Unidentified tandem mass spectra typically represent 50−90% of the spectra acquired in proteomics studies. This manuscript describes a novel algorithm, “Bonanza”, for clustering spectra without knowledge of peptide or protein identifications. Further analysis leverages existing peptide identifications to infer related, likely valid identifications. Significantly more spectra can be identified with this approach, including spectra with unexpected potential modifications or amino-acid substitutions.Keywords: bonanza; clustering; falkner; MS/MS; peak list library; proteomecommons.org; spectral clustering; spectral library; tranche;

Systems analysis of chromatin has been constrained by complex patterns and dynamics of histone post-translational modifications (PTMs), which represent major challenges for both mass spectrometry (MS) and immuno-based approaches (e.g., chromatin immuno-precipitation, ChIP). Here we present a proof-of-concept study demonstrating that crosstalk among PTMs and their functional significance can be revealed via systematic bioinformatic and proteomic analysis of steady-state histone PTM levels from cells under various perturbations. Using high resolution tandem MS, we quantified 53 modification states from all core histones and their conserved variants in the unicellular eukaryotic model organism Tetrahymena. By correlating histone PTM patterns across 15 different conditions, including various physiological states and mutations of key histone modifying enzymes, we identified 5 specific chromatin states with characteristic covarying histone PTMs and associated them with distinctive functions in replication, transcription, and DNA repair. In addition to providing a detailed picture on histone PTM crosstalk at global levels, this work has established a novel bioinformatic and proteomic approach, which can be adapted to other organisms and readily scaled up to allow increased resolution of chromatin states.Keywords: chromatin states; histone code; histone demethylase; histone H3 lysine 27 methylation; histone methyltransferase; mass spectrometry; posttranslational modification; ubiquitin E3 ligase;

Analysis of protein complexes by ion mobility-mass spectrometry is a valuable method for the rapid assessment of complex composition, binding stoichiometries, and structures. However, capturing labile, unknown protein assemblies directly from cells remains a challenge for the technology. Furthermore, ion mobility-mass spectrometry measurements of complexes, subcomplexes, and subunits are necessary to build complete models of intact assemblies, and such data can be difficult to acquire in a comprehensive fashion. Here, we present the use of novel mass spectrometry cleavable cross-linkers and tags to stabilize intact protein complexes for ion mobility-mass spectrometry. Our data reveal that tags and linkers bearing permanent charges are superior stabilizers relative to neutral cross-linkers, especially in the context of retaining compact forms of the assembly under a wide array of activating conditions. In addition, when cross-linked protein complexes are collisionally activated in the gas phase, a larger proportion of the product ions produced are often more compact and reflect native protein subcomplexes when compared with unmodified complexes activated in the same fashion, greatly enabling applications in structural biology.

Myelin basic protein (MBP) is an important component of the myelin sheath surrounding neurons, and it is directly affected in demyelinating diseases. MBP contains a relatively large number of post-translational modifications (PTMs), which have been reported to play a role in multiple sclerosis, while MBPs from lower vertebrates have been reported to be incapable of inducing multiple sclerosis or allergic encephalitis. This study reveals the extent of differences in PTM patterns for mammalian and nonmammalian MBPs. This included intact mass and de novo sequence analysis of approximately 85% of rattlesnake MBP, the first reptile MBP to be characterized, and of bovine MBP. We identified 12 PTMs at 11 sites in the five bovine MBP charge components, which include both previously reported and novel modifications. The most notable modification is an acetylation of lysine 121. Other modifications found in bovine MBP include N-terminal acetylation in components C1, C2, and C3; oxidation of methionine 19 in all five components; all charge isomers having both a mono- and dimethylated (symmetric) arginine at position 106; deimination in arginines 23 and 47 found only in component C8b; deimination of arginine 96 and deamidation in glutamine 102 found in components C2, C3, C8a, and C8b; phosphorylation in threonine 97 restricted to charge components C2 and C3; deimination in arginine 161 only found in component C3; deamidation of glutamine 120 was only observed in C3. All four deiminated arginines and one acetylated lysine were first experimentally revealed in this study for bovine MBP. Mascot database searching combined with de novo sequence analysis of rattlesnake MBP provided more than 85% sequence coverage. A few PTMs were also revealed in rattlesnake MBP: mono- and dimethylated Arg, protein N-terminal acetylation, and deiminated Arg. Overall, snake MBP was found to undergo less modification than bovine MBP on the basis of the mass heterogeneity of the intact protein, the bottom-up structure analysis, and the limited complexity of rattlesnake MBP chromatography. The combined data from this study and information from previous studies extend the known MBP PTMs, and PTMs unique to higher vertebrates are proposed.

Mapping protein interactions and their dynamics is crucial to defining physiologic states, building effective models for understanding cell function, and to allow more effective targeting of new drugs. Crosslinking studies can estimate the proximity of proteins, determine sites of protein–protein interactions, and have the potential to provide a snapshot of dynamic interactions by covalently locking them in place for analysis. Several major challenges are associated with the use of crosslinkers in mass spectrometry, particularly in complex mixtures. We describe the synthesis and characterization of a MS-cleavable crosslinker containing cyclic amines, which address some of these challenges. The DC4 crosslinker contains two intrinsic positive charges, which allow crosslinked peptides to fragment into their component peptides by collision-induced dissociation (CID) or in-source decay. Initial fragmentation events result in cleavage on either side of the positive charges so crosslinked peptides are identified as pairs of ions separated by defined masses. The structures of the component peptides can then be robustly determined by MS3 because their fragmentation products rearrange to generate a mobile proton. The DC4 crosslinking reagent is stable to storage, highly reactive, highly soluble (1 M solutions), quite labile to CID, and MS3 results in productive backbone fragmentation.

ProteomeCommons.org has implemented a resource that incorporates concepts of Web 2.0 social networking for collaborative annotation of data sets placed in the Tranche repository. The annotation tools are part of a project management resource that is effective for individual laboratories or large distributed groups. The creation of the resource was motivated by the need for a way to encourage annotation of data sets with high accuracy and compliance rates. The system is designed to respond to the dynamic nature of research in an easy-to-use fashion through the use of a dynamic data model that does not inhibit the innovation that is important for basic research. Placing the annotation tool within a project manager allows annotation to occur over the life of the project and provides the security and monitoring capabilities needed for large or small collaborative projects. The resource effectively supports distributed groups of investigators working on common data sets and is available immediately at https://ProteomeCommons.org. In addition, a silver compliant data resource based on ProteomeCommons.org has been developed for cancer Biomedical Informatics Grid (caBIG) to allow much broader access to the annotations describing data sets in the Tranche repository.

Unidentified tandem mass spectra typically represent 50−90% of the spectra acquired in proteomics studies. This manuscript describes a novel algorithm, “Bonanza”, for clustering spectra without knowledge of peptide or protein identifications. Further analysis leverages existing peptide identifications to infer related, likely valid identifications. Significantly more spectra can be identified with this approach, including spectra with unexpected potential modifications or amino-acid substitutions.

A current focus of proteomics research is the establishment of acceptable confidence measures in the assignment of protein identifications in an unknown sample. Development of new algorithmic approaches would greatly benefit from a standard reference set of spectra for known proteins for the purpose of testing and training. Here we describe an openly available library of mass spectra generated on an ABI 4700 MALDI TOF/TOF from 246 known, individually purified and trypsin-digested protein samples. The initial full release of the Aurum Dataset includes gel images, peak lists, spectra, search result files, decoy database analysis files, FASTA file of protein sequences, manual curation, and summary pages describing protein coverage and peptides matched by MS/MS followed by decoy database analysis using Mascot, Sequest, and X!Tandem. The data are publicly available for use at ProteomeCommons.org.

A current focus of proteomics research is the establishment of acceptable confidence measures in the assignment of protein identifications in an unknown sample. Development of new algorithmic approaches would greatly benefit from a standard reference set of spectra for known proteins for the purpose of testing and training. Here we describe an openly available library of mass spectra generated on an ABI 4700 MALDI TOF/TOF from 246 known, individually purified and trypsin-digested protein samples. The initial full release of the Aurum Dataset includes gel images, peak lists, spectra, search result files, decoy database analysis files, FASTA file of protein sequences, manual curation, and summary pages describing protein coverage and peptides matched by MS/MS followed by decoy database analysis using Mascot, Sequest, and X!Tandem. The data are publicly available for use at ProteomeCommons.org.

Quantitative measurement of specific protein phosphorylation sites is a primary interest of biologists, as site-specific phosphorylation information provides insights into cell signaling networks and cellular dynamics at a system level. Over the last decade, selective phosphopeptide enrichment methods including IMAC and metal oxides (TiO2 and ZrO2) have been developed and greatly facilitate large scale phosphoproteome analysis of various cells, tissues and living organisms, in combination with modern mass spectrometers featuring high mass accuracy and high mass resolution. Various quantification strategies have been applied to detecting relative changes in expression of proteins, peptides, and specific modifications between samples. The combination of mass spectrometry-based phosphoproteome analysis with quantification strategies provides a straightforward and unbiased method to identify and quantify site-specific phosphorylation. We describe common strategies for mass spectrometric analysis of stable isotope labeled samples, as well as two widely applied phosphopeptide enrichment methods based on IMAC(NTA–Fe3+) and metal oxide (ZrO2). Instrumental configurations for on-line LC–tandem mass spectrometric analysis and parameters of conventional bioinformatic analysis of large data sets are also considered for confident identification, localization, and reliable quantification of site-specific phosphorylation.

Mass spectrometry has made major contributions to recent discoveries in the field of epigenetics, particularly in the characterization of the myriad post-translational modifications (PTMs) of histones which are technically challenging to analyze. These new developments have further aroused great interest in development of robust, new mass spectrometric methods to quantitatively study the dynamics of histone modifications. This review covers quantitative analysis of histone PTMs and discuss an 15N metabolic labeling procedure for quantifying histone PTMs applied to the analysis of methyltransferase knockouts in the model organism, Tetrahymena thermophila.