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At least 163 records · Page 9Linked to original sources
PubMed2006

Tandem parallel fragmentation of peptides for mass spectrometry.

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.

Dynorphins↗
PubMed2004

Immunoglobulin E reactivity of native Blo t 5, a major allergen of Blomia tropicalis.

BACKGROUND: Blo t 5 is a major allergen of Blomia tropicalis and its complementary DNA (cDNA) has been expressed in both prokaryotic and eukaryotic expression systems. Although the recombinant Blo t 5 has been well characterized, relatively less is known about its native counterparts and the allergenicity comparison of the native and recombinant Blo t 5 allergens has not been reported. OBJECTIVE: The aims of this study are to characterize the native counterpart of Blo t 5, and compare the allergenicity of native and recombinant Blo t 5 by in vivo and in vitro assays. METHODS: Native Blo t 5 were purified by immuno-affinity chromatography and characterized by proteomic means. The allergenicity of the allergen was evaluated by skin prick tests, human IgE ELISA, ELISA inhibition and histamine release assays. RESULTS: Native Blo t 5 consists of at least five distinct isoforms, ranging from pI 3 to 5.5. Allergenicity assessment of recombinant and native Blo t 5 based on skin reaction, IgE-binding capacity and histamine release in allergic individuals indicated that there was a good correlation between both forms of Blo t 5 in general. However, data from IgE ELISA inhibition assay revealed the presence of additional unique IgE epitopes in native Blo t 5. CONCLUSIONS: At least five distinct isoforms of Blo t 5 have been identified. Comparative assessment of native and recombinant Blo t 5 revealed that the allergenicity of these two forms was similar but not completely identical suggesting that the various isoforms of native Blo t 5 may exhibit additional unique IgE epitopes.

Adolescent↗
PubMed2003

Qualitative and quantitative proteomics by two-dimensional gel electrophoresis, peptide mass fingerprint and a chemically-coded affinity tag (CCAT).

The chemically-coded affinity tag (CCAT) method combines standard electrophoresis protocols with MALDI-TOF-MS analysis to identify and quantify protein abundances in complex samples in one step. This method is designed to fit into the workflow of SDS-PAGE or two-dimensional electrophoresis (2-DE) only requiring basic proteome laboratory equipment. Prior to electrophoresis two protein samples are separately labelled with a heavy or a light version of the CCAT reagent via reduced cysteines in the proteins. Equal amounts are then combined and electrophoretically separated. Proteins can then be excised from the gel to obtain their peptide mass fingerprint by mass spectrometry. This fingerprint enabled not only identification, but also quantification by comparing relative peak intensities of CCAT-labelled peptides. In this article, we display how the CCAT method can be used to analyse two protein samples in one gel and that the peak intensities of labelled peptides reflect the abundance of a protein in it.

Affinity Labels↗
PubMed2005

Application of metal-chelate affinity chromatography to the study of the phosphoproteome.

With the increasing importance of proteome analysis, studying the phosphoproteome is a priority for functional studies. Therefore, a rational approach to simplifying the proteome is needed. In this work, we examined the use of immobilized metal affinity chromatography (IMAC) using ferric ions-chelated column for enriching crude cell extracts in phosphoproteins. The adsorption of the proteins on Fe(3+) was obtained at an acidic pH 5.6, and their elution at a more basic pH in Tris buffer. To evaluate the separation, western blots were performed with either anti-phosphotyrosine or anti-phosphoserine/threonine. The analysis of the eluates demonstrated the selectivity of the separation, particularly for proteins phosphorylated on serine or threonine. In conclusion, the advantages and the limits of this approach are discussed.

Blotting, Western↗
PubMed2005

Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli.

Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0 to 99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mgL(-1) culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was subsequently used to study three scFv variants engineered to determine structure-function relationships.

Cloning, Molecular↗
PubMed2006

Isotope-coded affinity tags for protein quantification.

An important goal in proteomics is to compare the relative amounts of different proteins in biological samples and to try to correlate these differences with changes in physiological state. The isotopecoded affinity tag technique pioneered in Aebersold's laboratory takes advantage of differential tagging of cysteine residues in proteins with stable isotopes to significantly reduce the complexity of peptide mixtures and increase the number of sequences that are identified in a single tandem mass spectrometry experiment. In this approach, two samples are isotopically labeled (one heavy, one light) through a reactive group that specifically binds to cysteine residues; the samples are combined, separated with chromatography, and analyzed by mass spectrometry. The results are then database searched and a list of hundreds of proteins and their heavy:light ratio is obtained.

Avidin↗
PubMed2001

Automated signature peptide approach for proteomics.

This paper addresses the issue of automating the multidimensional chromatographic, signature peptide approach to proteomics. Peptides were automatically reduced and alkylated in the autosampler of the instrument. Trypsin digestion of all proteins in the sample was then executed on an immobilized enzyme column and the digest directly transferred to an affinity chromatography column. Although a wide variety of affinity columns may be used, the specific column used in this case was a Ga(III) loaded immobilized metal affinity chromatography (IMAC) column. Ga(III)-IMAC is known to select phosphorylated peptides. Phosphorylated peptides selected by the affinity column from tryptic digests of milk were automatically transferred to a reversed-phase liquid chromatography (RPLC) column. Further fractionation of tryptic peptides on the RPLC column was achieved with linear solvent gradient elution. Effluent from the RPLC column was electrosprayed into a time-of-flight mass spectrometer. The entire process was controlled by software in the liquid chromatograph. With slight modification, it is possible to add multiple columns in parallel at any of the single column positions to further increase throughput. Total analysis time in the tandem column mode of operation was under 2 h.

Automation↗
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

Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem-loop-binding proteins that contributes to high-affinity RNA binding.

The stem-loop-binding protein (SLBP) is involved in multiple aspects of histone mRNA metabolism. To characterize the modification status and sites of SLBP, we combined mass spectrometric bottom-up (analysis of peptides) and top-down (analysis of intact proteins) proteomic approaches. Drosophilia SLBP is heavily phosphorylated, containing up to seven phosphoryl groups. Accurate M(r) determination by Fourier transform ion cyclotron resonance (FTICR)-MS and FTICR-MS top-down experiments using a variety of dissociation techniques show there is removal of the initiator methionine and acetylation of the N terminus in the baculovirus-expressed protein, and that T230 is stoichiometrically phosphorylated. T230 is highly conserved; we have determined that this site is also completely phosphorylated in baculovirus-expressed mammalian SLBP and extensively phosphorylated in both Drosophila and mammalian cultured cells. Removal of the phosphoryl group from T230 by either dephosphorylation or mutation results in a 7-fold reduction in the affinity of SLBP for the stem-loop RNA.

Amino Acid Motifs↗
PubMed2003

Peptidomics: A new approach to affinity protein microarrays.

Protein microarrays for diagnostic and proteomic analyses are being developed using a number of different techniques for each of the steps required including immobilisation methods, assay and detection systems. This is extremely different to the development of DNA microarrays which is now a well established technology that has demonstrated the capabilities of transcriptomics to deliver validated differential transcripts. As mRNA and protein levels do not always correlate, protein microarrays would seem to be an obvious successor to DNA arrays. Unlike nucleic acids, however, protein targets are typically nonhomogeneous in physicochemical properties and affinity capture agents are often poorly characterised making the experiments difficult to perfect and reproduce. Moreover, running multiple affinity assays in parallel (multiplexing) is compromised by the heterogeneity of antibody affinities to their protein targets. In the peptidomic approach presented here the assayed mixture of proteins is enzymatically digested prior to affinity capture to form a mixture of short peptides that are more similar in their physicochemical properties than intact proteins. These peptides can be predicted by in silico digestion of individual proteins, e.g. from protein databases allowing design of nonhomologous reagents for the screening of affinity agent libraries. The use of mass spectrometry (e.g. matrix-assisted laser desorption/ionization-time of flight mass spectrometry) for a direct confirmation of the identity of the species captured, provides a further advantage compared to the more usual method of detection in which fluorescently labelled captured species are scanned to give a spatially resolved image of the array.

Peptides↗
PubMed2004

Proteomic analysis of carbonylated proteins in two-dimensional gel electrophoresis using avidin-fluorescein affinity staining.

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.

Animals↗
PubMed2007

Quantitative proteomics by stable isotope labeling and mass spectrometry.

The goal of quantitative proteomics is to systematically study static state or perturbation-induced changes in protein profile. Most of the recently developed mass spectrometry (MS)-based quantitative proteomic methods employ stable isotope labeling to introduce signature mass tags to peptides/proteins that can be used by a mass spectrometer to quantify each analyte and to determine the sample from which it originates. In this chapter, we discuss several methods for the introduction of mass tags to proteins and peptides for MS-based quantitative proteomic analysis, including isotope-coded affinity tags, stable isotope labeling by amino acids in cell culture, global internal standard technology, and mass-coded abundance tagging.

Isotope Labeling↗
PubMed2000

Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes.

Ubiquitin-dependent proteolysis is catalyzed by the 26S proteasome, a dynamic complex of 32 different proteins whose mode of assembly and mechanism of action are poorly understood, in part due to the difficulties encountered in purifying the intact complex. Here we describe a one-step affinity method for purifying intact 26S proteasomes, 19S regulatory caps, and 20S core particles from budding yeast cells. Affinity-purified 26S proteasomes hydrolyze both model peptides and the ubiquitinated Cdk inhibitor Sic1. Affinity purifications performed in the absence of ATP or presence of the poorly hydrolyzable analog ATP-gamma-S unexpectedly revealed that a large number of proteins, including subunits of the skp1-cullin-F-box protein ligase (SCF) and anaphase-promoting complex (APC) ubiquitin ligases, copurify with the 19S cap. To identify these proteasome-interacting proteins, we used a recently developed method that enables the direct analysis of the composition of large protein complexes (DALPC) by mass spectrometry. Using DALPC, we identified more than 24 putative proteasome-interacting proteins, including Ylr421c (Daq1), which we demonstrate to be a new subunit of the budding yeast 19S cap, and Ygr232w (Nas6), which is homologous to a subunit of the mammalian 19S cap (PA700 complex). Additional PIPs include the heat shock proteins Hsp70 and Hsp82, the deubiquitinating enzyme Ubp6, and proteins involved in transcriptional control, mitosis, tubulin assembly, RNA metabolism, and signal transduction. Our data demonstrate that nucleotide hydrolysis modulates the association of many proteins with the 26S proteasome, and validate DALPC as a powerful tool for rapidly identifying stoichiometric and substoichiometric components of large protein assemblies.

Adenosine Triphosphate↗
PubMed2003

A comparative analysis of an orthologous proteomic environment in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The sequential application of protein tagging, affinity purification, and mass spectrometry enables highly accurate charting of proteomic environments by the characterization of stable protein assemblies and the identification of subunits that are shared between two or more protein complexes, termed here "proteomic hyperlinks." We have charted the proteomic environments surrounding the histone methyltransferase, Set1, in both yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Although the composition of these nonessential Set1 complexes is remarkably conserved, they differ with respect to their hyperlinks to their proteomic environments. We speculate that conservation of the core components of protein assemblies and variability of hyperlinks represents a general principle in the molecular organization of eukaryotic proteomes.

DNA-Binding Proteins↗
PubMed2002

Low-energy collision-induced dissociation fragmentation analysis of cysteinyl-modified peptides.

The development of methods to chemically modify and isolate cysteinyl-residue-containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotope-coded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolate-specific biotin group to modify and isolate Cys-containing peptides in conjunction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of Deinococcus radiodurans, the presence of these label-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys residue, and to differentiate identical Cys-peptides labeled with either ICAT-d0 or ICAT-d8.

Animals↗
PubMed2005

Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.

BACKGROUND & AIMS: Pancreatic cancer is a highly lethal disease that has seen little headway in diagnosis and treatment for the past few decades. The effective treatment of pancreatic cancer is critically relying on the diagnosis of the disease at an early stage, which still remains challenging. New experimental approaches, such as quantitative proteomics, have shown great potential for the study of cancer and have opened new opportunities to investigate crucial events underlying pancreatic tumorigenesis and to exploit this knowledge for early detection and better intervention. METHODS: To systematically study protein expression in pancreatic cancer, we used isotope-coded affinity tag technology and tandem mass spectrometry to perform quantitative proteomic profiling of pancreatic cancer tissues and normal pancreas. RESULTS: A total of 656 proteins were identified and quantified in 2 pancreatic cancer samples, of which 151 were differentially expressed in cancer by at least 2-fold. This study revealed numerous proteins that are newly discovered to be associated with pancreatic cancer, providing candidates for future early diagnosis biomarkers and targets for therapy. Several differentially expressed proteins were further validated by tissue microarray immunohistochemistry. Many of the differentially expressed proteins identified are involved in protein-driven interactions between the ductal epithelium and the extracellular matrix that orchestrate tumor growth, migration, angiogenesis, invasion, metastasis, and immunologic escape. CONCLUSIONS: Our study is the first application of isotope-coded affinity tag technology for proteomic analysis of human cancer tissue and has shown the value of this technology in identifying differentially expressed proteins in cancer.

Humans↗
PubMed2004

Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium.

Prostate cancer is unusual among neoplasms in that it may be diagnosed at a curable stage through detection of a protein in serum, the serine protease prostate-specific antigen (PSA). PSA is secreted by both normal and neoplastic prostate epithelial cells in response to androgenic hormones and has found widespread use in cancer screening. Because PSA screening is controversial due to sensitivity and specificity issues, efforts continue to focus on the identification and characterization of additional markers that may be used for diagnostic and therapeutic purposes. In this study, we report the application of quantitative proteomic techniques that incorporate isotope coded affinity tag reagents and tandem mass spectrometry to comprehensively identify secreted and cell surface proteins from neoplastic prostate epithelium. LNCaP cells, a prostate tumor-derived cell line that secretes PSA in response to androgen exposure, were grown in a low protein-defined media under androgen-stimulated (A+) and -starved (A-) conditions. Proteomic analysis of the media identified in excess of 600 proteins, 524 of which could be quantified. Nine percent of the proteins had A+/A- ratios > 2.0, including PSA, and 2.5% had ratios < 0.5. A subset of these androgen-regulated proteins appeared to be expressed in abundance. Of these, selected mass spectrometry observations were confirmed by Western analysis. The findings suggest that androgen-mediated release of proteins may occur through the activation of proteolytic enzymes rather than exclusively through transcriptional or translational control mechanisms. On the basis of their known functional roles, several of the abundant androgen-regulated proteins may participate in the progression of neoplastic epithelial cell growth and should be considered as potential serum markers of neoplastic prostate diseases.

Biomarkers, Tumor↗