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1.
Statistical identification of differentially labeled peptides from liquid chromatography tandem mass spectrometry 总被引:1,自引:0,他引:1
LC-MS/MS with certain labeling techniques such as isotope-coded affinity tag (ICAT) enables quantitative analysis of paired protein samples. However, current identification and quantification of differentially expressed peptides (and proteins) are not reliable for large proteomics screening of complex biological samples. The number of replicates is often limited because of the high cost of experiments and the limited supply of samples. Traditionally, a simple fold change cutoff is used, which results in a high rate of false positives. Standard statistical methods such as the two-sample t-test are unreliable and severely underpowered due to high variability in LC-MS/MS data, especially when only a small number of replicates are available. Using an advanced error pooling technique, we propose a novel statistical method that can reliably identify differentially expressed proteins while maintaining a high sensitivity, particularly with a small number of replicates. The proposed method was applied both to an extensive simulation study and a proteomics comparison between microparticles (MPs) generated from platelet (platelet MPs) and MPs isolated from plasma (plasma MPs). In these studies, we show a significant improvement of our statistical analysis in the identification of proteins that are differentially expressed but not detected by other statistical methods. In particular, several important proteins - two peptides for beta-globin and three peptides for von Willebrand Factor (vWF) - were identified with very small false discovery rates (FDRs) by our method, while none was significant when other conventional methods were used. These proteins have been reported with their important roles in microparticles in human blood cells: vWF is a platelet and endothelial cell product that binds to P-selectin, GP1b, and GP IIb/IIIa, and beta-globin is one of the peptides of hemoglobin involved in the transportation of oxygen by red blood cells. 相似文献
2.
Nadeau OW Anderson DW Yang Q Artigues A Paschall JE Wyckoff GJ McClintock JL Carlson GM 《Journal of molecular biology》2007,365(5):1429-1445
Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft. 相似文献
3.
N R Goltermann 《Peptides》1982,3(5):733-737
The synthesis of the COOH-terminal octa- and tetrapeptides of cholecystokinin (CCK) has been studied in rat cerebral cortex after intraventricular administration of radioactive amino acids characteristic of the porcine COOH-terminal octapeptide of CCK, CCK-8. After immunosorption with a COOH-terminal directed antibody, cortical CCK was fractionated on Sephadex G-50 columns. The experiments demonstrated newly synthesized CCK forms which coeluted with porcine CCK-8 and CCK-4. Except for threonine the amino acids employed, methionine, tryptophan, aspartic acid, glycine and phenylalanine were incorporated. The sequence-specific radioimmunoassay, the incorporation of the employed labeled amino acids, and the elution pattern by gel filtration, suggest an almost identical structure of porcine and rat cortical CCK-8, and a concomitant synthesis of CCK-8 and CCK-4 in rat cerebral cortex. 相似文献
4.
A. P. Il’ina O. G. Kulikova D. I. Maltsev M. S. Krasnov E. Yu. Rybakova V. S. Skripnikova E. S. Kuznetsova A. K. Buryak V. P. Yamskova I. A. Yamskov 《Applied Biochemistry and Microbiology》2011,47(2):118-122
We performed the matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF) analysis of the peptides entering into the composition of not yet explored bioregulators derived from the extracellular
matrix of the tissues of the various organs of the mammals, and also plants and fungi. The study included 15 different mammalian
tissues, 13 species of plants, and 2 species of fungi. Exploring the bioregulators derived from eye tissues, we demonstrated
that their composition includes peptide components with the same values of the molecular weight. The composition of the bioregulators
derived from the tissues of various organs of mammals or different species of plants and fungi includes the peptides with
different values of molecular weight. Obtained data indicate the growing evidence of the assumptions about the major function
of the bioregulators of this group—their involvement in the regulation of tissue-organ homeostasis in the biological systems. 相似文献
5.
John D. Glass 《Enzyme and microbial technology》1981,3(1):2-8
The use of enzymes to catalyse peptide bond formation and for manipulating blocking groups during peptide synthesis is discussed. The history of solubility-controlled peptide condensations in the presence of proteolytic enzymes is traced. General techniques for obtaining improved yields of soluble condensation products are outlined along with special conditions which sometimes favour the enzymatic condensations of peptide fragments. Progress in the use of enzymes to manipulate blocking groups on α-amino groups, α-carboxyl groups, and on the sidechain functional groups of amino acid residues are examined. Some anticipated developments in the use of enzymes as reagents in peptide synthesis and semisynthesis are discussed. 相似文献
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Noncompetitive and competitive intermolecular deuterium isotope effects were measured for the cytochrome P-450 catalyzed hydroxylation of a series of selectively deuterated chlorobenzenes. An isotope effect of 1.27 accompanied the meta hydroxylation of chlorobenzene-2H5 as determined by two totally independent methods (EC-LC and GC-MS assays). All isotope effects associated with the meta hydroxylation of chlorobenzenes-3,5-2H2 and -2,4,6-2H3 were approximately 1.1. In contrast, competitive isotope studies on the ortho and para hydroxylation of chlorobenzenes-4-2H1, -3,5-2H2, and -2,4,6-2H3 resulted in significant inverse isotope effects (approximately 0.95) when deuterium was substituted at the site of oxidation whereas no isotope effect was observed for the oxidation of protio sites. These results eliminate initial epoxide formation and initial electron abstraction (charge transfer) as viable mechanisms for the cytochrome P-450 catalyzed hydroxylation of chlorobenzene. The results, however, can be explained by a mechanism in which an active triplet-like oxygen atom adds to the pi system in a manner analogous to that for olefin oxidation. The resulting tetrahedral intermediate can then rearrange to phenol directly or via epoxide or ketone intermediates. 相似文献
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10.
This paper introduces the genome annotating proteomic pipeline (GAPP), a totally automated publicly available software pipeline for the identification of peptides and proteins from human proteomic tandem mass spectrometry data. The pipeline takes as its input a series of MS/MS peak lists from a given experimental sample and produces a series of database entries corresponding to the peptides observed within the sample, along with related confidence scores. The pipeline is capable of finding any peptides expected, including those that cross intron-exon boundaries, and those due to single nucleotide polymorphisms (SNPs), alternate splicing, and post-translational modifications (PTMs). GAPP can therefore be used to re-annotate genomes, and this is supported through the inclusion of a Distributed Annotation System (DAS) server, which allows the peptides identified by the pipeline to be displayed in their genomic context within the Ensembl genome browser. GAPP is freely available via the web, at www. gapp.info. 相似文献
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12.
The increased interest in chemical cross-linking for probing protein structure and interaction has led to a large increase in literature describing new cross-linkers and search programs. However, this has not led to a corresponding increase in the analysis of large and complex proteins. A major obstacle is that the new cross-linkers are either not readily available and/or have a low reactivity. In combination with aging search programs that are slow and have low sensitivity, or new search programs that are described but not released, these efforts do little to advance the field of cross-linking. Here we present a method pipeline for chemical cross-linking, using two standard cross-linkers, BS3 and BS2G, combined with our freely available CrossWork search program. By this approach we generate cross-link data sufficient to derive structural information for large and complex proteins. CrossWork searches batches of tandem mass-spectrometric data, and identifies cross-linked and non-cross-linked peptides using a standard PC. We tested CrossWork by searching mass-spectrometric datasets of cross-linked complement factor C3 against small (1 protein) and large (1000 proteins) search spaces, and show that the resulting distance constraints agree with the established structures. We further investigated the structure of the multi-domain ERp72, and combined the individual domains of ERp72 into a single structure. 相似文献
13.
The production of bioactive peptides and small protein fragments is commonly achieved via solid-phase chemical synthesis. However, such techniques become unviable and prohibitively expensive when the peptides are large (e.g., >30 amino acids) or when isotope labeling is required for NMR studies. Expression and purification of large quantities of unfolded peptides in E. coli have also proved to be difficult even when the desired peptides are carried by fusion proteins such as GST. We have developed a peptide expression system that utilizes a novel fusion protein (SFC120) which is highly expressed and directs the peptides to inclusion bodies, thereby minimizing in-cell proteolysis whilst maintaining high yields of peptide expression. The expressed peptides can be liberated from the carrier protein by CNBr cleavage at engineered methionine sites or through proteolysis by specific proteases for peptides containing methionine residues. In the present systems, we use CNBr, due to the absence of methionine residues in the target peptides, although other cleavage sites can be easily inserted. We report the production of six unfolded protein fragments of different composition and lengths (19 to 48 residues) derived from the virulent effector kinases, Cla4 and Ste20 of Candida albicans. All six peptides were produced with high yields of purified material (30–40 mg/l in LB, 15–20 mg/l in M9 medium), pointing to the general applicability of this expression system for peptide production. The enrichment of these peptides with 15N, 15N/13C and even 15N/13C/2H isotopes is presented allowing speedy assignment of poorly-resolved resonances of flexible peptides. 相似文献
14.
Initial studies have shown that fish lens protein can be modified in vitro to acquire a specific affinity for selected molecules, e.g., human hemoglobins, and cells, e.g., human erythrocytes. These studies were extended by preparing lens reagents that can distinguish bovine serum albumin from ovalbumin, and major groups of human erythrocytes in the ABO system from each other. Lens reagents appear to be potentially useful tools for the identification of specific molecules, either in solution or on cell surfaces. 相似文献
15.
Phenolic content is a primary parameter for vegetables oil quality evaluation, and directly involved in the prevention of oxidation and oil preservation. Several methods have been reported in the literature for polyphenols extraction from seed oil but the approaches commonly used remain manually handled. In this work, we propose a rapid and sensitive method for seed oil polyphenols extraction and identification. For this purpose, polyphenols were extracted from Opuntia stricta Haw seed oil, using high frequency agitation, separated, and then identified using a liquid chromatography–high resolution mass spectrometry method. Our results showed good sensitivity and reproducibility of the developed methods. 相似文献
16.
Fujii N Jacobsen RB Wood NL Schoeniger JS Guy RK 《Bioorganic & medicinal chemistry letters》2004,14(2):427-429
A new approach to the determination of moderate resolution protein structures, termed MS3-D-Mass Spectrometry in 3 Dimensions-has recently been disclosed. The method involves the formation of covalent crosslinks between reactive residues on the protein surface, the determination of the location of those crosslinks in primary sequence space by mass spectrometry, and then the imposition of a distance constraint upon the location of the respective side chains during distance geometry calculations of protein structure. MS3-D is rapid, requires small amounts of protein, and works in native biochemical conditions. Therefore, it offers the potential for determination of the structures of all proteins expressed by an organism in a high throughput manner. However, the methodology is completely dependent upon the production of chemical crosslinks and technical limitations of available crosslinkers have proven problematic in generalization and automation of the method for the determination of the structures of complete proteomes. Presented herein is the design, synthesis, and proofing of a novel modular protein crosslinking reagent designed to enhance hydrophilicity, provide an increased effective signal to noise ratio for MS3-D, and allow the sampling of a wider variety of side chains during the process. 相似文献
17.
Electrospray ionization mass spectrometry is a powerful tool for identification of biomolecules such as peptides, proteins, oligosaccharides and neurotransmitters. Recent development of the nanospray techniques, applied at ultralow flow-rates, allowed a sensitive analysis of compounds at femto/attomolar level. Here, we present application of a novel nanospray device for the analysis and fragmentation of peptides with high sensitivity on a sector instrument. The lowest applied flow-rate of the mobile phase was maintained at 50 nl/min with a sample load of 90 fmol. Nanospray also provided a complete analysis of 500 nl of the sample for over 10 min, including sequencing of as little as 40 pmol of a substance. Such analysis provides full structural information necessary to identify the molecules. 相似文献
18.
Covalent methionylation of Escherichia coli methionyl-tRNA synthethase: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry.
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S. Gillet C. Hountondji J. M. Schmitter S. Blanquet 《Protein science : a publication of the Protein Society》1997,6(11):2426-2435
Methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by methionyl-tRNA synthetase (MetRS) is capable of reacting with this synthetase or other proteins, by forming an isopeptide bond with the epsilon-NH2 group of lysyl residues. It is proposed that the mechanism for the in vitro methionylation of MetRS might be accounted for by the in situ covalent reaction of methionyl-adenylate with lysine side chains surrounding the active center of the enzyme, as well as by exchange of the label between donor and acceptor proteins. Following the incorporation of 7.0 +/- 0.5 mol of methionine per mol of a monomeric truncated methionyl-tRNA synthetase species, the enzymic activities of [32P]PPi-ATP isotopic exchange and tRNA(Met) aminoacylation were lowered by 75% and more than 90%, respectively. The addition of tRNA(Met) protected the enzyme against inactivation and methionine incorporation. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-114, -132, -142 (or -147), -270, -282, -335, -362, -402, -439, -465, and -547 of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. These lysyl residues are distributed at the surface of the enzyme between three regions [114-150], [270-362], and [402-465], all of which were previously shown to be involved in catalysis or to be located in the binding sites of the three substrates, methionine, ATP, and tRNA. 相似文献
19.
Chacon A Masterson DS Yin H Liebler DC Porter NA 《Bioorganic & medicinal chemistry》2006,14(18):6213-6222
Although genome databases have become the key for proteomic analyses, de novo sequencing remains essential for the study of organisms whose genomes have not been completed. In addition, post-translational modifications present a challenge in database searching. Recognition of the b or y-ion series in a peptide MS/MS spectrum as well as identification of the b1 - and yn-1 -ions can facilitate de novo analyses. Therefore, it is valuable to identify either amino-acid terminus. In previous work, we have demonstrated that peptides modified at the epsilon-amino group of lysine as a t-butyl peroxycarbamate derivative undergo free radical promoted peptide backbone fragmentation under low-energy collision-induced dissociation (CID) conditions. Here we explore the chemistry of the N-terminal amino group modified as a t-butyl peroxycarbamate. The conversion of N-terminal amines to peroxycarbamates of simple amino acids and peptides was studied with aryl t-butyl peroxycarbonates. ESI-MS/MS analysis of the peroxycarbamate adducts gave evidence of a product ion corresponding to the neutral loss of the N-terminal side chain (R), thus identifying this residue. Further fragmentation (MS3) of product ions formed by N-terminal residue side-chain loss (-R) exhibited an m/z shift of the b-ions equal to the neutral loss of R, therefore labeling the b-ion series. The study was extended to the analysis of a protein tryptic digest where the SALSA algorithm was used to identify spectra containing these neutral losses. The method for N-terminus identification presented here has the potential for improvement of de novo analyses as well as in constraining peptide mass mapping database searches. 相似文献
20.
A method for the determination of the stoichiometry of protein complexes has been developed, which is based on proteolytic digestion of the complex, labeling with a fluorescent reagent, specific for amino or sulfhydryl groups, and separation by liquid chromatography with fluorescence and mass spectrometric detection. The intensity of the fluorescence signal of the labeled peptides resulting from different proteins is directly proportional to the stoichiometry of these proteins in the complex. The performance of the method was evaluated with standard peptides and proteins to ensure that accurate molar ratios can be obtained from the fluorescence chromatogram. Standard deviations of the measured molar ratio from the expected molar ratio were below 10% for both peptides and proteins. The method was finally employed for the determination of the stoichiometry of the 1:1 complex of sFc gamma RIII and hFc1. Using the described methodology, a stoichiometry of 1:1.1 was measured, which agrees well with a 1:1 complex. 相似文献