Dataset Information

MESC shotgun and positional proteomics based on deep proteome sequence database (derived from RIBOseq data)

ABSTRACT: Shotgun and positional proteomics study of a mouse embryonic stem cell line. We devised a proteogenomic approach constructing a custom protein sequence search space, built from both SwissProt and RIBO-seq derived translation products, applicable for LC-MSMS spectrum identification. To record the impact of using the constructed deep proteome database we performed two alternative MS-based proteomic strategies: (I) a regular shotgun proteomic and (II) an N-terminal COFRADIC approach. The obtained fragmentation spectra were searched against the custom database (combination of UniProtKB-SwissProt and RIBO-seq derived translation sequences) using three different search engines: OMSSA (version 2.1.9), X!Tandem (TORNADO, version 2010.01.01.04) and Mascot (version 2.3). The first two were run from the SearchGUI graphical user interface (version 1.10.4). A combination of X!Tandem and Mascot was used for the N-terminal COFRADIC analysis, a combination of all three search engines for the shotgun proteome analysis. Note that OMMSA cannot cope with the protease setting semi-ArgC/P needed to analyze N-terminal COFRADIC data.For the shotgun proteome data, trypsin was set as cleavage enzyme allowing for one missed cleavage, and singly to triply charged precursors or singly to quadruple charged precursors were taken into account respectively for the Mascot or X!Tandem/OMSSA search engines, and the precursor and fragment mass tolerance were set to respectively 10 ppm and 0.5 Da. Methionine oxidation to methionine-sulfoxide, pyroglutamate formation of N-terminal glutamine and acetylation (protein N-terminus) were set as variable modifications. For the N-terminal COFRADIC analysis the protease setting semi-ArgC/P (Arg-C specificity with arginine-proline cleavage allowed) was used. No missed cleavages were allowed and the precursor and fragment mass tolerance were also set to respectively 10 ppm and 0.5 Da. Carbamidomethylation of cysteine and methionine oxidation to methionine-sulfoxide and 13C3D2-acetylation of lysines were set as fixed modifications. Peptide N-terminal acetylation or 13C3D2-acetylation and pyroglutamate formation of N-terminal glutamine were set as variable modifications and instrument setting was put on ESI-TRAP. Protein and peptide identification in addition to data interpretation was done using the PeptideShaker algorithm (http://code.google.com/p/peptide-shaker, version 0.18.3), setting the false discovery rate to 1% at all levels (protein, peptide, and peptide to spectrum matching). Aforementioned tools and algorithms (SearchGui, X!Tandem, OMSSA, and PeptideShaker) are freely available as open source.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Es-e14 Cell, Stem Cell, Cell Culture

SUBMITTER: Gerben Menschaert

LAB HEAD: Gerben Menschaert

PROVIDER: PXD000124 | Pride | 2013-02-25

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	L22595_2008_Petra_mESC_Nter_28-32-1.RAW	Raw
	L22596_2008_Petra_mESC_Nter_28-32-2.RAW	Raw
	L22597_2008_Petra_mESC_Nter_28-32-3.RAW	Raw
	L22598_2008_Petra_mESC_Nter_28-32-4.RAW	Raw
	L22599_2008_Petra_mESC_Nter_28-32-5.RAW	Raw

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Publications

Deep proteome coverage based on ribosome profiling aids mass spectrometry-based protein and peptide discovery and provides evidence of alternative translation products and near-cognate translation initiation events.

Menschaert Gerben G Van Criekinge Wim W Notelaers Tineke T Koch Alexander A Crappé Jeroen J Gevaert Kris K Van Damme Petra P

Molecular & cellular proteomics : MCP 20130221 7

An increasing number of studies involve integrative analysis of gene and protein expression data, taking advantage of new technologies such as next-generation transcriptome sequencing and highly sensitive mass spectrometry (MS) instrumentation. Recently, a strategy, termed ribosome profiling (or RIBO-seq), based on deep sequencing of ribosome-protected mRNA fragments, indirectly monitoring protein synthesis, has been described. We devised a proteogenomic approach constructing a custom protein se ...[more]

PMID: 23429522

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Project description:The use of internal calibrants (the so called lock mass approach) provides much greater accuracy in mass spectrometry based proteomics. However, the polydimethylcyclosiloxane (PCM) peaks commonly used for this purpose are quite unreliable, leading to missing calibrant peaks in spectra and correspondingly lower mass measurement accuracy. Therefore, we here introduce a universally applicable and robust internal calibrant, the tripeptide Asn3. We show that Asn3 is a substantial improvement over PCM both in terms of consistent detection and resulting mass measurement accuracy. Asn3 is also very easy to adopt in the lab, as it requires only minor adjustments to the analytical setup. Data analysis: For mass measurement accuracy (MMA) calculations and comparisons, the following Mascot workflow was used. From the MS/MS data in each LC run, Mascot Generic Files were created using Distiller software (version 2.4.3.3, Matrix Science, London, UK, www.matrixscience.com/distiller.html). These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.4.0, Matrix Science). Spectra were searched against the Swiss-Prot database (version 13_04 of UniProtKB/Swiss-Prot protein database containing 20,232 sequence entries of human proteins) concatenated with its reversed sequence database. Variable modifications were set to pyro-glutamate formation of amino terminal glutamine and acetylation of the protein N-terminus, whereas fixed modifications only included oxidation of methionine. Mass tolerance on peptide ions was set to 10 ppm (with Mascot’s C13 option set to 1), and the mass tolerance on peptide fragment ions was set to 20 millimass units (mmu), except for the space-charge effect experiment(LMA5) where an extra search was done with a setting of 3 mmu. The peptide charge was set to 1+,2+,3+ and instrument setting was put on ESI-QUAD. Enzyme was set to trypsin allowing for one missed cleavage, and cleavage was allowed when arginine or lysine is followed by proline. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. All data was processed and managed by ms_lims.

Project description:Abstract still has to be written. The obtained peptide mixtures were introduced into an LC-MS/MS system, the Ultimate 3000 (Dionex, Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples were first loaded on a trapping column (made in-house, 100 um internal diameter (I.D.) x 20 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 um I.D. x 150 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). Peptides were loaded with solvent A (0.1% trifluoroacetic acid, 2% acetonitrile), and were separated with a linear gradient from 2% solvent A' (0.05% formic acid) to 55% solvent B' (0.05% formic acid and 80% acetonitrile) at a flow rate of 300 nL/min followed by a wash reaching 100% solvent B'. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the six most abundant peaks in a given MS spectrum. Full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The six most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 60 s. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts. From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.3.01, Matrix Science). While generating these peak lists, grouping of spectra was allowed with a maximum intermediate retention time of 30 s and a maximum intermediate scan count of 5 was used where possible. Grouping was done with 0.005 Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal to noise limit was set at 2. These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.3, Matrix Science). Spectra were searched against the human (H. sapiens) Swiss-Prot database. 13C2D3-acetylation of lysine side-chains, carbamidomethylation of cysteine and methionine oxidation to methionine-sulfoxide were set as fixed modifications for the N-terminal COFRADIC analyses. Variable modifications were 13C2D3-acetylation and acetylation of protein N-termini. Pyroglutamate formation of N-terminal glutamine was additionally set as a variable modification. Mass tolerance on precursor ions was set to 10 ppm (with Mascot's C13 option set to 1) and on fragment ions to 0.5 Da. Endoproteinase semi-Arg-C/P (Arg-C specificity with arginine-proline cleavage allowed) was set as enzyme allowing no missed cleavages. The peptide charge was set to 1+, 2+, 3+ and instrument setting was put to ESI-TRAP. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. Quantification of the degree of Nt-Acetylation was performed as described previously (1). All data management was done in ms_lims (2).

Project description:Abstract: N-terminal acetylation (Nt-acetylation) occurs on the majority of eukaryotic proteins and is catalysed by N-terminal acetyltransferases (NATs). Nt- acetylation is increasingly recognized as a vital modification with functional implications ranging from protein degradation to protein localization. Very recently, the first human X-linked genetic disorder caused by a mutation in a NAT gene was reported; Ogden syndrome boys harbour a p.Ser37Pro variant in the gene encoding Naa10, the catalytic subunit of the NatA complex, and suffer from global developmental delays and lethality during infancy. Here, we developed a Saccharomyces cerevisiae model by introducing the human wildtype or mutant NatA complex into yeast lacking NatA (NatA-∆). The human NatA complex phenotypically complemented the NatA-∆ strain, while only a partial rescue was observed for the Ogden mutant NatA complex suggesting that hNaa10-S37P is only partially functional in vivo. Furthermore, we performed quantitative Nt-acetylome analyses on a control yeast strain (yNatA), a yeast NatA deletion strain (yNatA-∆), a yeast NatA deletion strain expressing human NatA (hNatA), and a yeast NatA deletion strain expressing mutant human NatA (hNatA-hNaa10-S37P). Interestingly, a reduced degree of Nt-acetylation was specifically observed among a large group of NatA substrates in the yeast expressing mutant hNatA as compared to yeast expressing wildtype hNatA. Furthermore, immunoprecipitated mutant NatA complex displayed a reduced catalytic activity in vitro as compared to the wildtype NatA complex. Combined, these data provide strong support for the functional impairment of hNaa10-S37P in vivo and suggest that reduced Nt-acetylation of one or more target substrates contributes to the pathogenesis of Ogden syndrome. Comparative analysis between human and yeast NatA also provided novel insights into the co-evolution of the NatA complexes and their substrates. For instance, (Met-)Ala- N- termini are more prevalent in the human proteome as compared to the yeast proteome, and hNatA displays a relative preference towards these N-termini as compared to yNatA. Methods: The obtained peptide mixtures were introduced into an LC-MS/MS system, the Ultimate 3000 (Dionex, Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap XL (Thermo Fisher Scientific, Bremen, Germany). Samples were first loaded on a trapping column (made in-house, 100 µm internal diameter (I.D.) x 20 mm, 5 µm beads C18 Reprosil-HD, Dr. Maisch). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in- house, 75 µm I.D. x 150 mm , 5 µm beads C18 Reprosil-HD, Dr. Maisch). Peptides were loaded with solvent A (0.1% trifluoroacetic acid, 2% acetonitrile), and were separated with a linear gradient from 2% solvent A’ (0.05% formic acid) to 55% solvent B’ (0.05% formic acid and 80% acetonitrile) at a flow rate of 300 nl/min followed by a wash reaching 100% solvent B’. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the six most abundant peaks in a given MS spectrum. Full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The six most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 60 s. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts. From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.3.01, Matrix Science). 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Variable modifications were 13C2D3- acetylation and acetylation of protein N-termini. Pyroglutamate formation of N-terminal glutamine was additionally set as a variable modification. Mass tolerance on precursor ions was set to 10 ppm (with Mascot’s C13 option set to 1) and on fragment ions to 0.5 Da. Endoproteinase semi-Arg-C/P (Arg-C specificity with arginine-proline cleavage allowed) was set as enzyme allowing no missed cleavages. The peptide charge was set to 1+, 2+, 3+ and instrument setting was put on ESI-TRAP. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. The estimated false discovery rate by searching decoy databases was typically found to lie between 2 and 4% on the spectrum level [33]. Quantification of the degree of Nt-Ac was performed as described previously (5). All data management was done in ms_lims ([49]). Please note!! the pride result files will show a lot of delta m/z deviations. The modification for the heavy acetylation (+47 Da) used in this experiment is not in Pride. So another heavy acetylation (+45 Da) modification was annotated when creating the xml files.