Preprocessing Significantly Improves the Peptide/Protein Identification Sensitivity of High-resolution Isobarically Labeled Tandem Mass Spectrometry Data
2014; Elsevier BV; Volume: 14; Issue: 2 Linguagem: Inglês
10.1074/mcp.o114.041376
ISSN1535-9484
AutoresQuanhu Sheng, Rongxia Li, Jie Dai, Qingrun Li, Zhiduan Su, Yan Guo, Chen Li, Yu Shyr, Rong Zeng,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoIsobaric labeling techniques coupled with high-resolution mass spectrometry have been widely employed in proteomic workflows requiring relative quantification. For each high-resolution tandem mass spectrum (MS/MS), isobaric labeling techniques can be used not only to quantify the peptide from different samples by reporter ions, but also to identify the peptide it is derived from. Because the ions related to isobaric labeling may act as noise in database searching, the MS/MS spectrum should be preprocessed before peptide or protein identification. In this article, we demonstrate that there are a lot of high-frequency, high-abundance isobaric related ions in the MS/MS spectrum, and removing isobaric related ions combined with deisotoping and deconvolution in MS/MS preprocessing procedures significantly improves the peptide/protein identification sensitivity. The user-friendly software package TurboRaw2MGF (v2.0) has been implemented for converting raw TIC data files to mascot generic format files and can be downloaded for free from https://github.com/shengqh/RCPA.Tools/releases as part of the software suite ProteomicsTools. The data have been deposited to the ProteomeXchange with identifier PXD000994. Isobaric labeling techniques coupled with high-resolution mass spectrometry have been widely employed in proteomic workflows requiring relative quantification. For each high-resolution tandem mass spectrum (MS/MS), isobaric labeling techniques can be used not only to quantify the peptide from different samples by reporter ions, but also to identify the peptide it is derived from. Because the ions related to isobaric labeling may act as noise in database searching, the MS/MS spectrum should be preprocessed before peptide or protein identification. In this article, we demonstrate that there are a lot of high-frequency, high-abundance isobaric related ions in the MS/MS spectrum, and removing isobaric related ions combined with deisotoping and deconvolution in MS/MS preprocessing procedures significantly improves the peptide/protein identification sensitivity. The user-friendly software package TurboRaw2MGF (v2.0) has been implemented for converting raw TIC data files to mascot generic format files and can be downloaded for free from https://github.com/shengqh/RCPA.Tools/releases as part of the software suite ProteomicsTools. The data have been deposited to the ProteomeXchange with identifier PXD000994. Mass spectrometry-based proteomics has been widely applied to investigate protein mixtures derived from tissue, cell lysates, or from body fluids (1Yates 3rd, J.R. Gilchrist A. Howell K.E. Bergeron J.J. Proteomics of organelles and large cellular structures.Nat. Rev. Mol. Cell Biol. 2005; 6: 702-714Crossref PubMed Scopus (344) Google Scholar, 2Walther T.C. Mann M. Mass spectrometry-based proteomics in cell biology.J. Cell Biol. 2010; 190: 491-500Crossref PubMed Scopus (307) Google Scholar). Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) 1The abbreviations used are:MS/MSTandem Mass SpectrometryLCLiquid Chromatographym/zmass-to-charge ratiosSILACstable isotope labeling by amino acids in cell cultureiTRAQisobaric tag for relative and absolute quantificationTMTtandem mass tag. is the most popular strategy for protein/peptide mixtures analysis in shotgun proteomics (3Wolters D.A. Washburn M.P. Yates 3rd, J.R. An automated multidimensional protein identification technology for shotgun proteomics.Anal. Chem. 2001; 73: 5683-5690Crossref PubMed Scopus (1566) Google Scholar). Large-scale protein/peptide mixtures are separated by liquid chromatography followed by online detection by tandem mass spectrometry. The capabilities of proteomics rely greatly on the performance of the mass spectrometer. With the improvement of MS technology, proteomics has benefited significantly from the high-resolution and excellent mass accuracy (4Mann M. Kelleher N.L. Precision proteomics: the case for high-resolution and high mass accuracy.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 18132-18138Crossref PubMed Scopus (353) Google Scholar). In recent years, based on the higher efficiency of higher energy collision dissociation (HCD), a new "high–high" strategy (high-resolution MS as well as MS/MS(tandem MS)) has been applied instead of the "high–low" strategy (high-resolution MS, i.e. in Orbitrap, and low-resolution MS/MS, i.e. in ion trap) to obtain high quality tandem MS/MS data as well as full MS in shotgun proteomics. Both full MS scans and MS/MS scans can be performed, and the whole cycle time of MS detection is very compatible with the chromatographic time scale (5Olsen J.V. Schwartz J.C. Griep-Raming J. Nielsen M.L. Damoc E. Denisov E. Lange O. Remes P. Taylor D. Splendore M. Wouters E.R. Senko M. Makarov A. Mann M. Horning S. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed.Mol. Cell Proteomics. 2009; 8: 2759-2769Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). Tandem Mass Spectrometry Liquid Chromatography mass-to-charge ratios stable isotope labeling by amino acids in cell culture isobaric tag for relative and absolute quantification tandem mass tag. High-resolution measurement is one of the most important features in mass spectrometric application. In this high–high strategy, high-resolution and accurate spectra will be achieved in tandem MS/MS scans as well as full MS scans, which makes isotopic peaks distinguishable from one another, thus enabling the easy calculation of precise charge states and monoisotopic mass. During an LC-MS/MS experiment, a multiply charged precursor ion (peptide) is usually isolated and fragmented, and then the multiple charge states of the fragment ions are generated and collected. After full extraction of peak lists from original tandem mass spectra, the commonly used search engines (i.e. Mascot (6Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6763) Google Scholar), Sequest (7Eng J.K. McCormack A.L. Yates J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectr. 1994; 5: 976-989Crossref PubMed Scopus (5420) Google Scholar)) have no capability to distinguish isotopic peaks and recognize charge states, so all of the product ions are considered as all charge state hypotheses during the database search for protein identification. These multiple charge states of fragment ions and their isotopic cluster peaks can be incorrectly assigned by the search engine, which can cause false peptide identification. To overcome this issue, data preprocessing of the high-resolution MS/MS spectra is required before submitting them for identification. There are usually two major preprocessing steps used for high-resolution MS/MS data: deisotoping and deconvolution (8Carvalho P.C. Xu T. Han X. Cociorva D. Barbosa V.C. Yates 3rd, J.R. YADA: a tool for taking the most out of high-resolution spectra.Bioinformatics. 2009; 25: 2734-2736Crossref PubMed Scopus (64) Google Scholar, 9Liu X. Inbar Y. Dorrestein P.C. Wynne C. Edwards N. Souda P. Whitelegge J.P. Bafna V. Pevzner P.A. Deconvolution and database search of complex tandem mass spectra of intact proteins: a combinatorial approach.Mol. Cell Proteomics. 2010; 9: 2772-2782Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Deisotoping of spectra removes all isotopic peaks except monoisotopic peaks from multi-isotopic peaks. Deconvolution of spectra translates multiply charged ions to singly charged ions and also accumulates the intensity of fragment ions by summing up all the intensities from their multiply charged states. After performing these two data-preprocessing steps, the resulting spectra is simpler and cleaner and allows more precise database searching and accurate bioinformatics analysis. With the capacity to analyze multiple samples simultaneously, stable isotope labeling approaches have been widely used in quantitative proteomics. Stable isotope labeling approaches are categorized as metabolic labeling (SILAC, stable isotope labeling by amino acids in cell culture) and chemical labeling (10Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar, 11Bantscheff M. Schirle M. Sweetman G. Rick J. Kuster B. Quantitative mass spectrometry in proteomics: a critical review.Anal. Bioanal. Chem. 2007; 389: 1017-1031Crossref PubMed Scopus (1256) Google Scholar). The peptides labeled by the SILAC approach are quantified by precursor ions in full MS spectra, whereas peptides that have been isobarically labeled using chemical means are quantified by reporter ions in MS/MS spectra. There are two similar isobaric chemical labeling methods: (1) isobaric tag for relative and absolute quantification (iTRAQ), and (2) tandem mass tag (TMT) (12Thompson A. Schafer J. Kuhn K. Kienle S. Schwarz J. Schmidt G. Neumann T. Johnstone R. Mohammed A.K. Hamon C. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.Anal. Chem. 2003; 75: 1895-1904Crossref PubMed Scopus (1709) Google Scholar, 13Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3680) Google Scholar). These reagents contain an amino-reactive group that specifically reacts with N-terminal amino groups and epilson-amino groups of lysine residues to label digested peptides in a typical shotgun proteomics experiment. There are four different channels of isobaric tags: TMT two-plex, iTRAQ four-plex, TMT six-plex, and iTRAQ eight-plex (12Thompson A. Schafer J. Kuhn K. Kienle S. Schwarz J. Schmidt G. Neumann T. Johnstone R. Mohammed A.K. Hamon C. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.Anal. Chem. 2003; 75: 1895-1904Crossref PubMed Scopus (1709) Google Scholar, 13Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. 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Peptides labeled with different isotopic variants of the tag show identical or similar mass and appear as a single peak in full scans. This single peak may be selected for subsequent MS/MS analysis. In an MS/MS scan, the mass of reporter ions (114 to 117 for iTRAQ four-plex, 113 to 121 for iTRAQ eight-plex, and 126 to 131for TMT six-plex upon CID or HCD activation) are associated with corresponding samples, and the intensities represent the relative abundances of the labeled peptides. Meanwhile, the other ions from the MS/MS spectra can be used for peptide identification. Because of the multiplexing capability, isobaric labeling methods combined with bottom-up proteomics have been widely applied for accurate quantification of proteins on a global scale (14Aggarwal K. Choe L.H. Lee K.H. Shotgun proteomics using the iTRAQ isobaric tags.Brief. Funct. Genomics Proteomics. 2006; 5: 112-120Crossref PubMed Scopus (292) Google Scholar, 17Leitner A. Lindner W. Chemical tagging strategies for mass spectrometry-based phospho-proteomics.Methods Mol. Biol. 2009; 527: 229-243Crossref PubMed Scopus (29) Google Scholar, 18Treumann A. Thiede B. Isobaric protein and peptide quantification: perspectives and issues.Expert Rev. Proteomics. 2010; 7: 647-653Crossref PubMed Scopus (86) Google Scholar, 19Coombs K.M. Quantitative proteomics of complex mixtures.Expert Rev. Proteomics. 2011; 8: 659-677Crossref PubMed Scopus (52) Google Scholar). Although mostly associated with peptide labeling, these isobaric labeling methods have also been applied at protein level (20Wiese S. Reidegeld K.A. Meyer H.E. Warscheid B. Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research.Proteomics. 2007; 7: 340-350Crossref PubMed Scopus (594) Google Scholar, 21Prudova A. auf dem Keller U. Butler G.S. Overall C.M. Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.Mol. Cell Proteomics. 2010; 9: 894-911Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 22Sinclair J. Timms J.F. Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS.Methods. 2011; 54: 361-369Crossref PubMed Scopus (53) Google Scholar, 23Hung C.W. Tholey A. Tandem mass tag protein labeling for top-down identification and quantification.Anal. Chem. 2012; 84: 161-170Crossref PubMed Scopus (55) Google Scholar). For the proteomic analysis of isobarically labeled peptides/proteins in "high–high" MS strategy, the common consensus is that accurate reporter ions can contribute to more accurate quantification. However, there is no evidence to show how the ions related to isobaric labeling affect the peptide/protein identification and what preprocessing steps should be taken for high-resolution isobarically labeled MS/MS. To demonstrate the effectiveness and importance of preprocessing, we examined how the combination of preprocessing steps improved peptide/protein sensitivity in database searching. Several combinatorial ways of data-preprocessing were applied for high-throughput data analysis including deisotoping to keep simple monoisotopic mass peaks, deconvolution of ions with multiple charge states, and preservation of top 10 peaks in every 100 Dalton mass range. After systematic analysis of high-resolution isobarically labeled spectra, we further processed the spectra and removed interferential ions that were not related to the peptide. Our results suggested that the preprocessing of isobarically labeled high-resolution tandem mass spectra significantly improved the peptide/protein identification sensitivity. The Goto-Kakizaki (GK) rat liver tissue was respectively mixed with SDT-lysis buffer (2% SDS, 0.1 m DTT, and 0.1 m Tris-HCl, pH = 7.6) and then heated for 5 min at 100 °C. After that, the tissue layers were cooled to room temperature, sonicated 60 s at 100 w, and then centrifuged at 16,000 × g for 30 min at 20 °C for removing cell debris. The protein concentration was detected by measurements of tryptophan fluorescence as described (24Nielsen P.A. Olsen J.V. Podtelejnikov A.V. Andersen J.R. Mann M. Wisniewski J.R. Proteomic mapping of brain plasma membrane proteins.Mol. Cell Proteomics. 2005; 4: 402-408Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Briefly, 1 μl of sample or tryptophan standard (100 ng/μl) was added into 3 ml of 8 m urea buffer (8 m urea and 20 mm Tris-HCl, pH = 7.6). Fluorescence was excited at 295 nm and measured at 350 nm. The slits were set at 10 nm. Six hundred micrograms of liver tissue from GK rat was digested by the FASP procedure as described (25Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar) with small modifications. Each sample was transferred to a 10k filter (Pall Corporation, Port Washington, NY) and centrifuged at 10,000 × g for 20 min at 20 °C. 200 μl of UA buffer (8 m urea and 0.1 m Tris-HCl, pH = 8.5) was added and centrifuged at 10,000 × g for 20 min again. This step was repeated once. Then, the concentrate was mixed with 100 μl of 50 mm IAA in UA buffer and incubated for an additional 40 min at room temperature in darkness. After that, IAA was removed by centrifugation at 10,000 × g for 20 min. Following dilution with 200 μl of UA buffer and centrifugation twice, 200 μl of 200 mm triethylammonium bicarbonate (TEAB) buffer (pH 8.5) was added and centrifuged at 10,000 × g for 20 min. This step was repeated four times. Finally, 100 μl of 50 mm TEAB buffer (pH 8.5) and Trypsin (1:50, enzyme to protein) was added to the filter, and after 4 h, another 50 μg trypsin was added. The samples were digested 20 h at 37 °C and peptides were collected by centrifugation at 16,000 × g. To increase the yield of peptides, the filter was washed twice with 500 μl 0.5 m TEAB buffer (pH 8.5). The peptide solutions were dried in a vacuum concentrator. The trypsin digestion of 100 μg protein from each sample was processed as described elsewhere. iTRAQ labeling was done following the manufacturer's instructions (AB SCIEX, Foster City, CA). Briefly, for each four- or eight-plex experiment, 100 μg of dried peptide mixture power from each digested sample was reconstituted with 30μl 0.5 mm TEAB Buffer (pH 8.5). Each peptide solution was labeled at room temperature for 2 h with one iTRAQ reagent vial (four-plex mass tag 114, 115, 116, 117 or eight-plex mass tag 113,114, 115, 116, 117, 118, 119, 121) previously reconstituted with 70 μl of anhydrous acetonitrile (ACN). After 2 h, 100 μl ddH2O were added to each tube to quench the iTRAQ reaction and incubated at room temperature for 30 min. The contents of all iTRAQ reagent-labeled sample tubes were combined into one tube for four or eight-plex experiments, respectively. Then, labeled samples were dried down by evaporation in a SpeedVac to obtain a brown pellet. 100 μl of water was added to the tube and the sample was dried completely. Prior to MS analysis, samples were desalted onto Empore C18 47 mm Disk (3 m). Just prior nano-LC, the fractions were resuspended in 20 μl of H2O with 0.1% (v/v) TFA. The reverse phase-high performance liquid chromatography (RP-HPLC) separation was achieved on an UltiMate 3000 RSLC nanoLC Systems (Dionex, now ThermoFisher Scientific) equipped with a self-packed tip column (75 μm × 240 mm; C18, 1.9 μm) using a 180 min gradient at a flow rate of 150 nl/min. An LTQ-Orbitrap Velos instrument (Thermo Fisher Scientific) was operated in data-dependent mode. MS full scans were acquired in ranges m/z 300–2000. The mass spectrometer was set so that each full MS scan was followed by the ten most intense ions for MS/MS with charge ≥ +2 with the following Dynamic Exclusion™ settings: repeat counts, 1; repeat duration, 30 s; exclusion duration, 180 s. The normalized collision energy for MS2 was 45.0%. Full MS scans and MS/MS scans were acquired at a resolution of 30,000 for profile-mode and 7500 for centroid-mode respectively, with a lock mass option enabled for the 445.120025 ion. Data were acquired using Xcalibur software. b/y free windows are two mass windows for a specific mass spectrum that no B ion or Y ion would be in. With the assumption that the mass of an isobaric tag was M, trypsin was used as protease and the isobaric tag was attached at both the N-terminal of peptide and lysine (K), for a spectrum with singly charged precursor mass MH+, the b/y free windows of that spectrum can be calculated as below. Because only full-tryptic peptides are considered in data analysis, the latest amino acid of the peptide will be either arginine (R) with mass 156 or lysine with mass 128. Given the fact that glycine (G) is the smallest amino acid with mass 57, the minimum and maximum mass of B and Y ions can be calculated as formula (1–4): minimum (B)=M+ mass ( glycine )+H minimum (Y)= minimum(mass(arginine) +H2O+H,M+ mass(lysine) +H2O+H maximum(B) =MH+−mass (arginine) −H2O maximum(Y) =MH+-mass ( glycine )-M-H2O where H2O is the mass of water and H is the mass of hydrogen. Then, the b/y free window in the low mass range is from 0 to minimum (minimum (B), minimum (Y)) and the b/y free window in the high mass range is from maximum (maximum (B), maximum (Y)) to infinite. Only the spectra with precursor charges 2, 3, and 4 were used to detect high frequency ions. The ion frequency and ion abundance distribution in each sample were generated by software "Raw Ion Frequency Statistic Builder," which was also a part of ProteomicsTools. The charge, mass to charge (m/z), and abundance of each ion were extracted from each MS/MS spectrum through Thermo's MS File Reader interface. The abundance of ions in each MS/MS was normalized to a uniform distribution [0..1]. The ions with relative abundance less than 0.01 were discarded. All remaining ions were deconvoluted to corresponding singly charged ions by formula (5). The ions without charge information were treated as a single charge state. singly charged mass =m/Z⋆ charge −( charge −1)⋆H where H is the mass of hydrogen. The ions in different deconvoluted spectra but with difference in masses less than 20 parts per million (ppm) were considered identical ions. The ion frequency and ion average relative abundance were calculated from all the MS/MS spectra in the sample. The ions with frequency larger than 0.3 and average relative abundance larger than 0.05 were defined as high frequency ions and classified to five categories: "Rep+," "Label+," "Y1," "b/y free," and "Unknown." "Rep+" denotes that an ion is a reporter ion. "Label+" denotes that an ion is an isobaric tag ion with both reporter group and balance group. "Y1" denotes that an ion is a first Y series ion. Because trypsin was used in the sample preparation, a Y1 ion was produced from either lysine (K) or arginine (R). b/y free denotes that the mass of the ion is located in the b/y free windows of that spectrum. All other ions belonged to the "Unknown" category. An ion within one of the first four categories "Rep+, Label+, Y1, and b/y free) was considered annotated. For each deconvoluted tandem mass spectrum (forward spectrum), a backward spectrum was generated by using the mass of the precursor minus the mass of each forward ion. The backward ions were also filtered and annotated in the same fashion as the forward ions except that the ions with mass equal to "Label+" were marked as "Precursor-Label+." "Precursor-Label+" denotes a precursor ion without the isobaric tag. The ions annotated as Rep+, Label+, and Precursor-Label+ are not related to the peptide and therefore can be confidently removed during data preprocessing. For the ions annotated as b/y free in low mass range, they are very likely not related to the peptide as well. But it is still possible that those ions are actually multiply charged ions that lack charge information in the spectrum. The tandem mass spectra were extracted by TurboRaw2MGF (v1.3.4) for database searching. Four fixed criteria were used to filter out low quality spectra: (1) the required precursor mass weight range was 400 to 5000 Daltons, (2) the minimum ion absolute abundance was 1.0, 3) the minimum ion count of a spectrum was 15, and 4) the minimum total ion absolute abundance of a spectrum was 100. Four processing options were also provided in TurboRaw2MGF including deisotoping to keep monoisotopic mass peaks, deconvolution of ions with multiple charge states, preservation of the top 10 peaks in every 100 Dalton mass range, and removing the ions that may not be related to the peptide. The spectra that passed the fixed criteria and were processed with a combination of the four options were saved in mascot generic format for further database searching. Five engines were used for database searching, including Mascot (v2.2.2) (6Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6763) Google Scholar), Comet (2014.01 rev. 1) (26Eng J.K. Jahan T.A. Hoopmann M.R. Comet: an open-source MS/MS sequence database search tool.Proteomics. 2013; 13: 22-24Crossref PubMed Scopus (781) Google Scholar), MyriMatch (v2.2.140) (27Tabb D.L. Fernando C.G. Chambers M.C. MyriMatch: highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis.J. Proteome Res. 2007; 6: 654-661Crossref PubMed Scopus (445) Google Scholar), OMSSA (v2.1.9) (28Geer L.Y. Markey S.P. Kowalak J.A. Wagner L. Xu M. Maynard D.M. Yang X. Shi W. Bryant S.H. Open mass spectrometry search algorithm.J. Proteome Res. 2004; 3: 958-964Crossref PubMed Scopus (1164) Google Scholar), and X! Tandem (2013.09.01.1) (29Craig R. Beavis R.C. TANDEM: matching proteins with tandem mass spectra.Bioinformatics. 2004; 20: 1466-1467Crossref PubMed Scopus (1987) Google Scholar). All MS/MS spectra were searched against a composite target-decoy rat Uniprot database (Version 20120222), in which each protein sequence was followed by a reversed amino acid sequence. Trypsin was set as protease. Carbamidomethylation on cysteine (+57.021464), iTRAQ-labeling on N-terminal, and lysine were set as fixed modifications. Oxidation on methionine (+15.994915) was set as a variable modification. One missing cleavage site was allowed. The tolerances of peptides and fragment ions were set at 10 ppm and 0.02 Daltons respectively. SearchGUI (30Vaudel M. Barsnes H. Berven F.S. Sickmann A. Martens L. SearchGUI: An open-source graphical user interface for simultaneous OMSSA and X!Tandem searches.Proteomics. 2011; 11: 996-999Crossref PubMed Scopus (277) Google Scholar) was used for MyriMatch and OMSSA searching. BuildSummary (31Sheng Q. Dai J. Wu Y. Tang H. Zeng R. BuildSummary: using a group-based approach to improve the sensitivity of peptide/protein identification in shotgun proteomics.J. Proteome Res. 2012; 11: 1494-1502Crossref PubMed Scopus (44) Google Scholar) was used to generate a confident protein list for both peptide and protein with a false discovery rate ≤ 0.01. We implemented our preprocessing steps in a user friendly software package named TurboRaw2MGF (v2.0). The previous version of TurboRaw2MGF was developed for low-resolution tandem mass spectra and was integrated into the package ProtQuantSuite (32Mann B. Madera M. Sheng Q. Tang H. Mechref Y. Novotny M.V. ProteinQuant Suite: a bundle of automated software tools for label-free quantitative proteomics.Rapid Commun. Mass Spectrom. 2008; 22: 3823-3834Crossref PubMed Scopus (50) Google Scholar). TurboRaw2MGF (v2.0) was developed using the C# programming language and was compiled in the Microsoft Visual Studio 2012 Professional Edition. The software is fully compatible with Windows-based operating systems with dotNET framework v4.5. TurboRaw2MGF (v2.0) and its source code can be downloaded freely from ln]https://github.com/shengqh/RCPA.Tools/releases/. The manual of TurboRaw2MGF (v2.0) can be viewed at https://github.com/shengqh/RCPA.Tools/wiki/. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (33Vizcaino J.A. Deutsch E.W. Wang R. Csordas A. Reisinger F. Rios D. Dianes J.A. Sun Z. Farrah T. Bandeira N. Binz P.A. Xenarios I. Eisenacher M. Mayer G. Gatto L. Campos A. Chalkley R.J. Kraus H.J. Albar J.P. Martinez-Bartolome S. Apweiler R. Omenn G.S. Martens L. Jones A.R. Hermjakob H. ProteomeXchange provides globally coordinated proteomics data submission and dissemination.Nat. Biotechnol. 2014; 32: 223-226Crossref PubMed Scopus (2071) Google Scholar) via the PRIDE partner repository with the data set identifier PXD000994 and DOI 10.6019/PXD000994. To access the data please visit: http://tinyurl.com/pdbkesj Username: Password: jWjYoiuT Table I illustrates some important ion properties in isobaric labeling methods. For iTRAQ4 spectra, the mass of a Label+ ion is within the low mass b/y free window, and the mass of a Precursor-Label+ ion is also within the high mass b/y free window. The isobaric related mass ranges include both low and high b/y free windows. For iTRAQ8 spectra, the mass of a Label+ ion is not within the low mass b/y free window and the mass of a Precursor-Label+ ion is also not within the high mass b/y free window. The isobaric related mass ranges not only include both low and high b/y free windows but also include the mass range around Label+ ion and Precursor-Label+ ion within a specific tolerance, which was 20 ppm in our study.Table IIon characteristics of isobaric labeling methodsPropertyiTRAQ4iTRAQ8aRep+: reporter ions.Rep+114–117113–119,121bLabel+: isobaric tag ion.Label+145305Isobaric ion mass144304Minimum B ion202362Minimum Y ion175175Low mass b/y free window0∼1750∼175Maximum B ionMH+-174MH+-174Maximum Y ionMH+-201MH+-361High mass b/y free windowMH+-174∼INFMH+-174∼INFcPrecursor-Label+: the precursor ion without the isobaric tag.Precursor-Label+MH+-144MH+-304a Rep+: reporter ions
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