共查询到20条相似文献,搜索用时 9 毫秒
1.
The rate of false positive sequence matches of peptides profiled by MALDI MS and identified by MS/MS
Stoop MP Lamers RJ Burgers PC Sillevis Smitt PA Hintzen RQ Luider TM 《Journal of proteome research》2008,7(11):4841-4847
In most MALDI peptide profiling cases, sequencing is required to identify peptides of interest, preferentially by using different mass spectrometry techniques. Using identical samples, we determined the number of false positive matches in sequence of peptide identification using different mass spectrometers. This paper demonstrates that the reliability of the identification phase greatly benefits from concerted MS-technologies and determines the influence of mass accuracy, signal-to-noise and statistical score on peptide identification. 相似文献
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Pan C Ye M Liu Y Feng S Jiang X Han G Zhu J Zou H 《Journal of proteome research》2006,5(11):3114-3124
Fe3+-immobilized mesoporous molecular sieves MCM-41 with particle size of ca. 600 nm and pore size of ca. 3 nm is synthesized and applied to selectively trap and separate phosphopeptides from tryptic digest of proteins. For the capture of phosphopeptides, typically 10 microL of tryptic digest solution was first diluted to 1 mL by solution of ACN/0.1% TFA (50:50, v/v) and incubated with 10 microL of 0.1% acetic acid dispersed Fe3+-immobilized MCM-41 for 1 h under vibration. Fe3+-immobilized MCM-41 with trapped phosphopeptides was separated by centrifugation. The deposition was first washed with a volume of 300 microL of solution containing 100 mM NaCl in ACN/0.1% TFA (50:50, v/v) and followed by a volume of 300 microL of solution of 0.1% acetic acid to remove nonspecifically bound peptides. The nanoparticles with trapped phosphopeptides are mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) and deposited onto the target for analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). It was found that phosphopeptides from tryptic digest of alpha-casein and beta-casein are effectively and specifically trapped on Fe3+-immobilized MCM-41 with few peptides nonspecifically adsorbed. After the extraction by Fe3+-immobilized MCM-41, the suppression to the detection of phosphopeptides caused by abundant nonphosphopeptides from tryptic digest is effectively eliminated, and the detection of phosphopeptides by MALDI is greatly enhanced with the value of signal-to-noise (S/N) increased by more than an order of magnitude. It is demonstrated that the mechanism of the adsorption of phosphopeptides on Fe3+-immobilized MCM-41 is based on the interaction between the Fe3+ and the phosphate group. Finally, Fe3+-immobilized MCM-41 is applied to extract phosphopeptides from tryptic digest of the lysate of mouse liver for phosphoproteome analysis by nano-LC-MS/MS. 相似文献
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Janice L. Joss Mark P. Molloy Lyn A. Hinds Elizabeth M. Deane 《International journal of peptide research and therapeutics》2006,12(3):225-235
In proteomic studies, assigning protein identity from organisms whose genomes are yet to be completely sequenced remains a challenging task. For these organisms, protein identification is typically based on cross species matching of amino acid sequence obtained from collision induced dissociation (CID) of peptides using mass spectrometry. The most direct approach of de novo sequencing is slow and often difficult, due to the complexity of the resultant CID spectra. For MALDI-MS, this problem has been addressed by using chemical derivatisation to direct peptide fragmentation, thereby simplifying CID spectra and facilitating de novo interpretation. In this study, milk whey proteins from the tammar wallaby (Macropus eugenii) were used to evaluate three chemical derivatisation methods compatible with MALDI MS/MS. These methods included (i) guanidination and sulfonation using chemically-assisted fragmentation (CAF), (ii) guanidination and sulfonation using 4-sulfophenyl isothiocyanate (SPITC) and (iii) derivatising the epsilon-amino group of lysine residues with Lys Tag 4H. Derivatisation with CAF and SPITC resulted in more protein identification than Lys Tag 4H. Sulfonation using SPITC was the preferred method due to the low cost per experiment, the reactivity with both lysine and arginine terminated peptides and the resultant simplified MS/MS spectra.*Australian Peptide Conference Issue.**This project was funded by an ARC Linkage grant to Deane supported by TGR Biosciences and facilitated by access to the Australian Proteome Analysis Facility established under the Australian Government’s Major National Research Facilities program. 相似文献
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Anna Olejnik Izabela Nowak Krystian Eitner Grzegorz Schroeder 《International journal of peptide research and therapeutics》2013,19(3):217-224
The hexapeptide ALA-ASP-LEU-LYS-PRO-THR is currently widely used as an active ingredient in commercially available creams. Therefore, the proper analytical procedure to detect this compound is a very important issue. This paper presents the determination of the hexapeptide in solution and in cosmetic formulations by matrix assisted laser desorption ionisation mass spectrometry. We developed a procedure for the selective binding of the low molecular weight peptide by using a two-component matrix which enabled us to characterize the hexapeptide directly without any initial processing. Furthermore, the extensive computer simulations were carried out to assist in this analysis of the MS spectra. 相似文献
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Phosphopeptides can be identified by ion spray mass spectrometry. The method was tested with phosphokemptide and with a proteolytic digest of one subunit (delta-subunit) of the nicotinic acetylcholine receptor. In the latter one peptide containing tyrosine phosphate, one with two serine phosphates, and two different peptides each containing one serine phosphate were unambiguously identified. Thus it is proven that ion spray mass spectrometry can be applied for the localization of phosphorylation sites in a known primary structure. 相似文献
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Oxidation processes of lipids are of paramount interest from many viewpoints. For instance, oxidation processes are highly important under in vivo conditions because molecules with regulatory functions are generated by oxidation of lipids or free fatty acids. Additionally, many inflammatory diseases are accompanied by lipid oxidation and, therefore, oxidation products are also useful disease (bio)markers. Thus, there is also considerable interest in methods of (oxidized) lipid analysis.Nowadays, soft ionization mass spectrometric (MS) methods are regularly used to study oxidative lipid modifications due to their high sensitivities and the extreme mass resolution. Although electrospray ionization (ESI) MS is so far most popular, applications of matrix-assisted laser desorption and ionization (MALDI) MS are increasing. This review aims to summarize the so far available data on MALDI analyses of oxidized lipids. In addition to model systems, special attention will be paid to the monitoring of oxidized lipids under in vivo conditions, particularly the oxidation of (human) lipoproteins. It is not the aim of this review to praise MALDI as the “best” method but to provide a critical survey of the advantages and drawbacks of this method. 相似文献
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Identification and analysis of phosphopeptides 总被引:1,自引:0,他引:1
Chalmers MJ Kolch W Emmett MR Marshall AG Mischak H 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,803(1):111-120
Reversible phosphorylation of serine, threonine and tyrosine residues in proteins is one of the key events in signal transduction. To understand the process of signal transduction on a molecular level, it is imperative to identify phosphorylation sites in proteins. In this review, we offer an overview of the different methods/technologies currently available to identify protein phosphorylation sites. 相似文献
9.
Valentina Bonetto Ann-Charlotte Bergman Hans Jörnvall Rannar Sillard 《Journal of Protein Chemistry》1997,16(5):371-374
Peptides, cleaved by a mixture of carboxypeptidases CPP and CPY, can be detected by MALDI MS and the amino acid sequence thereby determined by calculation of the differences between consecutive peaks. In the present study we have used derivatizations of Lys and Cys to facilitate identification of these residues. Since the mass values do not readily distinguish Lys from Gln, we have converted Lys to homoarginine by guanidination, allowing simple detection of Lys. To identify the Cys positions in peptides that contain cystine, cysteic acid, or carboxymethylcysteine is not possible using CPY and CPP because of the lack of proteolytic cleavage. Instead we find that identification of Cys residues within the sequence can be achieved after conversion to a basic derivative, 4-thialaminine (Thi), by trimethylaminoethylation. 相似文献
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Baogang J. Xu Jiaqing Li R. Daniel Beauchamp Yu Shyr Ming Li M. Kay Washington Timothy J. Yeatman Robert H. Whitehead Robert J. Coffey Richard M. Caprioli 《Molecular & cellular proteomics : MCP》2009,8(5):936-945
Obtaining protein profiles from a homogeneous cell population in tissues can significantly improve our capability in protein biomarker discovery. In this study, unique protein profiles from the top and bottom sections of mouse crypts and ApcMin+/− adenomas were obtained using laser capture microdissection (LCM) combined with MALDI MS. Statistically significant protein peaks with differential expression were selected, and a set of novel protein biomarkers were identified. Immunohistochemistry was performed to confirm the differentially expressed protein biomarkers found by LCM combined with MALDI MS. To validate the relevance of the findings in human colorectal cancer (CRC), S100A8 was further confirmed in human CRC using immunohistochemistry. In addition, S100A8 was found to have an increased expression at different human CRC stages (Duke''s A–D) compared with controls at both protein (n = 168 cases) and RNA (n = 215 cases) levels. Overall LCM combined with MALDI MS is a promising method to identify intestinal protein biomarkers from minute amounts of tissue. The novel protein biomarkers identified from the top and bottom crypts will increase our knowledge of the specific protein changes taking place during cell migration from the crypt bottom to top. In addition, the identified cancer protein biomarkers will aid in the exploration of colorectal tumorigenesis mechanisms as well as in the advancement of molecularly based diagnosis of colorectal cancer.Obtaining protein profiles from a pure cell population can significantly improve our capability in protein biomarker discovery. Laser capture microdissection (LCM)1 is an important tool for acquisition of specific cells of interest from heterogeneous tissue sections (1,2). Obtaining protein profiles from laser-microdissected cells has been explored using multidimensional liquid chromatography-mass spectrometry or gel electrophoresis (3–5). However, usually ∼30,000 cells are required for these approaches (6–8). Direct analysis of laser-microdissected cells using MALDI MS is a sensitive, accurate, and high throughput technique to obtain protein profiles from limited numbers of cells (∼100 cells) (9–11). Combining LCM and MALDI MS is a promising tool for discovery of protein biomarkers from minute tissue structures.Individuals with advanced colorectal cancer (CRC) continue to have a poor prognosis despite recent improvements in treatment. As a result, early diagnosis and improved understanding of the pathogenesis of CRC are vital for improved clinical outcomes. Identification of early CRC protein markers has the potential to result in earlier diagnosis, more accurate prognosis, and improved treatment for individuals with CRC. Colorectal neoplasia is believed to arise from the colonic crypt (12,13). The colon is a self-renewing epithelium that consists of an actively dividing, relatively undifferentiated crypt base and a non-dividing, differentiated surface compartment (14–16). Small numbers of stem cells reside in the base of the crypt where daughter cells differentiate into absorptive cells, goblet cells, tuft cells, and endocrine cells. Absorptive cells and goblet cells, in particular, migrate to the luminal surface where they are eventually shed into the colonic lumen. Because the top and bottom crypts contain cells at different stages of differentiation, the protein profiles of these cells are likely to be different.Adenomatous polyps originate from crypts and represent important precursor lesions in the adenoma-carcinoma sequence (12). The ApcMin+/− mouse model, and other similar models based on truncating mutations in Apc, have been widely used to study intestinal neoplasia. This model contains a germ line mutation in the adenomatous polyposis coli (Apc) gene (17), leading to numerous adenomas in the intestine (18). In humans, loss of APC is often an early event in the cascade of genetic mutations that lead to colorectal neoplasia (19). Approximately 80% of all sporadic human CRC have inactivating mutations in the APC gene (20). Similarly human familial adenomatous polyposis patients have germ line mutations in the APC gene, and loss of heterozygosity leads to the development of hundreds of adenomatous polyps in the colon and rectum. Thus, the ApcMin+/− mouse is a promising mouse model for the discovery of potential early CRC markers using a proteomics approach.As a discovery study, we aimed to identify a set of proteomic profiles that can differentiate murine top and bottom crypts and ApcMin+/− adenomas using LCM combined with MALDI MS. Specific protein biomarkers were identified using tandem mass spectrometry. Finally the relevance of the identified protein biomarkers was examined in human CRC at both protein and mRNA levels. 相似文献
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The identification of proteins separated on two-dimensional gels is most commonly performed by trypsin digestion and subsequent matrix-assisted laser desorption ionization (MALDI) with time-of-flight (TOF). Recently, atmospheric pressure (AP) MALDI coupled to an ion trap (IT) has emerged as a convenient method to obtain tandem mass spectra (MS/MS) from samples on MALDI target plates. In the present work, we investigated the feasibility of using the two methodologies in line as a standard method for protein identification. In this setup, the high mass accuracy MALDI-TOF spectra are used to calibrate the peptide precursor masses in the lower mass accuracy AP-MALDI-IT MS/MS spectra. Several software tools were developed to automate the analysis process. Two sets of MALDI samples, consisting of 142 and 421 gel spots, respectively, were analyzed in a highly automated manner. In the first set, the protein identification rate increased from 61% for MALDI-TOF only to 85% for MALDI-TOF combined with AP-MALDI-IT. In the second data set the increase in protein identification rate was from 44% to 58%. AP-MALDI-IT MS/MS spectra were in general less effective than the MALDI-TOF spectra for protein identification, but the combination of the two methods clearly enhanced the confidence in protein identification. 相似文献
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In this study, we used nanocomposite magnetic particles coated with alumina as the affinity probes to selectively concentrate phosphorylated peptides and proteins from a low volume of sample solution. Tryptic digest products of phosphoproteins including alpha and beta-caseins, human protein phosphatase inhibitor 1, nonfat milk, egg white, and a cell lysate were used as the samples to demonstrate the feasibility of this approach. In only 30 and 90 s, phosphopeptides and phosphoproteins sufficient for characterization by MALDI-MS were enriched by the particles, respectively. Proteins trapped on the particles could be directly digested on the particles. The same particles in the digest solution were employed for enrichment of phosphopeptides. We estimated the required time for performing the enrichment of phosphopeptides from complex samples and characterization by MALDI MS was within 5 min. A small volume (50 microL) and a low concentration (5 x 10(-10) M) of tryptic digest product of a phosphoprotein sample could be dramatically enriched and characterized using this approach. 相似文献
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H. Gasser W. Strohmaier G. Schlag E.R. Schmid G. Allmaier 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1991,562(1-2)
The relative distribution of intact diacylphosphatidylcholine species isolated from the lung lavage fluid of rabbits has been investigated by positive ion fast-atom bombardment (FAB) mass spectrometry. Two different isolation/purification methods were applied and evaluated prior to mass spectrometric analysis. The first method consisted of a Bligh and Dyer extraction of the lung lavage fluid followed by isocratic high-performance liquid chromatographic (HPLC) separation. In the second method a thin-layer chromatographic purification step was introduced between the extraction procedure and the HPLC separation. Further, the FAB matrices glycerol and 3-nitrobenzyl alcohol were used, and their influence on the diacylphosphatidylcholine molecular ion species was studied. The Bligh and Dyer extraction followed by the simple HPLC separation was the method of choice to obtain stable, long-lasting protonated molecular ions and diagnostic fragment ions, which permitted the identification of the polar head-group. In combination with 3-nitrobenzyl alcohol as liquid matrix we established a procedure that yielded a fast sample preparation method, a good signal-to-noise ratio for detecting minor species, and reduced formation of [M + H − 2H]+ ion species. The relative fatty acid composition of the diacylphosphatidylcholine fractions isolated from rabbit lung lavage fluid was determined by negative ion FAB mass spectrometry using the carboxylate anions. The mass spectrometric results were compared with those acquired by gas chromatographic determination of the fatty acid methyl esters. Close agreement was found between the data obtained by the two independent methods. 相似文献
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Differentiating proteomic biomarkers in breast cancer by laser capture microdissection and MALDI MS 总被引:1,自引:0,他引:1
Sanders ME Dias EC Xu BJ Mobley JA Billheimer D Roder H Grigorieva J Dowsett M Arteaga CL Caprioli RM 《Journal of proteome research》2008,7(4):1500-1507
We assessed proteomic patterns in breast cancer using MALDI MS and laser capture microdissected cells. Protein and peptide expression in invasive mammary carcinoma versus normal mammary epithelium and estrogen-receptor positive versus estrogen-receptor negative tumors were compared. Biomarker candidates were identified by statistical analysis and classifiers were developed and validated in blinded test sets. Several of the m/ z features used in the classifiers were identified by LC-MS/MS and two were confirmed by immunohistochemistry. 相似文献
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Yu LR Zhu Z Chan KC Issaq HJ Dimitrov DS Veenstra TD 《Journal of proteome research》2007,6(11):4150-4162
Enrichment is essential for phosphoproteome analysis because phosphorylated proteins are usually present in cells in low abundance. Recently, titanium dioxide (TiO2) has been demonstrated to enrich phosphopeptides from simple peptide mixtures with high specificity; however, the technology has not been optimized. In the present study, significant non-specific bindings were observed when proteome samples were applied to TiO2 columns. Column wash with an NH4Glu solution after loading peptide mixtures significantly increased the efficiency of TiO2 phosphopeptide enrichment with a recovery of up to 84%. Also, for proteome samples, more than a 2-fold increase in unique phosphopeptide identifications has been achieved. The use of NH4Glu for a TiO2 column wash does not significantly reduce the phosphopeptide recovery. A total of 858 phosphopeptides corresponding to 1034 distinct phosphosites has been identified from HeLa cells using the improved TiO2 enrichment procedure in combination with data-dependent neutral loss nano-RPLC-MS2-MS3 analysis. While 41 and 35% of the phosphopeptides were identified only by MS2 and MS3, respectively, 24% was identified by both MS2 and MS3. Cross-validation of the phosphopeptide assignment by MS2 and MS3 scans resulted in the highest confidence in identification (99.5%). Many phosphosites identified in this study appear to be novel, including sites from antigen Ki-67, nucleolar phosphoprotein p130, and Treacle protein. The study also indicates that evaluation of confidence levels for phosphopeptide identification via the reversed sequence database searching strategy might underestimate the false positive rate. 相似文献
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Identification of negative and positive regulatory elements in the human renin gene 总被引:10,自引:0,他引:10
Renin gene expression is tissue-specific and under complex hormonal control. To investigate which DNA elements are involved in the control of human renin gene expression, we performed transient DNA transfer experiments with renin-chloramphenicol acetyltransferase fusions. We have mapped a complex arrangement of positive and negative control sequences in the 5' flanking region of the human renin gene. One positive control element is active in either orientation and defines a renin gene enhancer. The negative element is also active in either orientation and defines a renin gene silencer. Mapping in the same region as the silencer is a cAMP-responsive element, a sequence conserved in mouse, rat, and human renin genes. 相似文献
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Acidic PTMs such as phosphorylation and sulfonation of proteins are known to play important roles in many cellular processes including signal transductions and protein-protein interactions. In MS, the acidic modified peptides, that have negative charge, are observable in negative ion mode rather than in positive ion mode. Moreover, addition of ammonium salt into MALDI matrix solution improves the relative intensity of ionization of the phosphorylated peptide to unmodified one. We demonstrate that a combination of the negative ion mode and addition of ammonium salt is more effective in the ionization of the acidic modified peptides. We applied this method to 2-DE separated proteins of Caenorhabditis elegans. As a result, 42 spots were identified as modified proteins, of which 34 proteins were nonoverlapping unique proteins. Furthermore, our study revealed that pI shifts of the DIM-1 and MLC-1 proteins in the 2-DE gel were attributed to the presence of the acidic modifications. The negative ion mode together with the addition of ammonium salt provides us a useful method to detect the phosphorylation and/or sulfonation of protein in a simple manner. 相似文献