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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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Articles by Fred E. Regnier in JoVE

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A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples


JoVE 1398 10/01/2009

1, 2, 1, 1, 1, 2, 1, 1, 1, 3, 2, 1, 1

1Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco - UCSF, 2Buck Institute for Age Research, 3Department of Chemistry, Purdue University

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Other articles by Fred E. Regnier on PubMed

Capillary Electrochromatography of Peptides on Microfabricated Poly(dimethylsiloxane) Chips Modified by Cerium(IV)-catalyzed Polymerization

Journal of Chromatography. A. Mar, 2002  |  Pubmed ID: 12831199

Vinylsulfonic acid, acrylic acid, 2-acrylamido-2-methylpropanesulfonic acid (AMPS), 4-styrenesulfonic acid, and stearyl methacrylate were used for successful modification of the surface of poly(dimethylsiloxane) (PDMS) by cerium(IV) catalyzed polymerization on microfabricated collocated monolith support structures microchips. Reproducible and stable coatings were obtained allowing highly efficient separations of a peptide mixture with RSD for retention times below 2.6%. AMPS-coated PDMS channels were shown to give a reproducible separation of a synthetic peptide mixture for over a month. Subsequent modification of microchip channels by AMPS and methoxydimethyloctadecylsilane allowed selective separation of complex bovine serum albumin digest with high reproducibility, and efficiency of about 620,000 plates/m.

Quantitative Proteomics Strategy Involving the Selection of Peptides Containing Both Cysteine and Histidine from Tryptic Digests of Cell Lysates

Journal of Chromatography. A. Mar, 2002  |  Pubmed ID: 11999731

This paper describes a procedure for quantitative proteomics that selects peptides containing both cysteine and histidine residues from tryptic digests of cell lysates. Cysteine-containing peptides were selected first by covalent chromatography using thiol disulfide exchange. Following the release of cysteine-containing peptides from the covalent chromatography column with reductive cleavage, histidine-containing peptides were captured by passage through an immobilized metal affinity chromatography column loaded with copper. Quantification was achieved in a four-step process involving (i) differential labeling of control and experimental samples with isotopically differing forms of succinic anhydride, (ii) mixing the two globally labeled samples, (iii) fractionating the labeled peptides by reversed-phase liquid chromatography, and (iv) determining the isotope ratio in individual peptides by mass spectrometry. The results of these studies indicate that by selecting peptides containing both cysteine and histidine, the complexity of protein digests could be substantially reduced. Up-regulated proteins from plasmid bearing Escherichia coli that had been induced with isopropyl beta-thiogalacto-pyranoside were identified and quantified by the global internal standard technology (GIST) described above. Database searches were greatly simplified because the number of possible peptide candidates was reduced more than 95%.

Comparative Proteomics Based on Stable Isotope Labeling and Affinity Selection

Journal of Mass Spectrometry : JMS. Feb, 2002  |  Pubmed ID: 11857757

Disease, external stimuli (such as drugs and toxins), and mutations cause changes in the rate of protein synthesis, post-translational modification, inter-compartmental transport, and degradation of proteins in living systems. Recognizing and identifying the small number of proteins involved is complicated by the complexity of biological extracts and the fact that post-translational alterations of proteins can occur at many sites in multiple ways. It is shown here that a variety of new tools and methods based on internal standard technology are now being developed to code globally all peptides in control and experimental samples for quantification. The great advantage of these stable isotope-labeling strategies is that mass spectrometers can rapidly target those proteins that have changed in concentration for further analysis. When coupled to stable isotope quantification, targeting can be further focused through chromatographic selection of peptide classes on the basis of specific structural features. Targeting structural features is particularly useful when they are unique to types of regulation or disease. Differential displays of targeted peptides show that stimulus-specific markers are relatively easy to identify and will probably be diagnostically valuable tools.

Quantifying Peptides in Isotopically Labeled Protease Digests by Ion Mobility/time-of-flight Mass Spectrometry

Analytical Chemistry. Mar, 2002  |  Pubmed ID: 11924997

Ion mobility/time-of-flight techniques have been used to analyze mixtures of isotopically labeled peptides. The isotopic labels were generated by treatment of peptides with N-acetoxysuccinimide (or the deuterated analogue), which results in acetylation (or deuterioacetylation) of the primary amines (i.e., the N-terminus and lysine residues). The relative concentrations of a peptide in each sample are determined by comparing the peak intensities for isotopic pairs. An important consideration is that as mixtures become increasingly complex, isotopic pairs of peaks may overlap with other peaks in the mass spectrum. The influence of the acetyl and deuterioacetyl groups on the mobilities of peptides is considered. The coincidence in mobilities of isotopic pairs provides a means of distinguishing isotopic pairs from other isobaric interferences.

Global Internal Standard Technology for Comparative Proteomics

Journal of Chromatography. A. Mar, 2002  |  Pubmed ID: 11999733

The work described in this paper tests the efficacy of a global isotope labeling (global internal standard technology, GIST) strategy for quantification in proteomics. Using GIST, overexpression of beta-galactosidase in Escherichia coli was identified and quantified. The GIST protocol involved tryptic digestion of proteins from control and experimental samples followed by differential isotopic labeling of the resulting tryptic peptides, mixing the differentially labeled control and experimental digests, fractionation of the peptide mixture by reversed-phase chromatography, and isotope ratio analysis by mass spectrometry. N-Acetoxysuccinimide and N-acetoxy-[2H3]succinimide were used to differentially derivatize primary amino groups in peptides from experimental and control samples, respectively. The relative concentration of isotopically labeled peptides was determined by isotope ratio analysis with both matrix-assisted laser desorption ionization mass spectrometry and tandem mass spectrometry (MS-MS). Peptide masses and sequences obtained by MS-MS were used to identify proteins. MS-MS was found to be uniquely suited for isobaric peptide quantification.

Controlling Deuterium Isotope Effects in Comparative Proteomics

Analytical Chemistry. Aug, 2002  |  Pubmed ID: 12175151

This paper focuses on identifying structural features responsible for resolution of heavy isotope coded peptides during reversed-phase chromatography. This was achieved by using labeled coding agents that varied in structure, number of deuterium atoms, placement of deuterium in the coding agent, and the functional group targeted by the reagent. Six coding agents were examined. Deuterated versions of the coding agents studied included succinic anhydride-2H4, acetic acid 2,5-dioxopyrrolidin-1-yl ester-2H3, propionic acid 2,5-dioxopyrrolidin-1-yl ester-2H5, pentanoic acid 2,5-dioxopyrrolidin-1-yl ester-2H9, [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)-propyl]-trimethylammonium chloride-2H9, and the commercial ICAT-2H8 reagent. It was found that these labeling agents vary widely in both their absolute and relative contribution to the chromatographic isotope effect. Relative effects were evaluated by normalizing resolution for the number of deuterium atoms in the derivatized peptide. The single, most dominant effect was the placement of deuterium atoms relative to hydrophilic functional groups in the coding agent. It was concluded that the probability of a deuterium atom interacting with the stationary phase of a reversed-phase chromatography (RPC) column and impacting resolution is greatly diminished by placing it adjacent to a hydrophilic group, as explained by solvophobic theory. But peptide size and coding agent size were also seen to correlate inversely with the magnitude of the isotope effect. This effect was explained as being due to the relative size of the coding agent versus that of the coding agent-peptide conjugate.

Minimizing Resolution of Isotopically Coded Peptides in Comparative Proteomics

Journal of Proteome Research. Mar-Apr, 2002  |  Pubmed ID: 12643534

Stable isotopes are now widely used to quantify concentration changes in proteomics. This paper focuses on the resolution of isotopically coded peptides and how isotope effects occurring during chromatographic separations can be minimized. Heavy isotope derivatizing agents used in this work were the commercially available 2H8-ICAT reagent and 13C4-succinic anhydride. The ICAT reagent derivatizes cysteine-containing peptides, whereas the succinic anhydride reacts with primary amine groups in peptides. It was observed during reversed-phase chromatography of peptides from a BSA tryptic digest differentially labeled with the 2Hr and 2H8-ICAT reagents that resolution of the isoforms exceeded 0.5 with 20% of the peptides in the digest. Three-fourths of the peptides in this group contained two cysteine residues and were doubly labeled. Only 23% of the peptides labeled with a single ICAT residue had a resolution greater than 0.4. The resolution of peptides differentially labeled with 13C- and 12C-succinate never exceeded +/- 0.01, even in the case of peptides from the BSA digest labeled with 2 mol of succinate. Because this value is within the limits of the method used to determine resolution, it was concluded the 13C- and 12C-coded isoforms of labeled peptides did not resolve. The isotope ratio in the case of 13C/12C coding could be determined from a single mass spectrum taken at any point in the elution profile. This enabled isotope ratio analysis to be completed early in the elution of a peptide from chromatography columns.

An Isotope Coding Strategy for Proteomics Involving Both Amine and Carboxyl Group Labeling

Journal of Proteome Research. Sep-Oct, 2002  |  Pubmed ID: 12645916

This paper describes a heavy isotope coding strategy for the analysis of all types of tryptic peptides, including those that are N-terminally blocked and from the C-terminus of proteins. The method exploits differential derivatization of amine and carboxyl groups generated during proteolysis as a means of coding. Carboxyl groups produced during proteolysis incorporate 18O from H218O. Peptides from the C-terminus of proteins were not labeled with 18O unless they contained a basic C-terminal amino acid. Primary amines from control and experimental samples were differentially acylated after proteolysis with either 1H3- or 2H3-N-acetoxysuccinamide. When these two types of labeling were combined, unique coding patterns were achieved for peptides arising from the C-termini and blocked N-termini of proteins. This method was used to (1) distinguish C-terminal peptides in model proteins, (2) recognize N-terminal peptides from proteins in which the amino terminus is acylated, and (3) identify primary structure variations between proteins from different sources.

Stopped-flow Enzyme Assays on a Chip Using a Microfabricated Mixer

Analytical Chemistry. Apr, 2003  |  Pubmed ID: 12713034

This paper describes a microfabricated enzyme assay system including a micromixer that can be used to perform stopped-flow reactions. Samples and reagents were transported into the system by electroosmotic flow (EOF). Streams of reagents were merged and passed through the 100-pL micromixer in < 1 s. The objective of the work was to perform kinetically based enzyme assays in the stopped-flow mode using a system of roughly 6 nL volume. Beta-galactosidase (beta-Gal) was chosen as a model enzyme for these studies and was used to convert the substrate fluorescein mono-beta-D-galactopyranoside (FMG) into fluorescein. Results obtained with microfabricated systems using the micromixer compared well to those obtained with an external T mixing device. In contrast, assays performed in a microfabricated device by merging two streams and allowing mixing to occur by lateral diffusion did not compare well. Using the microfabricated mixer, Km and kcat values of 75 +/- 13 microM and 44 +/- 3 s(-1) were determined. These values compare well to those obtained with the conventional stopped-flow apparatus for which Km was determined to be 60 +/- 6 microM and kcat was 47 +/- 4 s(-1). Enzyme inhibition assays with phenylethyl-beta-D-thiogalactoside (PETG) were also comparable. It was concluded that kinetically based, stopped-flow enzyme assays can be performed in 60 s or less with a miniaturized system of roughly 6 nL liquid volume when mixing is assisted with the described device.

Regio-specific Adsorption of Cytochrome C on Negatively Charged Surfaces

Analytical Chemistry. Apr, 2003  |  Pubmed ID: 12713053

Studies are reported on the identification of the chromatographic contact domain of equine cytochrome c during its interaction with negatively charged sorbents. A negatively charged resin was designed that would simultaneously adsorb the protein electrostatically and covalently bind it through amide bond formation to succinate groups coupled to the support in an ester linkage. Protein immobilization occurred through lysine residues participating in electrostatic adsorbed cytochrome c to the resin surface. After covalent bond formation in the interface between the protein and the sorbent, ester linkages coupling succinate groups to the support were hydrolyzed, and the protein was released. Lysine residues on the protein that had participated in covalent capture were labeled with succinate residues. The tagged protein was then tryptic-mapped and the peptides were examined by matrix-assisted laser desorption ionization mass spectrometry to determine the position of the amino acids that had been tagged. Comparing the tagged sites with the X-ray crystallographic structure of cytochrome c, it was concluded that a single face of the protein dominated the adsorption process and the 3-D structure of the protein remained largely undisturbed during adsorption.

Protein Proteolysis and the Multi-dimensional Electrochromatographic Separation of Histidine-containing Peptide Fragments on a Chip

Journal of Chromatography. A. Jan, 2003  |  Pubmed ID: 12564680

This paper reports a system for three-dimensional electrochromatography in a chip format. The steps involved included trypsin digestion, copper(II)-immobilized metal affinity chromatography [Cu(II)-IMAC] selection of histidine-containing peptides, and reversed-phase capillary electrochromatography of the selected peptides. Trypsin digestion and affinity chromatography were achieved in particle-based columns with a microfabricated frit whereas reversed-phase separations were executed on a column of collocated monolithic support structures. Column frits were designed to maintain constant cross sectional area and path length in all channels and to retain particles down to a size of 3 microm. Cu(II)-IMAC selection of histidine-containing peptides from standard peptide mixtures and protein digests followed by reversed-phase chromatography of the selected peptides was demonstrated in the electrochromatography mode. The possibility to run a comprehensive proteomic analysis by combining trypsin digestion, affinity selection, and a reversed-phase separation on chips was shown using fluorescein isothiocyanate-labeled bovine serum albumin as an example.

Histidine-rich Peptide Selection and Quantification in Targeted Proteomics

Journal of Proteome Research. Jan-Feb, 2004  |  Pubmed ID: 14998161

Agarose based immobilized copper (II) affinity chromatography (Cu(II)-IMAC) in tandem with reversed-phase chromatography was applied to a yeast protein extract. Histidine-rich peptides were selected and, in the process, samples were substantially simplified prior to mass spectral analysis. Samples of proteins from the yeast extract at fermentation time periods of 2.5 and 10 h were compared quantitatively used the GIST protocol. Acylation of the N-terminus of tryptic peptides with N-acetoxysuccinamide was used to globally label and quantify relative protein concentration changes. Together with N-terminal acylation, an imidazole elution procedure allowed histidine-rich peptides to be preferentially selected by Cu(II)-IMAC. An inverse labeling strategy was applied to increase reliability in determinations of up- and down-regulation. It was found that the concentration of some histidine-rich proteins changed in excess of 4-fold during fermentation. These proteins covered a wide range of molecular weight and pI values.

Effect of Microenvironment PH of Aluminum Hydroxide Adjuvant on the Chemical Stability of Adsorbed Antigen

Vaccine. Mar, 2004  |  Pubmed ID: 15003645

The rate of acid-catalyzed hydrolysis of glucose-1-phosphate (G1P) when adsorbed to aluminum hydroxide adjuvant was significantly slower than the rate of hydrolysis of a solution of G1P at the same pH. It was concluded that the positively charged aluminum hydroxide adjuvant (iep 11.4) electrostatically attracted anions including hydroxyls to form a double layer surrounding the adjuvant particles. Thus, the pH of the microenvironment surrounding the aluminum hydroxide adjuvant was higher than the bulk pH. Adsorbed G1P hydrolyzed at a rate associated with the pH of the microenvironment of the surface of the adjuvant rather than with the pH of the bulk solution. Comparison of the rate constant for the hydrolysis of adsorbed G1P to the pH-stability profile of G1P in solution revealed that adsorbed G1P hydrolyzed at a rate associated with a pH that was approximately two pH units higher than the bulk pH. The results suggest that the chemical stability of antigens that degrade by pH-dependent mechanisms can be optimized by modifying the surface charge of the aluminum-containing adjuvant to produce the pH of maximum stability in the microenvironment of the adjuvant.

Potential of Silica Monolithic Columns in Peptide Separations

Journal of Chromatography. A. Mar, 2004  |  Pubmed ID: 15043268

The objective of the work described here was to evaluate the efficacy of silica monolith supports in high-speed reversed-phase liquid chromatography (RPLC) of peptides. This was done using a commercial Chromolith column with an octadecylsilane stationary phase and a tryptic digest of cytochrome c. Columns (100 mm x 4.6 mm) were operated at mobile phase velocities ranging from 1 ml/min (2.0 mm/s) to 10 ml/min (25 mm/s). There was little noticeable change over this flow rate range in either resolution, peak elution volume, or analyte concentration in collected fractions. It was concluded that capillary columns in this silica monolith format would be particularly valuable in peptide separations for proteomics. There was, however, a small, but perceptible contamination of peaks at high mobile phase velocity with earlier eluting analytes. Based on the fact that peak shape did not change at high mobile phase velocity, it is suggested that this phenomena might be due to the presence of peptide conformers in structural equilibrium on the sorbent surface. When elution rate exceeds the rate of conformer interchange, conformers could elute as broadened or even separate peaks.

Contributions of Commercial Sorbents to the Selectivity in Immobilized Metal Affinity Chromatography with Cu(II)

Journal of Chromatography. A. Mar, 2004  |  Pubmed ID: 15058570

Immobilized copper(II) affinity chromatography [Cu(II)-immobilized metal affinity chromatography (IMAC)] has been used in proteomics to simplify sample mixtures by selecting histidine-containing peptides from proteolytic digests. This paper examines the specificity of four different support materials with an iminodiacetic acid (IDA) stationary phase in the selection of only histidine-containing peptides in the single step capture-release mode. Three of the sorbents examined were commercially available: HiTrap Chelating HP (agarose), TSK Chelate-5PW, and Poros 20MC. IDA was also immobilized on CIM discs (monolithic glycidylmethacrylate-ethylene dimethacrylate). Tryptic digests of transferrin and beta-galactosidase were used as model samples to evaluate these sorbents. It was found that among the examined matrices, the TSK Chelate-5PW sorbent bound histidine-containing peptides the strongest, while Poros matrix was found to have a high degree of non-specific bindings. Agarose-based columns showed relatively high selectivity and specificity.

Proteomic Analysis of Arabidopsis Glutathione S-transferases from Benoxacor- and Copper-treated Seedlings

The Journal of Biological Chemistry. Jun, 2004  |  Pubmed ID: 15069083

Glutathione S-transferases (GSTs) are involved in many stress responses in plants, for example, participating in the detoxification of xenobiotics and limiting oxidative damage. Studies examining the regulation of this gene family in diverse plant species have focused primarily on RNA expression. A proteomics method was developed to identify GSTs expressed in Arabidopsis seedlings and to determine how the abundance of these proteins changed in response to copper, a promoter of oxidative stress, and benoxacor, a herbicide safener. Eight GSTs were identified in seedlings grown under control conditions, and only one, AtGSTU19, was induced by benoxacor. In contrast, four GSTs, AtGSTF2, AtGSTF6, AtGSTF7, and AtGSTU19, were significantly more abundant in copper-treated seedlings. The different responses to these treatments may reflect the potential for copper to affect many more aspects of plant growth and physiology compared with a herbicide safener. Differences between RNA and protein expression of GSTs indicate that both transcriptional and translational mechanisms are involved in regulation of GSTs under these conditions.

High-speed Label-free Detection by Spinning-disk Micro-interferometry

Biosensors & Bioelectronics. Jun, 2004  |  Pubmed ID: 15093207

Spinning-disk interferometers are a new class of analytic sensors to detect immobilized biomolecules with high speed and high sensitivity. The disks are composed of a large number of surface-normal self-referencing interferometers, analogous to an optical CD, but operating on the principle of microdiffraction quadrature that achieves sensitive linear detection of bound molecules. The surface-normal structures have a small footprint of only 20 microm each, allowing potential integration to over a million interferometric elements per disk. We have fabricated interferometric microstructures on silicon and on dielectric mirror disks to demonstrate the basic principles of the BioCD. We have detected the presence of immobilized anti-mouse IgG and the specific binding of 10 femtomol of mouse IgG at a sampling rate of 100 kilo-samples/s, while also demonstrating negligible non-specific binding. This technique provides a label-free method that could potentially screen hundreds to thousands of proteins per disk.

Proteomic Analysis of Carbonylated Proteins in Two-dimensional Gel Electrophoresis Using Avidin-fluorescein Affinity Staining

Electrophoresis. May, 2004  |  Pubmed ID: 15174056

A method for detecting carbonylated proteins in two-dimensional electrophoresis (2-DE) was developed using biotinylation and avidin-fluorescein isothiocyanate (FITC) affinity staining. The method was used to examine oxidatively modified proteins associated with oxidative stress. Carbonyl formation in proteins was first examined in a model system by subjecting bovine serum albumin (BSA) and ribonuclease A (RNase A) to metal-catalyzed oxidation (MCO). Carbonyl group formation was found to occur at multiple sites along with a small amount of polypeptide chain cleavage. In vivo studies were conducted in yeast cell cultures using 5 mM hydrogen peroxide to induce oxidative stress. Biotinylation of yeast protein was accomplished during extraction at 4 degrees C in a lysis buffer containing 5 mM biotin-hydrazide. Biotin-hydrazide forms a Schiff base with a carbonyl group on an oxidized protein that is subsequently reduced before electrophoresis. Proteins were separated by either 2-DE or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biotinylated species were detected using avidin-FITC affinity staining. Detection sensitivity with biotinylated proteins was five times higher than achieved by silver staining. The limit of detection with avidin-FITC staining approached 0.64 pmol of protein-associated carbonyls. Twenty carbonylated proteins were identified in the proteome of yeast following oxidative stress with hydrogen peroxide. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis of tryptic peptides was used to identify peptides extracted from gels. Aconitase, heat shock protein SSA1 and SSC1, pyruvate decarboxylase isozyme 1, pyruvate kinase 1, enolase 1 and 2, phosphoglycerate kinase, fructose-bisphosphate aldorase, and glyceraldehyde-3-phosphate dehydrogenase were among the major targets of oxidative stress.

Enrichment of Cysteine-containing Peptides from Tryptic Digests Using a Quaternary Amine Tag

Analytical Chemistry. Aug, 2004  |  Pubmed ID: 15283597

A new strategy for specifically targeting cysteine-containing peptides in a tryptic digest is described. The method is based on quantitatively derivatizing cysteine residues with a quaternary amine tag (QAT). Tags were introduced into proteins following reduction of disulfide bonds through derivatization of cysteine residues with (3-acrylamidopropyl)trimethylammonium chloride. After trypsin digestion, derivatized cysteine-containing peptides were enriched by strong cation exchange chromatography. The method was validated using model peptides and a protein. The QAT strategy has several advantages over other methods for the selection of cysteine-containing peptides. One is that it increases the ionization efficiency of cysteine-containing peptides. The other is that chromatographic selection is achieved with simple, robust cation exchange chromatography columns. As a result, this new strategy provides a simple way to facilitate enrichment of cysteine-containing peptides, thereby reducing sample complexity in bottom-up proteomics.

New Approach for Analysis of the Phosphotyrosine Proteome and Its Application to the Chicken B Cell Line, DT40

Journal of Proteome Research. Jul-Aug, 2004  |  Pubmed ID: 15359727

In this study, we have begun to analyze phosphotyrosyl and associated proteins present in a DT40 chicken B cell line overexpressing the nonreceptor protein-tyrosine kinase, Syk. An anti-phosphotyrosine antibody was used to select tyrosine-phosphorylated proteins. After tryptic digestion, peptides were subjected to a beta-elimination reaction and phosphotyrosine-containing peptides were enriched via immobilized metal affinity chromatography. Several known substrates and candidate substrates for Syk and the location of 22 tyrosine phosphorylation sites were identified.

Benzoyl Derivatization As a Method to Improve Retention of Hydrophilic Peptides in Tryptic Peptide Mapping

Analytical Chemistry. Oct, 2004  |  Pubmed ID: 15456300

This study exploits the increase in chromatographic retention that accrues from benzoyl derivatization of primary amines as a tool to increase sequence coverage in tryptic peptide mapping. N-hydroxysuccinamide sulfonyl benzoate quantitatively derivatizes primary amines of peptides. Introduction of the hydrophobic benzoyl moiety into peptides increased retention of peptides during reversed-phase chromatography (RPC), particularly in the case of smaller hydrophilic peptides. Short chain (1-6 amino acids) tryptic fragments of model proteins lysozyme, myoglobin, and cytochrome c derivatized with N-hydroxysuccinamide sulfonyl benzoate eluted in the linear acetonitrile gradient. Application of benzoyl derivatization was further extended to achieve complete sequence coverage of a therapeutic protein, recombinant human growth hormone, and in detection of single amino acid polymorphism.

Optimization of Diagonal Chromatography for Recognizing Post-translational Modifications

Journal of Chromatography. A. Aug, 2004  |  Pubmed ID: 15460252

Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents a formidable analytical challenge. This paper examines the potential of diagonal chromatography for recognizing post-translational modifications. Diagonal chromatography is the process of using the same chromatographic separation in two dimensions. Between the two dimensions, a chemical modification is applied to all fractions. Substances that have been modified are revealed by the change in their chromatographic properties between the two dimensions. When the modification is specific to a particular type of post-translational modification, peptides that carry the modification have the potential to be revealed. Changes in the retention time of modified peptides have to be large enough to be different from unmodified peptides. Tyrosine, serine, and threonine phosphorylation were identified with diagonal chromatography. Heptafluorobutyric acid was used as an ion-pairing agent to improve the selectivity between serine and threonine phosphorylated peptides and parent peptides after dephosphorylation. The diagonal chromatography method was also examined in the recognition of glycopeptides. However, changes of retention time after deglycosylation were considered to be too small to make this an unequivocal method for the study of glycosylation.

Processing of Data Generated by 2-dimensional Gel Electrophoresis for Statistical Analysis: Missing Data, Normalization, and Statistics

Journal of Proteome Research. Nov-Dec, 2004  |  Pubmed ID: 15595730

Several high-throughput statistical methods were evaluated for processing data generated by two-dimensional polyacrylamide gel electrophoresis, including how to handle missing data, normalization, and statistical analysis of data obtained from 2-D gels. Quantile normalization combined with a nonparametric permutation test based on minimizing false discover rates gave the highest yield of proteins that changed with genotype and detected the anticipated 50% decrease in Mn-superoxide dismutase (MnSOD) protein levels in mitochondrial extracts obtained from MnSOD-deficient mice.

Use of Multidimensional Lectin Affinity Chromatography in Differential Glycoproteomics

Analytical Chemistry. May, 2005  |  Pubmed ID: 15859596

This paper reports studies comparing the relative degree of sialylation among human serum glycoproteins carrying complex biantennary N-linked, hybrid, and high-mannose oligosaccharides. Comparisons were made by coupling lectin affinity selection with stable isotope coding of peptides from tryptic digests of serum. After proteolysis, samples were split and differentially acetylated with stable isotope coding agents according to either origin or the separation method by which they would be fractionated. A lectin column prepared from Sambucus nigra agglutinin (SNA) was used to select and compare the concentration of sialic acid containing glycopeptides. The relative standard deviation in quantification using this method was 4%. Using this method the concentration of sialic acid containing glycoproteins from a normal individual were compared to those in a pooled serum sample from a large number of normal individuals. It was found that sialylation varied less than 2-fold in all but four or five glycoproteins. Further studies were done on the degree of sialylation within glycoproteins. Samples labeled with the light isoform of the coding agent were applied to a set of serial lectin columns consisting of a concanavalin A (Con A) column coupled to an SNA column for selecting sialic acid appended to glycopeptides with complex biantennary N-linked, hybrid, and high-mannose glycans. In contrast, samples labeled with the heavy isoform of the coding agent were applied to a Con A lectin column alone to select glycopeptides containing complex biantennary N-linked, hybrid, and high-mannose glycans, without regard to sialylation. Glycopeptides thus selected were mixed, deglycosylated by PNGase F, and fractionated by reversed-phase chromatography (RPC). The RPC fractions were then analyzed by ESI-MS. The relative standard deviation of the method was 4%. All glycopeptides identified contained sialic acid except one. Peptides in which the relative abundance of isotopic isoforms was equal were considered to indicate that the protein parent was fully sialylated at that specific glycosylation site.

An Automated Method for the Analysis of Stable Isotope Labeling Data in Proteomics

Journal of the American Society for Mass Spectrometry. Jul, 2005  |  Pubmed ID: 15922621

An algorithm is presented for the generation of a reliable peptide component peak table from liquid chromatography-mass spectrometry (LC-MS) and subsequent quantitative analysis of stable isotope coded peptide samples. The method uses chemical noise filtering, charge state fitting, and deisotoping toward improved analysis of complex peptide samples. Overlapping peptide signals in mass spectra were deconvoluted by correlation with modeled peptide isotopic peak profiles. Isotopic peak profiles for peptides were generated in silico from a protein database producing reference model distributions. Doublets of heavy and light labeled peak clusters were identified and compared to provide differential quantification of pairs of stable isotope coded peptides. Algorithms were evaluated using peptides from digests of a single protein and a seven-protein mixture that had been differentially coded with stable isotope labeling agents and mixed in known ratios. The experimental results correlated well with known mixing ratios.

Recent Advancements in Differential Proteomics Based on Stable Isotope Coding

Briefings in Functional Genomics & Proteomics. Jul, 2005  |  Pubmed ID: 16102271

Stable isotope coding continues to be a powerful approach in comparative proteomics. This review focuses on recent developments in stable isotope coding-based strategies targeted towards protein expression, protein interactions with other biomolecules, post-translational modifications and absolute quantification. The focus of the bulk of proteomics studies is still on protein expression. An important recent application of isotope coding has been in organelle proteomics. The review ends with the conclusion that isotope coding remains an integral part of quantitative proteomics. There is, however, a need to develop coding strategies which can differentiate changes in protein expression and post-translational modification, address issues of protein dynamic range and facilitate real-time detection of proteins which show a statistically significant change after stimulus.

Comparative Glycoproteomics of N-linked Complex-type Glycoforms Containing Sialic Acid in Human Serum

Analytical Chemistry. Nov, 2005  |  Pubmed ID: 16285669

This study describes a simple and efficient approach for comparative analysis of sialylated glycoforms of proteins containing differentially branched complex-type glycans. The analytical protocol is based on glycopeptide selection from tryptic digests with serial lectin affinity chromatography (SLAC), quantification with global internal standard technology, fractionation of deglycosylated peptides with reversed-phase chromatography, and peptide sequencing with tandem mass spectrometry. Fractionation of complex tri- and tetraantennary N-linked glycoforms from biantennary N-linked glycoforms bearing terminal sialic acid residues was achieved using a set of serial lectin columns with immobilized Sambucus nigra agglutinin and concanavalin A. These two fractions from the affinity selection were differentially labeled, mixed, and then deglycosylated with the enzyme PNGase F. The deglycosylated sample was further fractionated by reversed-phase chromatography and analyzed by electrospray ionization mass spectrometry. The SLAC strategy was applied to tryptic digests of human serum, and it was found that most sialylated glycopeptides identified carry more biantennary glycans than tri- and tetraantennary glycans, and the relative amount of biantennary glycan versus tri- and tetraantennary glycans was different at separate glycosylation sites within the same glycoprotein.

Primary Amine Coding As a Path to Comparative Proteomics

Proteomics. Jul, 2006  |  Pubmed ID: 16800033

Various isotope coding strategies are being used today in the field of comparative proteomics. This article specifically reviews the strengths and limitations of various N-termini-directing strategies. N-termini-directed coding strategy allows for use of different chromatographic enrichment techniques. Since N-termini-directed coding strategies are global in nature, they can be utilized in studying PTMs as well as protein expression. Using different N-termini-directed coding strategies, both relative and absolute quantification of proteins can be achieved either in the MS mode or in the MS/MS mode. The review ends with the conclusion that significant improvements have been made in the last decade. Among various issues, a need still exists for a better understanding of the kinetic issues in proteomics, relative protein pool sizes for different proteins and the issue of stimulus-induced changes in protein aggregation. Another critical issue that needs to be addressed in great detail is the role of PTMs in regulation.

Enhancement of Amino Acid Detection and Quantification by Electrospray Ionization Mass Spectrometry

Analytical Chemistry. Jul, 2006  |  Pubmed ID: 16808485

A new strategy for amino acid analysis is reported involving derivatization with an N-hydroxysuccinimide ester of N-alkylnicotinic acid (Cn-NA-NHS) followed by reversed-phase chromatography and electrospray ionization mass spectrometry (RPC-MS). Detection sensitivity increased as the N-alkyl chain length of the nicotinic acid derivatizing agent was increased from 1 to 4. N-Acylation of amino acids with the Cn-NA-NHS reagents in water produced a stable product in roughly 1 min using a 4-fold molar excess of derivatizing agent in 0.1 M sodium borate buffer at pH values ranging from 8.5 to 10. Some O-acylation of tyrosine was also observed, but the product hydrolyzed within a few minutes at pH 10. The cystine product also degraded slowly over the course of a few days from reduction of the disulfide bond to form cysteine. The retention time of Cn-NA derivatized amino acids was lengthened in reversed-phase chromatography to the extent that polar amino acids were retained beyond the solvent peak, particularly in the cases of the C3-NA and C4-NA derivatives. Complete resolution of 18 amino acids was achieved in 28 min using the C4-NA-NHS reagent. Compared to N-acylation with benzoic acid, derivatization with C4-NA-NHS increased MS detection sensitivity 6-80-fold. This was attributed to the surfactant properties of the Cn-NA-NHS reagents. The quaternary amine increased the charge on amino acid conjugates while the presence of an adjacent alkyl chain further increased ionization efficiency by apparently enhancing amino acid migration to the surface of electrospray droplets. Further modification of the Cn-NA-NHS reagents with deuterium was used to prepare coded sets of derivatizing agents. These coding agents were used to differentially code samples and after mixing carry out comparative concentration measurements between samples using extracted ion chromatograms to estimate relative peak areas of derivatized amino acids.

Targeted Glycoproteomics: Serial Lectin Affinity Chromatography in the Selection of O-glycosylation Sites on Proteins from the Human Blood Proteome

Journal of Chromatography. A. Nov, 2006  |  Pubmed ID: 16919642

Although lectin selection is gaining increasing acceptance as a tool for targeting glycosylation in glycoproteomics, most of the work has been directed at N-glycosylation. The work reported here focuses on the use of lectins in the study of O-glycosylation. The problem with using lectins for studying O-glycosylation is that they are not sufficiently specific. This paper reports that through the use of serial lectin affinity chromatography (SLAC) it is possible to select predominantly O-glycosylated peptides from tryptic digests of human serum. Jacalin is relatively specific for O-glycosylation but has the problem that it also selects high mannose N-type glycans. This problem was addressed by using a concanavalin A affinity column to first remove high mannose, hybrid-type and biantennary complex-type N-type glycans before application of the Jacalin columns. When used in a serial format, concanavalin A and Jacalin together provide essentially O-glycosylated peptides. The glycoprotein parents of glycopeptides were identified by deglycosylating the selected O-glycopeptides by oxidative elimination. These peptides were then separated by RPC and further analyzed using ESI-MS/MS and MALDI-MS/MS. Using this approach all the O-glycosylated sites in a model protein (fetuin) and over thirty glycoprotein parents from human serum were identified. It is concluded that a serial combination of Con A and Jacalin can be of utility in the study of O-glycosylation in glycoproteomics.

Neural Network Prediction of Peptide Separation in Strong Anion Exchange Chromatography

Bioinformatics (Oxford, England). Jan, 2007  |  Pubmed ID: 17092987

The still emerging combination of technologies that enable description and characterization of all expressed proteins in a biological system is known as proteomics. Although many separation and analysis technologies have been employed in proteomics, it remains a challenge to predict peptide behavior during separation processes. New informatics tools are needed to model the experimental analysis method that will allow scientists to predict peptide separation and assist with required data mining steps, such as protein identification.

Destabilization of DJ-1 by Familial Substitution and Oxidative Modifications: Implications for Parkinson's Disease

Biochemistry. May, 2007  |  Pubmed ID: 17451229

Parkinson's disease (PD) is a neurodegenerative disorder characterized by oxidative stress and protein aggregation. Both toxic phenomena are mitigated by DJ-1, a homodimeric protein with proposed antioxidant and chaperone activities. The neuroprotective function of DJ-1 is modulated by oxidation of cysteine 106, a residue that may act as an oxidative stress sensor. Loss-of-function mutations in the DJ-1 gene have been linked to early onset PD, and age-dependent over-oxidation of DJ-1 is thought to contribute to sporadic PD. The familial mutant L166P fails to dimerize and is rapidly degraded, suggesting that protein destabilization accounts for the dysfunction of this mutant. In this study, we investigated how the structure and stability of DJ-1 are impacted by two other pathogenic substitutions (M26I and E64D) and by over-oxidation with H2O2. Whereas the recombinant wild-type protein and E64D both adopted a stable dimeric structure, M26I showed an increased propensity to aggregate and decreased secondary structure. Similar to M26I, over-oxidized wild-type DJ-1 exhibited reduced secondary structure, and this property correlated with destabilization of the dimer. The engineered mutant C106A had a greater thermodynamic stability and was more resistant to oxidation-induced destabilization than the wild-type protein. These results suggest that (i) the M26I substitution and over-oxidation destabilize dimeric DJ-1, and (ii) the oxidation of cysteine 106 contributes to DJ-1 destabilization. Our findings provide a structural basis for DJ-1 dysfunction in familial and sporadic PD, and they suggest that dimer stabilization is a reasonable therapeutic strategy to treat both forms of this disorder.

An Isotope Coding Strategy for Proteomics Involving Both Amine and Carboxyl Group Labeling

Methods in Molecular Biology (Clifton, N.J.). 2007  |  Pubmed ID: 17484114

A stable isotope coding strategy is described for the analysis of all types of tryptic peptides, including those that are N-terminally blocked and from the C-terminus of proteins. The method exploits differential derivatization of amine and carboxyl groups generated during proteolysis as a means of coding. Carboxyl groups produced during proteolysis incorporate 18O from H2(18)O. Peptides from the C-terminus of proteins were not labeled with 18O unless they contained a basic C-terminal amino acid. Primary amines form controls, and experimental samples were differentially acylated after proteolysis with either 1H3- or 2H3-N-acetoxysuccinamide. When these two types of labeling were combined, unique coding patterns were achieved for peptides arising from the C-termini and blocked N-termini of proteins.

Enhancement of the LC/MS Analysis of Fatty Acids Through Derivatization and Stable Isotope Coding

Analytical Chemistry. Jul, 2007  |  Pubmed ID: 17492837

This paper focuses on the development of an enhanced LC/ESI-MS method for the identification and quantification of fatty acids through derivatization. Fatty acids were derivatized with 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide, forming 3-acyloxymethyl-1-methylpyridinium iodide (AMMP). This process attaches a quaternary amine to analytes and enabled ESI-MS in the positive mode of ionization with common LC mobile phases. Moreover, detection sensitivity was generally 2500-fold higher than in the negative mode of ionization used with underivatized fatty acids. The limits of detection were roughly 1.0-4.0 nM (or 10 pg/injection) for standard fatty acids from C10 to C24 and spanned approximately 2 orders of magnitude in linearity. AMMP derivatives had unique tandem mass spectra characterized by common ions at m/z 107.0, 124.0, and 178.0. Individual fatty acids also had unique fingerprint regions that allowed identification of their carbon skeleton number, number of double bonds, and double bond position. The derivatization method also allowed coding of analytes as a means of recognizing derivatives and enhancing quantification. 2H-Coding was achieved through derivatization with deuterated 3-carbinol-1-methyl-d3-pyridinium iodide. The 2H-coded derivatization reagent, 3-acyloxymethyl-1-methyl-d3-pyridinium iodide, was used in two ways. One was to differentially label equal fractions of a sample such that after being recombined and analyzed by ESI-MS all fatty acids appeared as doublet clusters of ions separated by roughly 3 amu. This greatly facilitated identification of fatty acids in complex mixtures. Another use of stable isotope coding was in comparative quantification. Control and experimental samples were differentially labeled with nondeuterated and deuterated isotopomers of CPM, respectively. After mixing the two samples, they were analyzed by ESI-MS. The abundance of a fatty acid in an experimental sample relative to the control was established by the isotope ratio of the isotopomeric fatty acids. Absolute quantification was achieved by adding differentially labeled fatty acid standards to experimental samples containing unknown quantities of fatty acids. Utility of the method was examined in the analysis of human serum samples.

Adaptive Interferometry of Protein on a BioCD

Applied Optics. Aug, 2007  |  Pubmed ID: 17676154

Adaptive spinning-disk interferometry is capable of measuring surface profiles of a thin biolayer with subnanometer longitudinal resolution. High-speed phase modulation in the signal beam arises from the moving surface height profile on the spinning disk and is detected as a homodyne signal via dynamic two-wave mixing. A photorefractive quantum-well device performs as an adaptive mixer that compensates disk wobble and vibration while it phase-locks the signal and reference waves in the phase quadrature condition (pi/2 relative phase between the signal and local oscillator). We performed biosensing of immobilized monolayers of antibodies on the disk in both transmission and reflection detection modes. Single- and dual-analyte adaptive spinning-disk immunoassays were demonstrated with good specificity and without observable cross-reactivity. Reflection-mode detection enhances the biosensing sensitivity to one-twentieth of a protein monolayer, creates a topographic map of the protein layer, and can differentiate monolayers of different species by their effective optical thicknesses.

Differential Metabolomics Using Stable Isotope Labeling and Two-dimensional Gas Chromatography with Time-of-flight Mass Spectrometry

Analytical Chemistry. Jan, 2008  |  Pubmed ID: 18052339

This work describes an approach to differential metabolomics that involves stable isotope labeling for relative quantification as part of sample analysis by two-dimensional gas chromatography/mass spectrometry (GCxGC/MS). The polar metabolome in control and experimental samples was extracted and differentially derivatized using isotopically light and heavy (D6) forms of the silylation reagent N-methyl-N-tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA). MTBSTFA derivatives are of much greater hydrolytic stability than the more common trimethylsilyl derivatives, thus diminishing the possibility of isotopomer scrambling during GC analysis. Subsequent to derivatization with MTBSTFA, differentially labeled samples were mixed and analyzed by GCxGC/MS. Metabolites were identified, and the isotope ratio of isotopomers was quantified. The method was tested using three classes of metabolites; amino acids, fatty acids, and organic acids. The relative concentration of isotopically labeled metabolites was determined by isotope ratio analysis. The accuracy and precision, respectively, in quantification of standard mixtures was 9.5 and 4.77% for the 16 amino acids, 9.7 and 2.83% for the mixture of 19 fatty acids, and 14 and 4.53% for the 20 organic acids. Suitability of the method for the examination of complex samples was demonstrated in analyses of the spiked blood serum samples. This differential isotope coding method proved to be an effective means to compare the concentration of metabolites between two samples simultaneously.

Comprehensive Two-dimensional Gas Chromatography/time-of-flight Mass Spectrometry Peak Sorting Algorithm

Journal of Chromatography. A. Feb, 2008  |  Pubmed ID: 18093607

We report a novel peak sorting method for the two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/TOF-MS) system. The objective of peak sorting is to recognize peaks from the same metabolite occurring in different samples from thousands of peaks detected in the analytical procedure. The developed algorithm is based on the fact that the chromatographic peaks for a given analyte have similar retention times in all of the chromatograms. Raw instrument data are first processed by ChromaTOF (Leco) software to provide the peak tables. Our algorithm achieves peak sorting by utilizing the first- and second-dimension retention times in the peak tables and the mass spectra generated during the process of electron impact ionization. The algorithm searches the peak tables for the peaks generated by the same type of metabolite using several search criteria. Our software also includes options to eliminate non-target peaks from the sorting results, e.g., peaks of contaminants. The developed software package has been tested using a mixture of standard metabolites and another mixture of standard metabolites spiked into human serum. Manual validation demonstrates high accuracy of peak sorting with this algorithm.

Two-dimensional Correlation Optimized Warping Algorithm for Aligning GC X GC-MS Data

Analytical Chemistry. Apr, 2008  |  Pubmed ID: 18351753

A two-dimensional (2-D) correlation optimized warping (COW) algorithm has been developed to align 2-D gas chromatography coupled with time-of-flight mass spectrometry (GC x GC/TOF-MS) data. By partitioning raw chromatographic profiles and warping the grid points simultaneously along the first and second dimensions on the basis of applying a one-dimensional COW algorithm to characteristic vectors, nongrid points can be interpolatively warped. This 2-D algorithm was directly applied to total ion counts (TIC) chromatographic profiles of homogeneous chemical samples, i.e., samples including mostly identical compounds. For heterogeneous chemical samples, the 2-D algorithm is first applied to certain selected ion counts chromatographic profiles, and the resultant warping parameters are then used to warp the corresponding TIC chromatographic profiles. The developed 2-D COW algorithm can also be applied to align other 2-D separation images, e.g., LC x LC data, LC x GC data, GC x GC data, LC x CE data, and CE x CE data.

Use of Glycan Targeting Antibodies to Identify Cancer-associated Glycoproteins in Plasma of Breast Cancer Patients

Analytical Chemistry. Jul, 2008  |  Pubmed ID: 18558770

Immunoaffinity chromatography (IAC) was used to isolate and identify potential cancer biomarker glycoproteins by targeting disease-associated glycans. Glycoproteins were selected from plasma of disease-free and breast cancer patients with an anti-Lewis x (Le(x)) IAC column. After extensive washing of the IAC column to remove abundant proteins, the selected proteins were eluted with an acidic mobile phase and identified in two ways. The protocol used in route A involved the steps of tryptic digestion, reversed-phase chromatographic fractionation of the digest, and identification of peptides in collected RPC fractions by MALDI-MS/MS. Route B differed in that IAC-selected proteins were further fractionated by reversed-phase chromatography before proteolysis of individual chromatographic fractions and identification by MALDI-MS/MS. Route A was the more efficacious of the two protocols in total number of proteins identified. Of the 26 proteins identified, 9 were found to be potential breast cancer marker candidates based on their elevation in breast cancer patients. The potential of these candidates as cancer markers remains to be validated in much larger, more diverse populations of breast cancer patients.

Simultaneous Quantification of Metabolites Involved in Central Carbon and Energy Metabolism Using Reversed-phase Liquid Chromatography-mass Spectrometry and in Vitro 13C Labeling

Analytical Chemistry. Dec, 2008  |  Pubmed ID: 19007244

Comprehensive analysis of intracellular metabolites is a critical component of elucidating cellular processes. Although the resolution and flexibility of reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) makes it one of the most powerful analytical tools for metabolite analysis, the structural diversity of even the simplest metabolome provides a formidable analytical challenge. Here we describe a robust RPLC-MS method for identification and quantification of a diverse group of metabolites ranging from sugars, phosphosugars, and carboxylic acids to phosphocarboxylics acids, nucleotides, and coenzymes. This method is based on in vitro derivatization with a (13)C-labeled tag that allows internal standard based quantification and enables separation of structural isomer pairs like glucose 6-phosphate and fructose 6-phosphate in a single chromatographic run. Calibration curves for individual metabolites showed linearity ranging over more than 2 orders of magnitude with correlation coefficients of R(2) > 0.9975. The detection limits at a signal-to-noise ratio of 3 were below 1.0 microM (20 pmol) for most compounds. Thirty common metabolites involved in glycolysis, the pentose phosphate pathway, and tricarboxylic acid cycle were identified and quantified from yeast lysate with a relative standard deviation of less than 10%.

Comparative Metabolite Profiling of Carboxylic Acids in Rat Urine by CE-ESI MS/MS Through Positively Pre-charged and (2)H-coded Derivatization

Electrophoresis. Nov, 2008  |  Pubmed ID: 19035407

A new approach to the selective comparative metabolite profiling of carboxylic acids in rat urine was established using CE-MS and a method for positively pre-charged and (2)H-coded derivatization. Novel derivatizing reagents, N-alkyl-4-aminomethyl-pyridinum iodide (alkyl = butyl, butyl-d9 or hexyl), containing quaternary amine and stable-isotope atoms (deuterium), were introduced for the derivatization of carboxylic acids. CE separation in positive polarity showed high reproducibility (0.99-1.32% RSD of migration time) and eliminated problems with capillary coating known in CE-MS anion analyses. Essentially complete ionization and increased hydrophobicity after the derivatization also enhanced MS detection sensitivity (e.g. formic acid was detected at 0.5 pg). Simultaneous derivatization of one sample using two structurally similar reagents, N-butyl-4-aminomethyl-pyridinum iodide (BAMP) and N-hexyl-4-aminomethyl-pyridinum iodide, provided additional information for recognizing a carboxylic acid in an unknown sample. Moreover, characteristic fragmentation acquired by online CE-MS/MS allowed for identification and categorization of carboxylic acids. Applying this method on rat urine, we found 59 ions matching the characteristic patterns of carboxylic acids. From these 59, 32 ions were positively identified and confirmed with standards. For comparative analysis, 24 standard carboxylic acids were derivatized by chemically identical but isotopically distinct BAMP and N-butyl-d9-4-aminomethyl-pyridinium iodide, and their derivatization limits and linearity ranges were determined. Comparative analysis was also performed on two individual urine samples derivatized with BAMP and N-butyl-d9-4-aminomethyl-pyridinium iodide. The metabolite profiling variation between these two samples was clearly visualized.

Glycoproteomics of Plasma Based on Narrow Selectivity Lectin Affinity Chromatography

Journal of Proteome Research. Feb, 2009  |  Pubmed ID: 19099503

Lectin affinity chromatography using concanavalin A (Con A), Helix pomatia agglutinin (HPA), Lycopersicon esculentum lectin (LEL), Aleuria aurantia lectin (AAL) and Lens culinaris agglutinin (LCA) was used to investigate the utility of narrow selectivity lectins in the characterization of plasma glycoproteome diversity and to recognize cancer associated aberrations in glycosylation. Following affinity chromatographic selection, proteins were tryptically digested, the peptide fragments separated by reversed phase chromatography (RPC), and fractions from RPC identified by tandem mass spectrometry. The diversity of glycosylation found with narrow selectivity lectins was generally 2/3 that of Con A and not related to protein abundance. Small groups of proteins were found with each of the affinity columns, HPA, LEL, AAL, and LCA, that changed 3-fold or more in concentration between normal and breast cancer patient plasma. Although the number of cancer patients examined was too small to validate cancer marker candidates, they are clearly worth examining in a larger, more diverse patient population.

Multi-site Assessment of the Precision and Reproducibility of Multiple Reaction Monitoring-based Measurements of Proteins in Plasma

Nature Biotechnology. Jul, 2009  |  Pubmed ID: 19561596

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low mug/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.

Performance Metrics for Liquid Chromatography-tandem Mass Spectrometry Systems in Proteomics Analyses

Molecular & Cellular Proteomics : MCP. Feb, 2010  |  Pubmed ID: 19837981

A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.

Interlaboratory Study Characterizing a Yeast Performance Standard for Benchmarking LC-MS Platform Performance

Molecular & Cellular Proteomics : MCP. Feb, 2010  |  Pubmed ID: 19858499

Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize pre-analytical and analytical variation in comparative proteomics experiments.

Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography-tandem Mass Spectrometry

Journal of Proteome Research. Feb, 2010  |  Pubmed ID: 19921851

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.

Oxidative Stress Studies in Yeast with a Frataxin Mutant: a Proteomics Perspective

Journal of Proteome Research. Feb, 2010  |  Pubmed ID: 19957947

Cellular response of wild-type Saccharomyces cerevisiae and the Delta yfh1 mutant to oxidative stress (OS) was examined by stressing cells through the addition of H(2)O(2) to the growth medium. The Delta yfh1 mutant is unusual in that it accumulates iron in it is mitochondria. Wild-type growth was immediately arrested and recovered in 2 h following H(2)O(2) treatment. No change in viability was observed. Growth of the mutant, on the other hand, was similar to wild-type yeast for 4 h but then rapidly declined, eventually reaching zero. Levels of carbonyl groups and reactive oxygen species (ROS) reached their maximum at 3 h following exposure. The impact of OS on protein function was also evaluated by proteomic techniques targeting protein carbonylation. Oxidized proteins were selected by affinity chromatography, and following trypsin digestion, peptide fragments were identified by RPLC-MS/MS. A total of 53 proteins were identified in both wild-type and mutant cells, respectively.

Sweetening the Pot: Adding Glycosylation to the Biomarker Discovery Equation

Clinical Chemistry. Feb, 2010  |  Pubmed ID: 19959616

Cancer has profound effects on gene expression, including a cell's glycosylation machinery. Thus, tumors produce glycoproteins that carry oligosaccharides with structures that are markedly different from the same protein produced by a normal cell. A single protein can have many glycosylation sites that greatly amplify the signals they generate compared with their protein backbones.

In Vitro Stable Isotope Labeling for Discovery of Novel Metabolites by Liquid Chromatography-mass Spectrometry: Confirmation of Gamma-tocopherol Metabolism in Human A549 Cell

Journal of Chromatography. A. Jan, 2010  |  Pubmed ID: 20006340

A general approach for discovering novel catabolic metabolites from a parent biocompound was developed and validated on the metabolism of gamma-tocopherol in human A549 cell. The method is based on LC-MS analysis of in vitro stable isotope-labeled metabolites and assumes that a parent compound and its metabolites share a common functional group that can be derivatized by well-documented reagents. In this method, two equal aliquots of extracted metabolites are separately derivatized with isotope-coded (heavy) and non-isotope-coded (light) form of derivatizing reagent, mixed at 1:1 ratio and analyzed using LC-MS. The metabolites with common functional group are then easily recognized by determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to the mass difference between heavy and light form of derivatization reagent. The feasibility of this approach was demonstrated and validated by the identification of products of gamma-tocopherol catabolism in human A549 cell culture media using N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NANHS) and N-methyl-d3-nicotinic acid N-hydroxysuccinimide ester (C1-d3-NANHS) derivatizing reagent. Overall four gamma-tocopherol metabolites were identified including 9'-COOH, 11'-COOH, 13'-COOH and 13'-OH. In addition, the developed LC-MS method can also be used for the fast and sensitive quantitative analysis of gamma-tocopherol and other forms of vitamin E related compounds.

Protein-based Multiplex Assays: Mock Presubmissions to the US Food and Drug Administration

Clinical Chemistry. Feb, 2010  |  Pubmed ID: 20007858

As a part of ongoing efforts of the NCI-FDA Interagency Oncology Task Force subcommittee on molecular diagnostics, members of the Clinical Proteomic Technology Assessment for Cancer program of the National Cancer Institute have submitted 2 protein-based multiplex assay descriptions to the Office of In Vitro Diagnostic Device Evaluation and Safety, US Food and Drug Administration. The objective was to evaluate the analytical measurement criteria and studies needed to validate protein-based multiplex assays. Each submission described a different protein-based platform: a multiplex immunoaffinity mass spectrometry platform for protein quantification, and an immunological array platform quantifying glycoprotein isoforms. Submissions provided a mutually beneficial way for members of the proteomics and regulatory communities to identify the analytical issues that the field should address when developing protein-based multiplex clinical assays.

Analytical Validation of Protein-based Multiplex Assays: a Workshop Report by the NCI-FDA Interagency Oncology Task Force on Molecular Diagnostics

Clinical Chemistry. Feb, 2010  |  Pubmed ID: 20007859

Clinical proteomics has the potential to enable the early detection of cancer through the development of multiplex assays that can inform clinical decisions. However, there has been some uncertainty among translational researchers and developers as to the specific analytical measurement criteria needed to validate protein-based multiplex assays. To begin to address the causes of this uncertainty, a day-long workshop titled "Interagency Oncology Task Force Molecular Diagnostics Workshop" was held in which members of the proteomics and regulatory communities discussed many of the analytical evaluation issues that the field should address in development of protein-based multiplex assays for clinical use. This meeting report explores the issues raised at the workshop and details the recommendations that came out of the day's discussions, such as a workshop summary discussing the analytical evaluation issues that specific proteomic technologies should address when seeking US Food and Drug Administration approval.

Profiling Carbonylated Proteins in Human Plasma

Journal of Proteome Research. Mar, 2010  |  Pubmed ID: 20121119

This study reports the first proteomic-based identification and characterization of oxidized proteins in human plasma. The study was conducted by isolating carbonylated proteins from the plasma of male subjects (age 32-36) with avidin affinity chromatography subsequent to biotinylation of carbonyl groups with biotin hydrazide and sodium cyanoborohydride reduction of the resulting Schiff's bases. Avidin selected proteins were digested with trypsin, and the peptide fragments were separated by C18 reversed phase chromatography and identified and characterized by both electrospray ionization and matrix assisted laser desorption ionization mass spectrometry. Approximately 0.2% of the total protein in plasma was selected with this method. Sixty-five high, medium, and low abundance proteins were identified, the majority appearing in all subjects. An interesting feature of the oxidized proteins isolated was that in addition to carbonylation they often bore other types of oxidative modification. Twenty-four oxidative modifications were mapped in 14 proteins. Fifteen carbonylation sites carried on 7 proteins were detected. Methionine oxidation was the most frequent single type of oxidative modification followed by tryptophan oxidation. Apolipoprotein B-100 had 20 oxidative modifications, the largest number for any protein observed in this study. Among the organs contributing oxidized proteins to plasma, kidney, liver, and soft tissues were the most frequent donors. One of the more important outcomes of this work was that mass spectral analysis allowed differentiation between different biological mechanisms of oxidation in individual proteins. For the first time, oxidation products arising from direct ROS oxidation of amino acid side chains in proteins, formation of advanced glycation endproducts (AGEs) adducts, and formation of adducts with lipid peroxidation products were simultaneously recognized and assigned to specific sites in proteins.

Multi-dimensional Liquid Chromatography in Proteomics--a Review

Analytica Chimica Acta. Apr, 2010  |  Pubmed ID: 20363391

Proteomics is the large-scale study of proteins, particularly their expression, structures and functions. This still-emerging combination of technologies aims to describe and characterize all expressed proteins in a biological system. Because of upper limits on mass detection of mass spectrometers, proteins are usually digested into peptides and the peptides are then separated, identified and quantified from this complex enzymatic digest. The problem in digesting proteins first and then analyzing the peptide cleavage fragments by mass spectrometry is that huge numbers of peptides are generated that overwhelm direct mass spectral analyses. The objective in the liquid chromatography approach to proteomics is to fractionate peptide mixtures to enable and maximize identification and quantification of the component peptides by mass spectrometry. This review will focus on existing multidimensional liquid chromatographic (MDLC) platforms developed for proteomics and their application in combination with other techniques such as stable isotope labeling. We also provide some perspectives on likely future developments.

Screening Antibody and Immunosorbent Selectivity by Two-dimensional Liquid Chromatography-MS/MS (2-D LC-MS/MS)

Journal of Separation Science. Jun, 2010  |  Pubmed ID: 20405482

Selectivity of both peptide- and glycan-targeting antibodies was examined by 2-D LC-MS/MS. Proteins selected from biological extracts immunospecifically in a first chromatography dimension using antibodies immobilized by either covalent coupling or adsorption to protein G were desorbed with a denaturing mobile phase and transferred to a 1.5 microm nonporous particle RP chromatography (NP-RPC) column in a second dimension. Protein peak capacity of the NP-RPC column was approximately 50. Peaks collected from the RPC column were tryptic digested and the peptide fragments were identified by MALDI-MS/MS. The objective of this analytical strategy was to discriminate between protein antigens and nonantigens through identification of their peptides, leading to an evaluation of the selectivity of antibodies and immunosorbents. Quantification of the relative amount of antigen and nonantigen species captured by immunosorbents was achieved by absorbance, along with the likely capture mechanism. A limitation of the approach was in discriminating between isoforms of an antigen in which neither the antibody nor the LC-MS system targeted the differentiating feature in the isoforms.

Proteomic Identification of Carbonylated Proteins and Their Oxidation Sites

Journal of Proteome Research. Aug, 2010  |  Pubmed ID: 20521848

Excessive oxidative stress leaves a protein carbonylation fingerprint in biological systems. Carbonylation is an irreversible post-translational modification (PTM) that often leads to the loss of protein function and can be a component of multiple diseases. Protein carbonyl groups can be generated directly (by amino acids oxidation and the alpha-amidation pathway) or indirectly by forming adducts with lipid peroxidation products or glycation and advanced glycation end-products. Studies of oxidative stress are complicated by the low concentration of oxidation products and a wide array of routes by which proteins are carbonylated. The development of new selection and enrichment techniques coupled with advances in mass spectrometry are allowing the identification of hundreds of new carbonylated protein products from a broad range of proteins located at many sites in biological systems. The focus of this review is on the use of proteomics tools and methods to identify oxidized proteins along with specific sites of oxidative damage and the consequences of protein oxidation.

Sialylated Lewis X Antigen Bearing Glycoproteins in Human Plasma

Journal of Proteome Research. Nov, 2010  |  Pubmed ID: 20858014

Recent studies have shown that antibodies targeting Lewis x (Le(x)) antigen are a valuable tool in the isolation and identification of glycoproteins in plasma. A focus of this study was to determine whether sialylated Lewis x (sLe(x)) antigen carrying glycoproteins occur in human plasma and whether an antibody targeting this antigen could be used to isolate and identify glycoproteins bearing this antigen. An additional objective was to determine the degree to which proteins conjugated to Le(x) and sLe(x) antigens are similar in structure. A specific anti-sLe(x) antibody (anti-sLe(x)Ab), CHO-131, immobilized in an immunoaffinity column was used to select a set of specific sLe(x) bearing proteins from human plasma, after which they were identified by either of two analytical strategies. One approach was to further resolve the affinity selected proteins by reversed phase chromatography (RPC), tryptic digest the RPC fractions, and identify peptide fragments by MALDI-MS/MS. The second was to tryptic digest the affinity selected protein fraction, further resolve the tryptic fragments by RPC, and identify peptides from RPC fractions by MALDI-MS/MS. Histidine-rich glycoprotein, plasminogen, apolipoprotein A-I, vitronectin, proteoglycan-4, clusterin, Ig gamma-2 chain C region, Ig mu chain C region, and interalpha-trypsin inhibitor heavy chain H4 were found to change three folds or more in association with breast cancer. Fifty percent of the glycoproteins carrying either sLe(x) antigen from CHO-131 selection, Le(x) antigen from selection with TG-1 antibody, or both were found to be changed three folds or more in concentration in breast cancer plasma relative to controls.

Dynamics of Protein Damage in Yeast Frataxin Mutant Exposed to Oxidative Stress

Omics : a Journal of Integrative Biology. Dec, 2010  |  Pubmed ID: 20958246

Oxidative stress and protein carbonylation is implicated in aging and various diseases such as neurodegenerative disorders, diabetes, and cancer. Therefore, the accurate identification and quantification of protein carbonylation may lead to the discovery of new biomarkers. We have developed a new method that combines avidin affinity selection of carbonylated proteins with iTRAQ labeling and LC fractionation of intact proteins. This simple LC-based workflow is an effective technique to reduce sample complexity, minimize technical variation, and enable simultaneous quantification of four samples. This method was used to determine protein oxidation in an iron accumulating mutant of Saccharomyces cerevisiae exposed to oxidative stress. Overall, 31 proteins were identified with 99% peptide confidence, and of those, 27 proteins were quantified. Most of the identified proteins were associated with energy metabolism (32.3%), and cellular defense, transport, and folding (38.7%), suggesting a drop in energy production and reducing power of the cells due to the damage of glycolytic enzymes and decrease in activity of enzymes involved in protein protection and regeneration. In addition, the oxidation sites of seven proteins were identified and their estimated position also indicated a potential impact on the enzymatic activities. Predicted 3D structures of peroxiredoxin (TSA1) and thioredoxin II (TRX2) revealed close proximity of all oxidized amino acid residues to the protein active sites.

Coupling Protein Complex Analysis to Peptide Based Proteomics

Journal of Chromatography. A. Dec, 2010  |  Pubmed ID: 21036361

Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200 mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at -20°C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at -80°C while lowest recovery was achieved at 4°C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes.

A Large, Consistent Plasma Proteomics Data Set from Prospectively Collected Breast Cancer Patient and Healthy Volunteer Samples

Journal of Translational Medicine. 2011  |  Pubmed ID: 21619653

Variability of plasma sample collection and of proteomics technology platforms has been detrimental to generation of large proteomic profile datasets from human biospecimens.

Differential Carbonylation of Proteins As a Function of in Vivo Oxidative Stress

Journal of Proteome Research. Sep, 2011  |  Pubmed ID: 21800835

This study reports for the first time qualitative and quantitative differences in carbonylated proteins shed into blood as a function of increasing levels of OS. Carbonylated proteins in freshly drawn blood from pairs of diabetic and lean rats were derivatized with biotin hydrazide, dialyzed, and enriched with avidin affinity chromatography. Proteins thus selected were used in several ways. Differences between control and diabetic subjects in relative concentration of proteins was achieved by differential labeling of tryptic digests with iTRAQ reagents followed by reversed phase chromatography (RPC) and tandem mass spectrometry (MS/MS). Identification and characterization of OS induced post-translational modification sites in contrast was achieved by fractionation of affinity selected proteins before proteolysis and RPC-MS/MS. Relative quantification of peptides bearing oxidative modifications was achieved for the first time by selective reaction monitoring (SRM). Approximately 1.7% of the proteins in Zucker diabetic rat plasma were selected by the avidin affinity column as compared to 0.98% in lean animal plasma. Among the 35 proteins identified and quantified, Apo AII, clusterin, hemopexin precursor, and potassium voltage-gated channel subfamily H member 7 showed the most dramatic changes in concentration. Seventeen carbonylation sites were identified and quantified, 11 of which changed more than 2-fold in oxidation state. Three types of carbonylation were identified at these sites: direct oxidative cleavage from reactive oxygen species, glycation and addition of advanced glycation end products, and addition of lipid peroxidation products. Direct oxidation was the dominant form of carbonylation observed while hemoglobin and murinoglobulin 1 homologue were the most heavily oxidized proteins.

Oxidative Stress Induced Carbonylation in Human Plasma

Journal of Proteomics. Oct, 2011  |  Pubmed ID: 21856457

The focus of this study was on the assessment of technology that might be of clinical utility in identification, quantification, characterization of carbonylation in human plasma proteins. Carbonylation is widely associated with oxidative stress diseases. Breast cancer patient samples were chosen as a stress positive case based on the fact that oxidative stress has been reported to be elevated in this disease. Measurements of 8-isoprostane in plasma confirmed that breast cancer patients in this study were indeed experiencing significant oxidative stress. Carbonyl groups in proteins from freshly drawn blood were derivatized with biotin hydrazide after which the samples were dialyzed and the biotinylated proteins subsequently selected, digested and labeled with iTRAQ™ heavy isotope coding reagent(s). Four hundred sixty proteins were identified and quantified, 95 of which changed 1.5 fold or more in concentration. Beyond confirming the utility of the analytical method, association of protein carbonylation was examined as well. Nearly one fourth of the selected proteins were of cytoplasmic, nuclear, or membrane origin. Analysis of the data by unbiased knowledge assembly methods indicated the most likely disease associated with the proteins was breast neoplasm. Pathway analysis showed the proteins which changed in carbonylation were strongly associated with Brca1, the breast cancer type-1 susceptibility protein. Pathway analysis indicated the major molecular functions of these proteins are defense, immunity and nucleic acid binding.

Determining the Effects of Antioxidants on Oxidative Stress Induced Carbonylation of Proteins

Analytical Chemistry. Dec, 2011  |  Pubmed ID: 21939227

There is potential that the pathological effects of oxidative stress (OS) associated diseases such as diabetes could be ameliorated with antioxidants, but this will require a clearer understanding of the pathway(s) by which proteins are damaged by OS. This study reports the development and use of methods that assess the efficacy of dietary antioxidant supplementation at a mechanistic level. Data reported here evaluate the impact of green tea supplementation on oxidative stress induced post-translational modifications (OSi-PTMs) in plasma proteins of Zucker diabetic fatty (ZDF) rats. The mechanism of antioxidant protection was examined through both the type and amount of OSi-PTMs using mass spectrometry based identification and quantification. Carbonylated proteins in freshly drawn blood samples were derivatized with biotin hydrazide. Proteins thus biotinylated were selected from plasma samples of green tea fed diabetic rats and control animals by avidin affinity chromatography, further fractionated by reversed phase chromatography (RPC); fractions from the RPC column were tryptic digested, and the tryptic digest was fractionated by RPC before being identified by tandem mass spectrometry (MS/MS). Relative quantification of peptides bearing carbonylation sites was achieved for the first time by RPC-MS/MS using selective reaction monitoring (SRM). Seventeen carbonylated peptides were detected and quantified in both control and treated plasma. The relative concentration of eight was dramatically different between control and green tea treated animals. Seven of the OSi-PTM bearing peptides had dropped dramatically in concentration with treatment while one increased, indicating differential regulation of carbonylation by antioxidants. Green tea antioxidants were found to reduce carbonylation of proteins by lipid peroxidation end products most, followed by advanced glycation end products to a slightly lower extent. Direct oxidation of proteins by reactive oxygen species (ROS) was protected the least by green tea.

A Lectin Affinity Workflow Targeting Glycosite-specific, Cancer-related Carbohydrate Structures in Trypsin-digested Human Plasma

Analytical Biochemistry. Jan, 2011  |  Pubmed ID: 20705048

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.

Effect of Single Amino Acid Substitution on Oxidative Modifications of the Parkinson's Disease-Related Protein, DJ-1

Molecular & Cellular Proteomics : MCP. Feb, 2012  |  Pubmed ID: 22104028

Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H(2)O(2) resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD.

A Lectin Chromatography/mass Spectrometry Discovery Workflow Identifies Putative Biomarkers of Aggressive Breast Cancers

Journal of Proteome Research. Feb, 2012  |  Pubmed ID: 22309216

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n = 5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ~90% of which were components of the cell surface or extracellular matrix. We observed 1011 unique glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNAseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were upregulated in triple negative vs. luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.

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