ProSight PTM (https://prosightptm.scs.uiuc.edu/) is a web application for identification and characterization of proteins using mass spectra data from 'top-down' fragmentation of intact protein ions (i.e. without any tryptic digestion). ProSight PTM has many tools and graphical features to facilitate analysis of single proteins, proteins in mixtures and proteins fragmented in parallel. Sequence databases from across the phylogenetic tree are supported, with a new database strategy of 'shotgun annotation' used to assist characterization of wild-type proteins. During a database search, data from divergent sources regarding potential mass differences such as polymorphisms, alternate splicing and post-translational modifications are utilized. The user can optionally control how much of this biological variability should be searched.

Intimately associated with DNA, histone proteins serve as both a structural scaffold for DNA packaging into the nucleus and an epigenetic means for the regulation of gene expression. One such histone-based mechanism for transcriptional regulation is posttranslational modification (PTM) of histones H2A, H2B, H3, and H4. 1,2 Combinations of modifications such as acetylation, methylation, and phosphorylation create a "Histone Code" that influences gene transcription, gene silencing, and chromatin formation. 3-5 Essential for complete understanding of this code is an efficient methodology for detection, exact localization, and quantitation of modifications at specific sites. We combine here gas-phase concentration and purification 6 inside a quadrupole-FTMS hybrid (Q-FTMS) with top down fragmentation using electron capture dissociation (ECD) 7,8 and large-scale PTM prediction. This prediction uses a new type of protein database that has been "shotgun annotated" by assigning site-specific posttranslational modifications (and all their combinations) prior to searching for best matches with ECD data. The approach considers PTMs during a database search and enables complete and automated characterization of human histones harboring 2-6 PTMs from asynchronous and butyrate-treated HeLa cells.With its sequence and modification sites extensively studied, human histone H4 was chosen as a model. We generated all possible protein forms by combinatorial modification of the seven known sites 1,2 according to following rules: arginine 3 can be mono-or dimethylated, lysines 5,8,12,16, and 20 can be mono-, di-, trimethylated or acetylated, and serine 1 can be phosphorylated. For a given rule set, the possible number of protein forms was calculated by the following equation:where n i is the number of possible PTMs for amino acid i, and f i is the number of occurrences of amino acid i in the sequence allowed to be modified. For histone H4, this generated 3 1 × 5 5 × 2 1 = 18 750 protein forms. Consideration of possible N-terminal acetylation and start methionine on/off increased the total to 46 875. Perl scripts were written to populate all these protein forms into a relational database (7.8 megabyte, MySQL) stored within the architecture of ProSight PTM, a software environment designed for Top Down Proteomics. searches (typically <6 min) were executed with MS/MS data from particular histone forms using ProSight Retriever, an algorithm for probability-based protein identification. 9An ESI/Q-FTMS spectrum of acid-extracted and RPLC-purified histone H4 10 from asynchronous human HeLa cells revealed many potentially modified forms (Figure 1b). An MS/MS spectrum from ECD of a species +112 Da above unmodified H4 gave 91 observed fragment ion masses (Figure 1c). The calibrated fragment ion masses were used to probe the heavily annotated database using tolerances of 5, 15, and 25 ppm (Supporting Information Table 1) with the top 10 hits from the 5 ppm search shown in Figure 2. Of the 91 fragment ions, 50 and 28 match c and z • ions, respec...

For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/).

Gas-phase fractionation (GPF) is an efficient and straightforward method to increase proteome coverage. In this report, optimal m/z ranges were calculated based on genomic complexity and experimental data. Then, theoretical precursor ion densities were calculated in silico from various organisms' genomes and found to corroborate the empirical selection of m/z ranges based on ion density mapping. According to both calculations, the choice of m/z range for most efficient GPF coverage in the lower m/z range should be very narrow and increase as m/z value increases. Next, a systematic LC-MS/MS analysis was performed to confirm this observation. The behavior of data-dependent precursor ion selection and the origin of the observed variability was investigated under three different scan modes of an LTQ-Orbitrap hybrid mass spectrometer. Finally, GPF combined with data-dependent analysis was compared to a targeted, pseudo-multiple reaction monitoring analysis of proteotypic peptides that should be, based on empirical observation of LC-ESI-MS/MS data, detectable. The result of the latter experiment supported our conclusion that data-dependent analysis using rational gas-phase fractionation was sufficient for comprehensive proteomic analysis of the proteotypic peptides in an unfractionated cell lysate.

For more complete characterization of DNA-predicted proteins (including their posttranslational modifications) a ''top-down'' approach using high-resolution tandem MS is forwarded here by its application to methanogens in both hypothesis-driven and discovery modes, with the latter dependent on new automation benchmarks for intact proteins. With proteins isolated from ribosomes and whole-cell lysates of Methanococcus jannaschii (Ϸ1,800 genes) using a 2D protein fractionation method, 72 gene products were identified and characterized with 100% sequence coverage via automated fragmentation of intact protein ions in a custom quadrupole͞Fourier transform hybrid mass spectrometer. Three incorrect start sites and two modifications were found, with one of each determined for MJ0556, a 20-kDa protein with an unknown methylation at Ϸ50% occupancy in stationary phase cells. The separation approach combined with the quadrupole͞Fourier transform hybrid mass spectrometer allowed targeted and efficient comparison of histones from M. jannaschii, Methanosarcina acetivorans (largest Archaeal genome, 5.8 Mb), and yeast. This finding revealed a striking difference in the posttranslational regulation of DNA packaging in Eukarya vs. the Archaea. This study illustrates a significant evolutionary step for the MS tools available for characterization of WT proteins from complex proteomes without proteolysis.T he development of mass spectrometry (MS) to spearhead large-scale protein analysis continues its long maturation toward the global sample coverage achieved routinely with DNA microarrays (1). Of course, the field of proteomics involves a far more complicated measurement challenge, with posttranslational modification (PTM) of proteins one possible source of extra complexity even in Bacterial and Archaeal proteomes. Although identification of thousands of proteins (2, 3) with information about their relative abundance changes (4) is now possible, the task of detecting and localizing protein modifications is far more difficult (5, 6). Recent proteome-scale methods can use tryptic digestion of entire cell lysates into pools of peptides (7), producing mixtures of staggering complexity. Before such ''shotgun'' digestion methods (8), the classical approach of using 2D gels gave a different perspective of the proteome by visualizing intact proteins before their proteolytic digestion (9). Robotic systems now allow fast identification of proteins from 2D gels, but do not readily provide characterization of modifications (10).Recent application of 2D gel technology to the proteome of a thermophilic (85°C) and barophilic methanogen, Methanococcus jannaschii (11), identified 170 proteins from 166 spots in multiple 2D gels. Few proteins ϾpI 8 (16 distinct proteins) or Ͻ15 kDa (22 distinct proteins) were identified. Furthermore, a few potential PTMs were postulated (from identifications of the same protein from multiple spots), but the peptide data from in-gel digestion did not provide direct evidence for the presence or absence of PTMs. In a sepa...