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For top-down protein database search and identification from tandem mass spectra, our isotopic envelope fingerprinting search algorithm and ProteinGoggle search engine have demonstrated their strength of efficiently resolving heavily overlapping data as well separating non-ideal data with non-ideal isotopic envelopes from ideal ones with ideal isotopic envelopes. Here we report our updated ProteinGoggle 2.0 for intact protein database search with full-capacity. The indispensable updates include users' optional definition of dynamic post-translational modifications and static chemical labeling during database creation, comprehensive dissociation methods and ion series, as well as a Proteoform Score for each proteoform. ProteinGoggle has previously been benchmarked with both collision-based dissociation (CID, HCD) and electron-based dissociation (ETD) data of either intact proteins or intact proteomes. Here we report our further benchmarking of the new version of ProteinGoggle with publically available photon-based dissociation (UVPD) data ( http://hdl.handle.net/2022/17316 ) of intact E. coli ribosomal proteins. Biological significance Protein species (aka proteoforms) function at their molecular level, and diverse structures and biological roles of every proteoform come from often co-occurring proteolysis, amino acid variation and post-translational modifications. Complete and high-throughput capture of this combinatorial information of proteoforms has become possible in evolving top-down proteomics; yet, various methods and technologies, especially database search and bioinformatics identification tools, in the top-down pipeline are still in their infancy stages and demand intensive research and development. [ABSTRACT FROM AUTHOR]

N-linked glycosylation is one of the most common protein PTMs, and the topological structure (monosaccharide composition and sequence as well as glycosidic linkages) of N-glycans is vital information to understand their biological functions and roles. Tandem mass spectrometry has been widely used for topological structure characterization of N-glycans, where comprehensive understanding of fragmentation pathways and characteristics of product ions are essential to achieve best interpretation of MS/MS data and highest confidence of identification. Here, we report our glycomic study of N-glycome of mouse brain as well as fragmentation pathway analysis of the identified N-glycans. With LC-MS/MS analysis at both the positive and negative ESI modes together with our recently developed N-glycan database search engine GlySeeker, 221 unique N-glycans with putative topological structures were identified with target-decoy searches and number of best hits of 1. Analysis of fragmentation pathways and characteristics of product ions of permethylated N-glycans in the positive mode and native N-glycans in the negative mode were further carried out. The reported N-glycans serve as a basic reference for future glycosylation study of mouse brain; and in general database search of tandem mass spectra of N-glycans, B/Y/Z ions should be preferentially considered for the permethylated form in the positive mode and B/C/Z ions for the native form in the negative mode. [ABSTRACT FROM AUTHOR]

Rationale: Most of the current popular tandem mass spectrometers have the capability of resolving the primary structures (monosaccharide composition, sequence and linkage) of N-glycans; however, compositions or putative structures have mostly been reported so far. Identification and visualization tools of N-glycans are needed. Methods: The isotopic mass-to-charge ratio and envelope fingerprinting algorithm, which has been successfully used for intact protein database search and identification, was adapted for N-glycan database search, and a stand-alone N-glycan database search engine, GlySeeker, for automated N-glycan identification and visualization was developed and successfully benchmarked. Both pseudo 2D graph and one-line text formats with one-letter symbols for monosaccharides were proposed for representing N-glycans. N-glycans were identified with comprehensive interpretation of product ions and false discovery rate (FDR) control. Results: In a database search of reversed-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS) datasets of the N-glycome enriched from OVCAR-3 ovarian cancer cells, with FDR ≤1% and number of best hits (NoBHs) = 1-30, 1525 N-glycans with comprehensive primary structural information (composition, sequence and linkage) were identified and visualized; among these 1525 N-glycans, 559 had NoBHs = 1, i.e. their structures were uniquely identified. This represents a large-scale identification and visualization of N-glycans with primary structures from tandem mass spectra. Conclusions: A stand-alone N-glycan database search engine called GlySeeker has been developed for large-scale identification and visualization of N-glycans with comprehensive interpretation of tandem mass spectra and FDR control. [ABSTRACT FROM AUTHOR]

Phosphorylation is one of the most important and widely studied protein post-translational modifications. Tandem mass spectrometry using higher-energy collisional dissociation has evolved into a state-of-the-art analytical platform for both phosphorylation identification and site localization. Tens of thousands of phosphopeptides can now be routinely identified from a single shotgun proteomics study; site localization, however, is much more complicated and many challenges still exist. Here, we report our development of P-bracket using direct experimental evidence of phospho-containing site-determining product ions for accurate site localization without the need for additional FLR control. A P-bracket is defined as a complementary product ion pair that forms a bracket to confine a phosphorylation event to a unique site. P-bracket has been successfully benchmarked with a set of six synthetic phosphopeptides with a single phosphorylation event, a set of 96 synthetic peptides and phosphopeptide reference libraries, and two HeLa phosphopeptide LC-MS/MS (HCD) datasets; Accurate phosphosite localization by P-bracket will greatly enhance identification confidence of phosphopeptides and contribute to structural and functional studies of phosphoproteins. [ABSTRACT FROM AUTHOR]

Neutral loss and internal product ions have been found to be significant in both peptide and protein tandem mass spectra and they have been proposed to be included in database search and for protein identification. In addition to common canonical b/y ions in collision-based dissociation or c/z ions in electron-based dissociation, inclusion of neutral loss and internal product ions would certainly make better use of tandem mass spectra data; however, their ultimate utility for protein identification with false discovery rate control remains unclear. Here we report our proteome-level utility benchmarking of neutral loss and internal product ions with tandem mass spectra of intact E. coli proteome. Utility of internal product ions was further evaluated at the protein level using selected tandem mass spectra of individual E. coli proteins. We found that both neutral loss and internal products ions do not have direct utility for protein identification when they were used for scoring of P Score; but they do have indirect utility for provision of more canonical b/y ions when they are included in the database search and overlapping ions between different ion types are resolved. Biological significance Tandem mass spectrometry has evolved to be a state-of-the-art method for characterization of protein primary structures (including amino acid sequence, post-translational modifications (PTMs) as well as their site location), where full study and utilization tandem mass spectra and product ions are indispensable. This primary structure information is essential for higher order structure and eventual function study of proteins. [ABSTRACT FROM AUTHOR]

Rationale The mass measurement accuracy (MMA) of Orbitrap mass spectrometers is 1-5 ppm according to the manufacturer's specification; yet, up to 50 ppm has been used as mass tolerance to interpret Orbitrap data in the literature. A systematic evaluation of MMA is thus necessary to find the optimal mass tolerance to be used. Methods Reversed-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS) analyses of the intact E. coli proteome were carried out on a Q Exactive Orbitrap mass spectrometer coupled to a Dionex UltiMate 3000 RSLCnano system. The analysis included three technical replicates each day and was repeated for six continuous days right after a mass calibration. The obtained raw datasets were searched using ProteinGoggle 2.0 under four different mass tolerances of 5, 10, 15, and 20 ppm. Results With both forward and random database searches and FDR ≤1% at the spectrum level, the most protein spectrum matches and protein IDs were obtained at a mass tolerance of 15 ppm. The average mass accuracy of both precursor and product ions from three representative high, medium, and low abundance proteins as well as the common proteins identified in all the 18 replicate runs was found to be 0-4 ppm; and no significant drift of measured mass accuracy was observed within the calibration period of 1 week. Conclusions Despite the mass measurement accuracy of 1-5 ppm of the Orbitrap stated by the manufacturer, the optimal mass tolerance for protein identification was found to be 15 ppm for both the precursor and product ions. Weekly mass calibration is appropriate because no significant drift in MMA was found within the 6-day period. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]