; Rahman, Jamshedur; Kikuchi, Takefume; Massion, Pierre P.; Carbone, David P.; Billheimer, Dean; Liebler, Daniel C.

2011-01-01

365

Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility.  

PubMed

Accurate and rapid protein quantitation is essential for screening biomarkers for disease stratification and monitoring, and to validate the hundreds of putative markers in human biofluids, including blood plasma. An analytical method that utilizes stable isotope-labeled standard (SIS) peptides and selected/multiple reaction monitoring-mass spectrometry (SRM/MRM-MS) has emerged as a promising technique for determining protein concentrations. This targeted approach has analytical merit, but its true potential (in terms of sensitivity and multiplexing) has yet to be realized. Described herein is a method that extends the multiplexing ability of the MRM method to enable the quantitation 142 high-to-moderate abundance proteins (from 31mg/mL to 44ng/mL) in undepleted and non-enriched human plasma in a single run. The proteins have been reported to be associated to a wide variety of non-communicable diseases (NCDs), from cardiovascular disease (CVD) to diabetes. The concentrations of these proteins in human plasma are inferred from interference-free peptides functioning as molecular surrogates (2 peptides per protein, on average). A revised data analysis strategy, involving the linear regression equation of normal control plasma, has been instituted to enable the facile application to patient samples, as demonstrated in separate nutrigenomics and CVD studies. The exceptional robustness of the LC/MS platform and the quantitative method, as well as its high throughput, makes the assay suitable for application to patient samples for the verification of a condensed or complete protein panel. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. PMID:23806606

Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Hardie, Darryl B; Borchers, Christoph H

2014-05-01

366

Monitoring folding/unfolding transitions of proteins by capillary zone electrophoresis: measurement of deltaG and its variation along the pH scale.  

PubMed

Free-solution capillary zone electrophoresis (CZE) can be used to monitor folding/unfolding transitions of proteins and to construct the classical sigmoidal transition curve describing this isomerization process. By performing a series of CZE experiments along the pH scale (here between pH 2.5 and 6.0) it is possible to measure the parameter [urea]1/2, which represents the concentration of urea at the midpoint of each transition curve, and its dependence from the local pH value. The [urea]1/2 parameter provides an idea of the stability of the protein at a given pH; in the case of cytochrome c, for example, it shows that at and below pH 2 the protein will spontaneously unfold even in the absence of a denaturant. The equation describing the sigmoidal folding/unfolding transition can be used for deriving the term deltaG degrees, which refers to the intrinsic difference in the Gibb's free energy between the (total or partial) denatured state and the reference state, taken usually as the native configuration of a protein. The variation of deltaG degrees between the two extremes of our measurements (pH 2.5 and 6.0) along the stated pH interval has been measured (and theoretically calculated) to be of the order of 7-10 kcal/mol and is here interpreted by assuming that at pH 2.5 and below there is an additionally stretching of the polypeptide coil due to coulombic repulsion, as the unfolded chain looses its zwitterionic character and assumes a pure (or very nearly so) cationic surface. Given the minute amounts of sample required, the fully automated state of the analysis, the rapidity and ease of operation, it is hoped that the CZE technique will become more and more popular in the years to come for monitoring folding/unfolding transitions of proteins. PMID:11699911

Verzola, B; Fogolari, F; Righetti, P G

2001-10-01

367

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.

1985-01-01

368

Reptation theories of electrophoresis.  

PubMed

In this review, we present the main aspects of the reptation theory, which has provided an essential insight into the processes at work during DNA electrophoretic separation in gels. We avoid mathematical developments, and rely as much as possible on an intuitive description. We first present the original biased reptation model, which assumes that the DNA threads its way as a "worm" of fixed length among the fibers of the gel. We then introduce a more recent version, the model of Biased Reptation with Fluctuations (BRF), which allows for longitudinal flexibility along the DNA. We then propose a quantitative comparison with experiments performed in constant field, and discuss the application of reptation theories to pulsed field techniques either with crossed fields or with field inversion. We also discuss at some length the different experiments that led to a criticism of reptation ideas, such as orientation measurements and videomicroscopy. Finally, we use these experiments together with various computer simulations developed recently for gel electrophoresis, to propose a more realistic qualitative description of DNA motion in gels, and we discuss what elements in this motion are relevant to reptation and what processes are not included in present analytical models. PMID:8887359

Viovy, J L

1996-08-01

369

Profiling of host cell proteins by two-dimensional difference gel electrophoresis (2D-DIGE): Implications for downstream process development.  

PubMed

Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two-dimensional difference gel electrophoresis (2D-DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D-DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D-DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample. PMID:19739084

Jin, Mi; Szapiel, Nicolas; Zhang, Jennifer; Hickey, John; Ghose, Sanchayita

2010-02-01

370

Quantitative Mass Spectrometry-based Proteomics Reveals the Dynamic Range of Primary Mouse Astrocyte Protein Secretion  

PubMed Central

Growing appreciation for astrocytes as active participants in nervous system development, neurovascular metabolic coupling, and neurological disease progression has stimulated recent investigation into specific astrocyte-secreted proteins that may mediate these functions. The current work utilized SILAC-generated isotope reference proteomes to quantify relative protein abundances between the astrocyte proteome and secretome. Multidimensional GeLC-MS/MS analysis of astrocyte conditioned media and cell lysates resulted in the relative quantification of 516 proteins, 92 of which were greater than 1.5-fold enriched in astrocyte-conditioned media (ACM). Eighty of the ACM-enriched proteins had N-terminal signal peptides, comprising well known classically secreted proteins, such as apolipoprotein E and SPARC, and several cathepsins that localize to endosomal/lysosomal compartments. The remaining twelve ACM-enriched proteins, such as vimentin, ferritins, and histones, lacked N-terminal signal peptides. Also, 47 proteins contained predicted N-terminal signal peptides but were not enriched in ACM (< 1.5-fold), 25 of which were localized to ER, Golgi, or mitochondria membrane-bound compartments. Overall, by combining quantitative proteomics with subcellular localization prediction, an informative description of protein distribution can be obtained, providing insights into protein secretion.

Greco, Todd M.; Seeholzer, Steven H.; Mak, Adrian; Spruce, Lynn; Ischiropoulos, Harry

2010-01-01

371

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

PubMed

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 (T(b)), 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 ((15)N) 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

2011-06-01

372

Protein quantitative trait loci identify novel candidates modulating cellular response to chemotherapy.  

PubMed

Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p ? 0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms. PMID:24699359

Stark, Amy L; Hause, Ronald J; Gorsic, Lidija K; Antao, Nirav N; Wong, Shan S; Chung, Sophie H; Gill, Daniel F; Im, Hae K; Myers, Jamie L; White, Kevin P; Jones, Richard Baker; Dolan, M Eileen

2014-04-01

373

Protein Quantitative Trait Loci Identify Novel Candidates Modulating Cellular Response to Chemotherapy  

PubMed Central

Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p?0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms.

Gorsic, Lidija K.; Antao, Nirav N.; Wong, Shan S.; Chung, Sophie H.; Gill, Daniel F.; Im, Hae K.; Myers, Jamie L.; White, Kevin P.; Jones, Richard Baker; Dolan, M. Eileen

2014-01-01

374

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.

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

2011-01-01

375

Huntingtin protein interactions altered by polyglutamine expansion as determined by quantitative proteomic analysis.  

PubMed

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of a polyglutamine repeat within the HD gene product, huntingtin. Huntingtin, a large (347 kDa) protein containing multiple HEAT repeats, acts as a scaffold for protein-protein interactions. Huntingtin-induced toxicity is believed to be mediated by a conformational change in expanded huntingtin, leading to protein misfolding and aggregation, aberrant protein interactions and neuronal cell death. While many non-systematic studies of huntingtin interactions have been reported, they were not designed to identify and quantify the changes in the huntingtin interactome induced by polyglutamine expansion. We used tandem affinity purification and quantitative proteomics to compare and quantify interactions of normal or expanded huntingtin isolated from a striatal cell line. We found that proteins preferentially interacting with expanded huntingtin are enriched for intrinsically disordered proteins, consistent with previously suggested roles of such proteins in neurodegenerative disorders. Our functional analysis indicates that proteins related to energy production, protein trafficking, RNA post-transcriptional modifications and cell death were significantly enriched among preferential interactors of expanded huntingtin. Expanded huntingtin interacted with many mitochondrial proteins, including AIFM1, consistent with a role for mitochondrial dysfunction in HD. Furthermore, expanded huntingtin interacted with the stress granule-associated proteins Caprin-1 and G3BP and redistributed to RNA stress granules under ER-stress conditions. These data demonstrate that a number of key cellular functions and networks may be disrupted by abnormal interactions of expanded huntingtin and highlight proteins and pathways that may be involved in HD cellular pathogenesis and that may serve as therapeutic targets. PMID:22580459

Ratovitski, Tamara; Chighladze, Ekaterine; Arbez, Nicolas; Boronina, Tatiana; Herbrich, Shelley; Cole, Robert N; Ross, Christopher A

2012-05-15

376

Huntingtin protein interactions altered by polyglutamine expansion as determined by quantitative proteomic analysis  

PubMed Central

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of a polyglutamine repeat within the HD gene product, huntingtin. Huntingtin, a large (347 kDa) protein containing multiple HEAT repeats, acts as a scaffold for protein-protein interactions. Huntingtin-induced toxicity is believed to be mediated by a conformational change in expanded huntingtin, leading to protein misfolding and aggregation, aberrant protein interactions and neuronal cell death. While many non-systematic studies of huntingtin interactions have been reported, they were not designed to identify and quantify the changes in the huntingtin interactome induced by polyglutamine expansion. We used tandem affinity purification and quantitative proteomics to compare and quantify interactions of normal or expanded huntingtin isolated from a striatal cell line. We found that proteins preferentially interacting with expanded huntingtin are enriched for intrinsically disordered proteins, consistent with previously suggested roles of such proteins in neurodegenerative disorders. Our functional analysis indicates that proteins related to energy production, protein trafficking, RNA post-transcriptional modifications and cell death were significantly enriched among preferential interactors of expanded huntingtin. Expanded huntingtin interacted with many mitochondrial proteins, including AIFM1, consistent with a role for mitochondrial dysfunction in HD. Furthermore, expanded huntingtin interacted with the stress granule-associated proteins Caprin-1 and G3BP and redistributed to RNA stress granules under ER-stress conditions. These data demonstrate that a number of key cellular functions and networks may be disrupted by abnormal interactions of expanded huntingtin and highlight proteins and pathways that may be involved in HD cellular pathogenesis and that may serve as therapeutic targets.

Ratovitski, Tamara; Chighladze, Ekaterine; Arbez, Nicolas; Boronina, Tatiana; Herbrich, Shelley; Cole, Robert N.; Ross, Christopher A.

2012-01-01

377

Quantitative surface studies of protein adsorption by infrared spectroscopy. II. Quantification of adsorbed and bulk proteins  

SciTech Connect

Attenuated total reflectance Fourier transform infrared spectra of surface-adsorbed proteins are correlated with concentration measurements determined by /sup 125/I-labeled proteins. This paper demonstrates that linear correlations between the intensity of the major bands of proteins and the quantity of proteins can be obtained for human albumin and immunoglobulin G up to surface concentrations of approximately 0.25 microgram/cm2. A poorer correlation was observed for human fibrinogen. A linear correlation was also observed between the concentration in the bulk solution and the major bands of albumin up to a concentration of 60 mg/ml.

Fink, D.J.; Hutson, T.B.; Chittur, K.K.; Gendreau, R.M.

1987-08-15

378

Quantitative assessment of protein interaction with methyl-lysine analogues by hybrid computational and experimental approaches.  

PubMed

In cases where binding ligands of proteins are not easily available, structural analogues are often used. For example, in the analysis of proteins recognizing different methyl-lysine residues in histones, methyl-lysine analogues based on methyl-amino-alkylated cysteine residues have been introduced. Whether these are close enough to justify quantitative interpretation of binding experiments is however questionable. To systematically address this issue, we developed, applied, and assessed a hybrid computational/experimental approach that extracts the binding free energy difference between the native ligand (methyl-lysine) and the analogue (methyl-amino-alkylated cysteine) from a thermodynamic cycle. Our results indicate that measured and calculated binding differences are in very good agreement and therefore allow the correction of measured affinities of the analogues. We suggest that quantitative binding parameters for defined ligands in general can be derived by this method with remarkable accuracy. PMID:21991995

Seeliger, Daniel; Soeroes, Szabolcs; Klingberg, Rebecca; Schwarzer, Dirk; Grubmüller, Helmut; Fischle, Wolfgang

2012-01-20

379

Combining SELEX with quantitative assays to rapidly obtain accurate models of protein-DNA interactions  

Microsoft Academic Search

Models for the specificity of DNA-binding transcrip- tion factors are often based on small amounts of qual- itative data and therefore have limited accuracy. In this study we demonstrate a simple and efficient method of affinity chromatography-SELEX followed by a quantitative binding (QuMFRA) assay to rapidly collect the data necessary for more accurate models. Using the zinc finger protein EGR

Jiajian Liu; Gary D. Stormo

2005-01-01

380

Targeted ion parking for the quantitation of biotherapeutic proteins: Concepts and preliminary data  

Microsoft Academic Search

Targeted ion parking (or TIPing) is the first quantitative application of ion\\/ion reactions for mass spectrometry. In TIPing,\\u000a intact biotherapeutic proteins are electrosprayed as intact molecules (no digestion) and, as expected, many multiply protonated\\u000a species are produced (e.g., (M + 7H)7+, (M + 8H)8+, etc.). Several of these multiply charged species are selectively isolated using a quadrupole mass analyzer and

J. Larry Campbell; J. C. Yves Le Blanc

2010-01-01

381

Quantitation of Heme Oxygenase-1: Heme Titration Increases Yield of Purified Protein  

PubMed Central

Free heme binds to heme oxygenase as a prosthetic group and substrate in the conversion of heme to biliverdin, carbon monoxide, and free iron. Current methods for quantifying heme oxygenase-1 (HO-1) involve reconstitution of the enzyme with heme, followed by a hydroxyapatite column to remove the excess heme. As a result of the hydroxyapatite chromatography, there are significant losses of purified protein. We have developed a method which allows accurate quantitation of HO-1 using a heme titration and elimination of the final hydroxyapatite column, increasing the amount of purified protein.

Huber, Warren J.; Backes, Wayne L.

2008-01-01

382

Enzyme-Linked Immunosorbent Assay for Quantitative Detection of Bacillus thuringiensis Crystal Protein  

PubMed Central

Accurate measurement of the toxic protein crystal produced during deep-tank fermentation of Bacillus thuringiensis is critical for optimum process yield. The currently accepted method is a bioassay that requires more time to generate data than to complete the fermentation itself. A noncompetitive enzyme-linked immunosorbent assay has been developed with purified B. thuringiensis crystals to generate rabbit antiserum. This technique gives a quantitative crystal protein value with a colorimetric endpoint for either liquids or powders within 4 h of sampling. Reproducibility of this enzyme-linked immunosorbent assay satisfies criteria for use in a commercial process. Images

Smith, Robert A.; Ulrich, J. Terry

1983-01-01

383

Monitoring endocytic trafficking of anthrax lethal factor by precise and quantitative protein labeling.  

PubMed

Coupling the genetic code expansion technique with bioorthogonal reactions enables precise control over the conjugation site as well as the choice of fluorescent probes during protein labeling. However, the advantages of this strategy over bulky and rigid fluorescent proteins (FPs) remain to be fully explored. Here we applied site-specific bioorthogonal labeling on anthrax lethal factor (LF) to visualize its membrane translocation inside live cells. In contrast to the previously reported FP tags that significantly perturbed LF's membrane trafficking, our precisely and quantitatively labeled LF exhibited an endocytic activity comparable to wild-type LF. This allowed time-lapse imaging of LF's natural translocation process from host cell membrane to cytosol, which revealed molecular details of its virulence mechanism. Our strategy is generally applicable for monitoring intracellular protein membrane translocation that is difficult to access using conventional protein labeling methodologies. PMID:24828812

Zheng, Siqi; Zhang, Gong; Li, Jie; Chen, Peng R

2014-06-16

384

Quantitative phosphoproteomic profiling of PINK1-deficient cells identifies phosphorylation changes in nuclear proteins.  

PubMed

The Parkinson's disease (PD) associated gene PINK1 encodes a protein kinase that mediates the phosphorylation of multiple proteins involved in mitochondrial homeostasis. The broader downstream signaling events mediated by PINK1 kinase activity have not been well documented. We combine quantitative phosphoproteomic strategies with siRNA mediated PINK1 knock down in mammalian cells to identify alterations of phosphorylation events downstream of PINK1. Although down-regulation of PINK1 has no major effect on the proteome expression in these cells, phosphorylation of over one hundred proteins was reduced reflecting basal levels of phosphorylation signaling events downstream of PINK1. Motif analysis of the residues flanking the phosphorylation sites indicates proline-directed kinase specificity. Surprisingly, we found that the downstream signaling nodes included many transcription factors, as well as nuclear proteins involved in DNA and RNA metabolism. Thus, PINK1 dependent phosphorylation signaling may regulate nuclear activities. PMID:24626860

Qin, Xiaoyan; Zheng, Chaoya; Yates Iii, John R; Liao, Lujian

2014-07-01

385

Quantitative study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy.  

PubMed

Dual-color FCS is a powerful method to monitor protein-protein interactions in living cells. The main idea is based on the cross-correlation analysis of temporal fluorescence intensity fluctuations of two fluorescent proteins to obtain their co-diffusion and relative concentration. But, when performing these experiments, the spectral overlap in the emission of the two colors produces an artifact that corrupts the cross-correlation data: spectral bleed-through. We have shown that problems with cross talk are overcome with Fluorescence Lifetime Correlation Spectroscopy (FLCS). FLCS applied to dual-color cross-correlation, utilizing for example eGFP and mCherry fluorescent proteins, allows the determination of protein-protein interactions in living cells without the need of spectral bleed-through calibration. Here, we present in detail how this methodology can be implemented using a commercial setup (Microtime from PicoQuant, SP8 SMD from Leica or any conventional confocal with PicoQuant TCSPC module, and also with a Becker and Hickl TCSPC module). The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during the experiment constitutes a very powerful technique to quantitatively study protein interactions in live samples. PMID:24108650

Padilla-Parra, Sergi; Audugé, Nicolas; Coppey-Moisan, Maïté; Tramier, Marc

2014-01-01

386

Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods  

PubMed Central

We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P2. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.

Busse, Ricarda A.; Scacioc, Andreea; Hernandez, Javier M.; Krick, Roswitha; Stephan, Milena; Janshoff, Andreas; Thumm, Michael; Kuhnel, Karin

2013-01-01

387

Simple luminescence detectors using a light-emitting diode or a Xe lamp, optical fiber and charge-coupled device, or photomultiplier for determining proteins in capillary electrophoresis: A critical comparison  

Microsoft Academic Search

The performance of two homemade fluorescence-induced capillary electrophoresis detectors, one based on light-emitting diode (LED) as the excitation source and a charge-coupled device (CCD) photodetector and the other based on a commercial luminescence spectrometer (Xe lamp) as the excitation source and a photomultiplier tube as a detector, were compared for the determination of fluorescent proteins R-phycoerythrin and B-phycoerythrin. Both devices

Silvia Casado-Terrones; Jorge F. Fernández-Sánchez; Antonio Segura-Carretero; Alberto Fernández-Gutiérrez

2007-01-01

388

Comparative Mutation Detection Screening of the Type VII Collagen Gene (COL7A1) Using the Protein Truncation Test, Fluorescent Chemical Cleavage of Mismatch, and Conformation Sensitive Gel Electrophoresis  

Microsoft Academic Search

Mutations in the type VII collagen gene, COL7A1, give rise to the blistering skin disease, dystrophic epidermolysis bullosa. We have developed two new mutation detection strategies for the screening of COL7A1 mutations in patients with dystrophic epidermolysis bullosa and compared them with an established protocol using conformational sensitive gel electrophoresis. The first strategy consisted of an RNA based protein truncation

Neil V. Whittock; Gabrielle H. S. Ashton; Rafik Mohammedi; Jemima E. Mellerio; Christopher G. Mathew; Stephen J. Abbs; Robin A. J. Eady; John A. McGrath

1999-01-01

389

Characterization of selenium incorporation into wheat proteins by two-dimensional gel electrophoresis-laser ablation ICP MS followed by capillary HPLC-ICP MS and electrospray linear trap quadrupole Orbitrap MS.  

PubMed

A method has been developed for a rapid and precise location of selenium-containing proteins in large two-dimensional (2D) electrophoresis gels. A sample was divided into four aliquots which were analyzed in parallel by 1D isoelectric focusing electrophoresis (IEF)-laser ablation (LA) inductively coupled plasma mass spectrometry (ICP MS), 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE)-LA ICP MS, and, in duplicate, by 2D IEF-PAGE. On the basis of the 1 D electropherograms obtained, areas supposed to contain the largest concentrations of Se were subjected to LA ICP MS imaging to locate precisely the position of Se-containing proteins which were then identified in the parallel 2D gel by electrospray Orbitrap MS/MS. The method was applied to the identification and semiquantitative determination of selenium storage proteins in wheat. MS evidence is presented for the Se-S substitution in plants not only in methionine but also in cysteine. PMID:23330978

Bianga, Juliusz; Govasmark, Espen; Szpunar, Joanna

2013-02-19

390

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.

1975-01-01

391

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.

1977-01-01

392

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.

YANG, CHIN-RANG [NHLBI, NIH] [NHLBI, NIH

2013-12-11

393

Quantitative detection of zeta-chain-associated protein 70 expression in chronic lymphocytic leukemia  

PubMed Central

Overexpression of zeta-chain-associated protein 70 (ZAP-70) was recently recognized as an independent prognostic marker for the aggressive form of chronic lymphocytic leukemia (CLL). The objective of this study was to demonstrate the feasibility and implementation of quantitative detection of ZAP-70 protein in B cells to clearly distinguish patients with CLL with the aggressive form of the disease. B cells were isolated from patient blood and lysed. Released ZAP-70 protein was detected using an immunomagnetic fluorescence assay. The assay protocol was developed using Jurkat cells and recombinant ZAP-70 (rZAP-70). The limit of detection was determined to be lower than 125 Jurkat cells and 39 pg of rZAP-70 protein. The signal response was linear over a wide dynamic range, from 125 to 40 000 Jurkat cells per test (R2 = 0.9987) and from 0 to 40 000 pg rZAP-70 protein per test (R2 = 0.9928). The results from 20 patients with CLL correlated strongly with flow cytometry analysis. Concordance between the two methods for positive and negative results was 100% (7/7) and 92% (12/13), respectively, while the overall concordance between the tw