Biomedical subjects

Fred E Regnier

Publications and source records attributed to Fred E Regnier.

At least 19 recordsLinked to original sources

PubMed2006

Neural network prediction of peptide separation in strong anion exchange chromatography.

MOTIVATION: 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. RESULTS: We developed a software package to predict the separation of peptides in strong anion exchange (SAX) chromatography using artificial neural network based pattern classification techniques. A multi-layer perceptron is used as a pattern classifier and it is designed with feature vectors extracted from the peptides so that the classification error is minimized. A genetic algorithm is employed to train the neural network. The developed system was tested using 14 protein digests, and the sensitivity analysis was carried out to investigate the significance of each feature. AVAILABILITY: The software and testing results can be downloaded from ftp://ftp.bbc.purdue.edu.

Algorithms↗

PubMed2006

A method for the identification of glycoproteins from human serum by a combination of lectin affinity chromatography along with anion exchange and Cu-IMAC selection of tryptic peptides.

This paper reports a method for identifying glycoproteins from human serum. Glycoproteins were selected with a concanavalin A (Con A) lectin column and then tryptically digested prior to sequential chromatographic selection of acidic and histidine containing peptides. Acidic peptides were selected with a strong anion exchange (SAX) column. Peptides captured by the SAX columns were then released and histidine-containing peptides in the mixture selected with a copper loaded immobilized metal affinity chromatography (Cu-IMAC) column. This serial chromatographic selection process reduced the complexity of proteolytic digests by more than an order of magnitude. Peptides selected by this serial process were then fractionated by reversed-phase chromatography (RPC) and identified by tandem mass spectrometry. The method was initially validated using human transferrin before application to human serum. The results show that all the peptides identified except one contained histidine and acidic amino acids.

Amino Acid Sequence↗

PubMed2006

Targeted glycoproteomics: serial lectin affinity chromatography in the selection of O-glycosylation sites on proteins from the human blood proteome.

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.

Chromatography, Affinity↗

PubMed2006

Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry.

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.

Amino Acids↗

PubMed2006

Primary amine coding as a path to comparative proteomics.

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.

Amines↗

PubMed2005

Comparative glycoproteomics of N-linked complex-type glycoforms containing sialic acid in human serum.

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.

Amino Acid Sequence↗

PubMed2005

Use of multidimensional lectin affinity chromatography in differential glycoproteomics.

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.

Amino Acid Sequence↗

PubMed2005

Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme.

The selectivity of immobilized metal affinity chromatography (IMAC) systems for the purification of phosphopeptides is poor. This is particularly a problem with tryptic digests of proteins where a large number of acidic peptides are produced that also bind during IMAC. The hypothesis examined in this work was that the selectivity of IMAC columns for phosphopeptides could be increased by using endoproteinase glu-C (glu-C) for protein digestion. Glu-C cleaves proteins at acidic residues and should reduce the number of acidic residues in peptides. This method was successfully applied to a mixture of model proteins and bovine milk. The percentage of phosphorylated peptides selected from proteolytic digests of the milk sample was increased from 40% with trypsin to 70% with glu-C. Additionally, this method was coupled with stable isotope coding methods to quantitatively compare the concentration of phosphoproteins between samples.

Amino Acid Sequence↗

PubMed2005

Quantification of phosphoproteins with global internal standard technology.

Global internal standard technology (GIST) is being developed for the quantification of all primary structure and post-translational variants of proteins in a proteome. This paper is directed at an analysis of phosphorylation, primarily of serine and threonine. Quantification was achieved by acylation of primary amino groups in peptide cleavage fragments of proteins with isotopically coded derivatizing agents. Peptides from controls were globally coded with an isotopically "light" form of the reagent while those from experimental samples were coded with a "heavy" form of the reagent. The two types coding reagents used in this work were N-hydroxyl succinimide derivatives of acetate and 4-trimethylammoniumbutyrate. Heavy isotope forms were produced by deuteration of methyl groups. Subsequent to coding and mixing, the two samples were passed through a Ga(III) immobilized metal affinity chromatography (IMAC) column and the selected peptide fraction was further resolved by reversed-phase chromatography (RPC) and analyzed by mass spectrometry (MS). Relative differences in phosphopeptide concentration between samples were derived from isotope ratio measurements of the peptide isoforms observed in mass spectra. The method was validated with model peptides.

Amino Acid Sequence↗

PubMed2005

An automated method for the analysis of stable isotope labeling data in proteomics.

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.

Algorithms↗

PubMed2005

Recent advancements in differential proteomics based on stable isotope coding.

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.

Amino Acids↗

PubMed2004

Benzoyl derivatization as a method to improve retention of hydrophilic peptides in tryptic peptide mapping.

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.

Amines↗

PubMed2004

Optimization of diagonal chromatography for recognizing post-translational modifications.

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.

Chromatography↗

PubMed2004

Enrichment of cysteine-containing peptides from tryptic digests using a quaternary amine tag.

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.

Amino Acid Sequence↗

PubMed2004

High-speed label-free detection by spinning-disk micro-interferometry.

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.

Biosensing Techniques↗

PubMed2004

Proteomic analysis of Arabidopsis glutathione S-transferases from benoxacor- and copper-treated seedlings.

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.

Arabidopsis↗

PubMed2004

Contributions of commercial sorbents to the selectivity in immobilized metal affinity chromatography with Cu(II).

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.

Absorption↗

PubMed2004

Potential of silica monolithic columns in peptide separations.

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.

Amino Acid Sequence↗