VEMS 3.0: algorithms and computational tools for tandem mass spectrometry based identification of post-translational modifications in proteins

Protein and peptide mass analysis and amino acid sequencing by mass spectrometry is widely used for identification and annotation of post-translational modifications (PTMs) in proteins. Modification-specific mass increments, neutral losses or diagnostic fragment ions in peptide mass spectra provide direct evidence for the presence of post-translational modifications, such as phosphorylation, acetylation, methylation or glycosylation. However, the commonly used database search engines are not always practical for exhaustive searches for multiple modifications and concomitant missed proteolytic cleavage sites in large-scale proteomic datasets, since the search space is dramatically expanded. We present a formal definition of the problem of searching databases with tandem mass spectra of peptides that are partially (sub-stoichiometrically) modified. In addition, an improved search algorithm and peptide scoring scheme that includes modification specific ion information from MS/MS spectra was implemented and tested using the Virtual Expert Mass Spectrometrist (VEMS) software. A set of 2825 peptide MS/MS spectra were searched with 16 variable modifications and 6 missed cleavages. The scoring scheme returned a large set of post-translationally modified peptides including precise information on modification type and position. The scoring scheme was able to extract and distinguish the near-isobaric modifications of trimethylation and acetylation of lysine residues based on the presence and absence of diagnostic neutral losses and immonium ions. In addition, the VEMS software contains a range of new features for analysis of mass spectrometry data obtained in large-scale proteomic experiments. Windows binaries are available at http://www.yass.sdu.dk/.     

Virtual Expert Mass Spectrometrist: iTRAQ tool for database‐dependent search,quantitation and result storage

A frequent goal of MS‐based proteomics experiments nowadays is to quantify changes in the abundance of proteins across several biological samples. The iTRAQ labeling method is a powerful technique; when combined with LC coupled to MS/MS it allows relative quantitation of up to eight different samples simultaneously. Despite the usefulness of iTRAQ current software solutions have limited functionality and require the combined use of several software programs for analysis of the data from different MS vendors. We developed an integrated tool, now available in the virtual expert mass spectrometrist (VEMS) program, for database‐dependent search of MS/MS spectra, quantitation and database storage for iTRAQ‐labeled samples. VEMS also provides useful alternative report types for large‐scale quantitative experiments. The implemented statistical algorithms build on quantitative algorithms previously used in proposed iTRAQ tools as described in detail herein. We propose a new algorithm, which provides more accurate peptide ratios for data that show an intensity‐dependent saturation. The accuracy of the proposed iTRAQ algorithm and the performance of VEMS are demonstrated by comparing results from VEMS, MASCOT and PEAKS Q obtained by analyzing data from a reference mixture of six proteins. Users can download VEMS and test data from “ http://www.portugene.com/software.html ”.     

Isotopically labeled crosslinking reagents: resolution of mass degeneracy in the identification of crosslinked peptides

Mass spectrometry in three dimensions (MS3D) is a newly developed method for the determination of protein structures involving intramolecular chemical crosslinking of proteins, proteolytic digestion of the resulting adducts, identification of crosslinks by mass spectrometry (MS), peak assignment using theoretical mass lists, and computational reduction of crosslinks to a structure by distance geometry methods. To facilitate the unambiguous identification of crosslinked peptides from proteolytic digestion mixtures of crosslinked proteins by MS, we introduced double 18O isotopic labels into the crosslinking reagent to provide the crosslinked peptides with a characteristic isotope pattern. The presence of doublets separated by 4 Da in the mass spectra of these materials allowed ready discrimination between crosslinked and modified peptides, and uncrosslinked peptides using automated intelligent data acquisition (IDA) of MS/MS data. This should allow ready automation of the method for application to whole expressible proteomes.     

Methods for on-chip protein analysis

The unambiguous identification of peptides/proteins is crucial for the definition of the proteome. Using ProteinChip Array technology also known as surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS), we developed experimental protocols and probed test conditions required for the protein identification on ProteinChip surfaces. We were able to directly digest peptides/proteins on-chip surfaces by specific proteases, such as trypsin, and to obtain the peptide mass fingerprint of the sample under investigation by its direct analysis on a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry was performed on several of the resulting tryptic peptides by using collision quadrupole time of flight (Qq-TOF) MS/MS via the ProteinChip interface, thus allowing the unambiguous identification of the protein(s) within the sample. In addition, we were able to identify the C-terminal sequence of peptides by their digestion with carboxypeptidase Y directly on ProteinChip surfaces coupled with SELDI-TOF MS analysis of the resulting peptide mass ladders employing the instrument's protein ladder sequence software. Moreover, the removal of up to nine amino acid residues from the C-terminal end of a peptide extends the functional range of Qq-TOF MS/MS sequence determination to over 3000 m/z. The utility of these procedures for the proteome exploration are discussed.     

Mitochondrial phosphoproteome revealed by an improved IMAC method and MS/MS/MS

IMAC in combination with mass spectrometry is a promising approach for global analysis of protein phosphorylation. Nevertheless this approach suffers from two shortcomings: inadequate efficiency of IMAC and poor fragmentation of phosphopeptides in the mass spectrometer. Here we report optimization of the IMAC procedure using (32)P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allowed recovery of phosphorylated tryptic peptides up to approximately 77% with only minor retention of unphosphorylated peptides. MS3 led to efficient fragmentation of the peptide backbone in phosphopeptides for sequence assignment. Proteomics of mitochondrial phosphoproteins using the resulting IMAC protocol and MS3 revealed 84 phosphorylation sites in 62 proteins, most of which have not been reported before. These results revealed diverse phosphorylation pathways involved in the regulation of mitochondrial functions. Integration of the optimized batchwise IMAC protocol with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.     

Increased protein identification capabilities through novel tandem MS calibration strategies

High mass measurement accuracy is critical for confident protein identification and characterization in proteomics research. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is a unique technique which can provide unparalleled mass accuracy and resolving power. However, the mass measurement accuracy of FTICR-MS can be affected by space charge effects. Here, we present a novel internal calibrant-free calibration method that corrects for space charge-induced frequency shifts in FTICR fragment spectra called Calibration Optimization on Fragment Ions (COFI). This new strategy utilizes the information from fixed mass differences between two neighboring peptide fragment ions (such as y(1) and y(2)) to correct the frequency shift after data collection. COFI has been successfully applied to LC-FTICR fragmentation data. Mascot MS/MS ion search data demonstrate that most of the fragments from BSA tryptic digested peptides can be identified using a much lower mass tolerance window after applying COFI to LC-FTICR-MS/MS of BSA tryptic digest. Furthermore, COFI has been used for multiplexed LC-CID-FTICR-MS which is an attractive technique because of its increased duty cycle and dynamic range. After the application of COFI to a multiplexed LC-CID-FTICR-MS of BSA tryptic digest, we achieved an average measured mass accuracy of 2.49 ppm for all the identified BSA fragments.     

毛细管区带电泳／串联质谱联用法鉴定多肽和蛋白质

建立了毛细管区带电泳－串联质谱联用（ＣＺＥ／ＭＳ／ＭＳ）对多肽和蛋白质高灵敏度鉴定方法,对Ｍｅｔ－脑啡肽和Ｌｅｕ－脑啡肽的混合物进行了分析,用ＣＺＥ／ＭＳ／ＭＳ方法验证了各自的序列,同样对细胞色素ｃ的胰蛋白酶酶解产物用ＣＺＥ／ＭＳ／ＭＳ方法进行了肽质谱分析,几科所有肽段的序列及其与在分子中的位置都得到了确定,通过ＳＥＱＵＥＳＴ软件进行蛋白质序列数据库搜索得到准确的鉴定结果,所消耗的样品量均在低皮可     

Validation of peptide MS/MS spectra using metabolic isotope labeling for spectral matching-based shotgun proteome analysis

We report an isotope labeling shotgun proteome analysis strategy to validate the spectrum-to-sequence assignments generated by using sequence-database searching for the construction of a more reliable MS/MS spectral library. This strategy is demonstrated in the analysis of the E. coli K12 proteome. In the workflow, E. coli cells were cultured in normal and (15)N-enriched media. The differentially labeled proteins from the cell extracts were subjected to trypsin digestion and two-dimensional liquid chromatography quadrupole time-of-flight tandem mass spectrometry (2D-LC QTOF MS/MS) analysis. The MS/MS spectra of the two samples were individually searched using Mascot against the E. coli proteome database to generate lists of peptide sequence matches. The two data sets were compared by overlaying the spectra of unlabeled and labeled matches of the same peptide sequence for validation. Two cutoff filters, one based on the number of common fragment ions and another one on the similarity of intensity patterns among the common ions, were developed and applied to the overlaid spectral pairs to reject the low quality or incorrectly assigned spectra. By examining 257,907 and 245,156 spectra acquired from the unlabeled and (15)N-labeled samples, respectively, an experimentally validated MS/MS spectral library of tryptic peptides was constructed for E. coli K12 that consisted of 9,302 unique spectra with unique sequence and charge state, representing 7,763 unique peptide sequences. This E. coli spectral library could be readily expanded, and the overall strategy should be applicable to other organisms. Even with this relatively small library, it was shown that more peptides could be identified with higher confidence using the spectral search method than by sequence-database searching.     

Proteolysis and mass spectrometric analysis of an integral membrane: aquaporin 0

Due to hydrophobicity, structural analysis of integral membrane proteins poses a formidable challenge for current mass spectrometry-based proteomics approaches. Herein, we demonstrate results from optimized sample preparation and enzymatic proteolysis procedures for the complete primary structure determination of a targeted integral membrane protein, lens aquaporin 0 (AQP0). Plasma membrane from bovine lens tissue was alkali treated and tryptic digestion was performed in optimized acetonitrile-ammonium bicarbonate solution. Full sequence coverage of AQP0 was observed as tryptic peptides using both matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS). An amino acid mutation of Thr to Ile/Leu at residue 199 was deduced based on MS/MS results. In a complementary effort to fully sequence the protein, peptic digestion was developed to take advantage of hydrophobic protein solubility in organic acid as well as the decreased activity of pepsin at low pH. Peptic digestion in 10% formic acid (pH 1.2) generated peptides of 500 to 3000 Da and gave 100% sequence coverage by cLC/MS/MS. In addition to post-translational modifications reported previously, a new phosphorylation site at serine 229 and two oxidation sites at tryptophan 202 and 205 were detected on the protein. These methodologies provide complementary detergent- and CNBr-free procedures for detailed analysis of this important membrane channel protein and offer promise for analysis of the integral membrane proteome.     

Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation

Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.     

The fate of b-ions in the two worlds of collision-induced dissociation

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss.     

The standard protein mix database: a diverse data set to assist in the production of improved Peptide and protein identification software tools

Tandem mass spectrometry (MS/MS) is frequently used in the identification of peptides and proteins. Typical proteomic experiments rely on algorithms such as SEQUEST and MASCOT to compare thousands of tandem mass spectra against the theoretical fragment ion spectra of peptides in a database. The probabilities that these spectrum-to-sequence assignments are correct can be determined by statistical software such as PeptideProphet or through estimations based on reverse or decoy databases. However, many of the software applications that assign probabilities for MS/MS spectra to sequence matches were developed using training data sets from 3D ion-trap mass spectrometers. Given the variety of types of mass spectrometers that have become commercially available over the last 5 years, we sought to generate a data set of reference data covering multiple instrumentation platforms to facilitate both the refinement of existing computational approaches and the development of novel software tools. We analyzed the proteolytic peptides in a mixture of tryptic digests of 18 proteins, named the "ISB standard protein mix", using 8 different mass spectrometers. These include linear and 3D ion traps, two quadrupole time-of-flight platforms (qq-TOF), and two MALDI-TOF-TOF platforms. The resulting data set, which has been named the Standard Protein Mix Database, consists of over 1.1 million spectra in 150+ replicate runs on the mass spectrometers. The data were inspected for quality of separation and searched using SEQUEST. All data, including the native raw instrument and mzXML formats and the PeptideProphet validated peptide assignments, are available at http://regis-web.systemsbiology.net/PublicDatasets/.     

Identification of a novel phosphorylation site in human androgen receptor by mass spectrometry

An N-terminal hexahistidine-tagged full-length human androgen receptor protein (His(6)-hAR) was overexpressed and purified to apparent homogeneity in the presence of dihydrotestosterone (DHT) in our previous studies. In-gel trypsin digestion of the purified DHT-bound His(6)-hAR, and tryptic peptide mapping using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), detected a total of 17 peptides (21% coverage of hAR) with 9 peptides originating from the ligand-binding domain (LBD, 31% coverage of LBD). Amino acid sequencing analysis of the tryptic peptides from a separate in-gel digestion of the His(6)-hAR, using HPLC-coupled electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS and MS/MS), unambiguously confirmed 21 peptides with 19% coverage of the hAR, of which 11 peptides originated from the LBD (35% coverage of LBD). These 21 peptides included 11 out of the 17 peptides detected by MALDI/TOF-MS. In addition, a novel serine phosphorylation site (Ser(308)) within the N-terminal transactivation domain of hAR was identified.     

Highly informative proteome analysis by combining improved N-terminal sulfonation for de novo peptide sequencing and online capillary reverse-phase liquid chromatography/tandem mass spectrometry

Recently, various chemical modifications of peptides have been incorporated into mass spectrometric analyses of proteome samples, predominantly in conjunction with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), to facilitate de novo sequencing of peptides. In this work, we investigate systematically the utility of N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate (SPITC) for proteome analysis by capillary reverse-phase liquid chromatography/tandem mass spectrometry (cRPLC/MS/MS). The experimental conditions for the sulfonation were carefully adjusted so that SPITC reacts selectively with the N-terminal amino groups, even in the presence of the epsilon-amino groups of lysine residues. Mass spectrometric analyses of the modified peptides by cRPLC/MS/MS indicated that SPITC derivatization proceeded toward near completion under the experimental conditions employed here. The SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra. Combining SPITC derivatization and cRPLC/MS/MS analyses facilitated the acquisition of sequence information for lysine-terminated tryptic peptides as well as arginine-terminated peptides without the need for additional peptide pretreatment, such as guanidination of lysine amino group. This process alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI MS experiments. We will discuss the utility of the technique as a viable method for proteome analyses and present examples of its application in analyzing samples having different levels of complexity.     

The spectral networks paradigm in high throughput mass spectrometry

High-throughput proteomics is made possible by a combination of modern mass spectrometry instruments capable of generating many millions of tandem mass (MS(2)) spectra on a daily basis and the increasingly sophisticated associated software for their automated identification. Despite the growing accumulation of collections of identified spectra and the regular generation of MS(2) data from related peptides, the mainstream approach for peptide identification is still the nearly two decades old approach of matching one MS(2) spectrum at a time against a database of protein sequences. Moreover, database search tools overwhelmingly continue to require that users guess in advance a small set of 4-6 post-translational modifications that may be present in their data in order to avoid incurring substantial false positive and negative rates. The spectral networks paradigm for analysis of MS(2) spectra differs from the mainstream database search paradigm in three fundamental ways. First, spectral networks are based on matching spectra against other spectra instead of against protein sequences. Second, spectral networks find spectra from related peptides even before considering their possible identifications. Third, spectral networks determine consensus identifications from sets of spectra from related peptides instead of separately attempting to identify one spectrum at a time. Even though spectral networks algorithms are still in their infancy, they have already delivered the longest and most accurate de novo sequences to date, revealed a new route for the discovery of unexpected post-translational modifications and highly-modified peptides, enabled automated sequencing of cyclic non-ribosomal peptides with unknown amino acids and are now defining a novel approach for mapping the entire molecular output of biological systems that is suitable for analysis with tandem mass spectrometry. Here we review the current state of spectral networks algorithms and discuss possible future directions for automated interpretation of spectra from any class of molecules.     

Improving MHC‐I Ligand Identifications from LC‐MS/MS Data by Incorporating Allelic Peptide Motifs

MHC class I (MHC‐I)‐bound ligands play a pivotal role in CD8 T cell immunity and are hence of major interest in understanding and designing immunotherapies. One of the most commonly utilized approaches for detecting MHC ligands is LC‐MS/MS. Unfortunately, the effectiveness of current algorithms to identify MHC ligands from LC‐MS/MS data is limited because the search algorithms used were originally developed for proteomics approaches detecting tryptic peptides. Consequently, the analysis often results in inflated false discovery rate (FDR) statistics and an overall decrease in the number of peptides that pass FDR filters. Andreatta et al. describe a new scoring tool (MS‐rescue) for peptides from MHC‐I immunopeptidome datasets. MS‐rescue incorporates the existence of MHC‐I peptide motifs to rescore peptides from ligandome data. The tool is demonstrated here using peptides assigned from LC‐MS/MS data with PEAKs software but can be deployed on data from any search algorithm. This new approach increased the number of peptides identified by up to 20–30% and promises to aid the discovery of novel MHC‐I ligands with immunotherapeutic potential.     

YPED:An Integrated Bioinformatics Suite and Database for Mass Spectrometry-based Proteomics Research

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database(YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a singlelaboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography–tandem mass spectrometry(LC–MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring(MRM)/selective reaction monitoring(SRM) assay development. We have linked YPED's database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results.     

Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Reversible phosphorylation is one of the most important posttranslational modifications of cellular proteins. Mass spectrometry is a widely used technique in the characterization of phosphorylated proteins and peptides. Similar to nonmodified peptides, sequence information for phosphopeptides digested from proteins can be obtained by tandem mass analysis using either electrospray ionization or matrix assisted laser desorption/ionization (MALDI) mass spectrometry. However, the facile loss of neutral phosphoric acid (H₃PO₄) or HPO₃ from precursor ions and fragment ions hampers the precise determination of phosphorylation site, particularly if more than one potential phosphorylation site or concensus sequence is present in a given tryptic peptide. Here, we investigated the fragmentation of phosphorylated peptides under laser-induced dissociation (LID) using a MALDI-time-of-flight mass spectrometer with a curved-field reflectron. Our data demonstrated that intact fragments bearing phosphorylated residues were produced from all tested peptides that contain at least one and up to four phosphorylation sites at serine, threonine, or tyrosine residues. In addition, the LID of phosphopeptides derivatized by N-terminal sulfonation yields simplified MS/MS spectra, suggesting the combination of these two types of spectra could provide an effective approach to the characterization of proteins modified by phosphorylation.     

iProphet: multi-level integrative analysis of shotgun proteomic data improves peptide and protein identification rates and error estimates

The combination of tandem mass spectrometry and sequence database searching is the method of choice for the identification of peptides and the mapping of proteomes. Over the last several years, the volume of data generated in proteomic studies has increased dramatically, which challenges the computational approaches previously developed for these data. Furthermore, a multitude of search engines have been developed that identify different, overlapping subsets of the sample peptides from a particular set of tandem mass spectrometry spectra. We present iProphet, the new addition to the widely used open-source suite of proteomic data analysis tools Trans-Proteomics Pipeline. Applied in tandem with PeptideProphet, it provides more accurate representation of the multilevel nature of shotgun proteomic data. iProphet combines the evidence from multiple identifications of the same peptide sequences across different spectra, experiments, precursor ion charge states, and modified states. It also allows accurate and effective integration of the results from multiple database search engines applied to the same data. The use of iProphet in the Trans-Proteomics Pipeline increases the number of correctly identified peptides at a constant false discovery rate as compared with both PeptideProphet and another state-of-the-art tool Percolator. As the main outcome, iProphet permits the calculation of accurate posterior probabilities and false discovery rate estimates at the level of sequence identical peptide identifications, which in turn leads to more accurate probability estimates at the protein level. Fully integrated with the Trans-Proteomics Pipeline, it supports all commonly used MS instruments, search engines, and computer platforms. The performance of iProphet is demonstrated on two publicly available data sets: data from a human whole cell lysate proteome profiling experiment representative of typical proteomic data sets, and from a set of Streptococcus pyogenes experiments more representative of organism-specific composite data sets.     

SwedCAD, a database of annotated high-mass accuracy MS/MS spectra of tryptic peptides

A database of high-mass accuracy tryptic peptides has been created. The database contains 15 897 unique, annotated MS/MS spectra. It is possible to search for peptides according to their mass, number of missed cleavages, and sequence motifs. All of the data contained in the database is downloadable, and each spectrum can be visualized. An example is presented of how the database can be used for studying peptide fragmentation. Fragmentation of different types of missed cleaved peptides has been studied, and the results can be used to improve identification of these types of peptides.