Dataset Information

Developing Soybean Seed Lysine-N-epsilon-acetylome

ABSTRACT: Intact proteins, as well as Glu-C, and Lys-C/trypsin-derived peptides extracted from developing soybean seeds were immunoenriched using antibodies against acetyllysine residues. Enriched proteins were separated by SDS-PAGE prior to tryptic digestions and enriched peptides were analyzed directly, in both cases using a Thermo LTQ-Orbitrap XL. In separate LC-MS/MS runs fragmentation was achieved using CID, ETD, or a combination of the two mediated by a data-dependent decision tree. Individual RAW files were converted to mzXML format for mining with MS-GFDB (v2012.06.07) using default settings of the msconvert executable within the Trans-Proteomic Pipeline v4.6. Searches with MS-GFDB were performed with default methods and parameters, except as noted: number of allowed isotope errors was set at zero, and fully enzymatic peptides were specified, i.e. no non-enzymatic termini. Data mining was additionally performed with a local Mascot server, v2.3.1, and ZCore v1.17, both accessed through Proteome Discoverer v1.3 (Thermo Scientific), as well as Sequest HT v1.3, a reimplementation of SEQUEST within Proteome Discoverer v1.4. Tandem mass spectra were searched in Proteome Discoverer with allowed missed cleavages set to four. Prior to Mascot and Sequest searches, ETD spectra were further filtered to remove peaks resulting from precursors, charge-reduced precursors, and neutral losses thereof with mass windows of 4 Da, 2 Da, and 2 Da, respectively.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Glycine Max

SUBMITTER: Colin Smith-Hammond

PROVIDER: PXD000192 | Pride | 2013-12-02

REPOSITORIES: Pride

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Publications

Initial description of the developing soybean seed protein Lys-N(ε)-acetylome.

Smith-Hammond Colin L CL Swatek Kirby N KN Johnston Mark L ML Thelen Jay J JJ Miernyk Ján A JA

Journal of proteomics 20131107

Characterization of the myriad protein posttranslational modifications (PTM) is a key aspect of proteome profiling. While there have been previous studies of the developing soybean seed phospho-proteome, herein we present the first analysis of non-histone lysine-N(Ɛ)-acetylation in this system. In recent years there have been reports that lysine acetylation is widespread, affecting thousands of proteins in diverse species from bacteria to mammals. Recently preliminary descriptions of the protein ...[more]

PMID: 24211405

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Project description:Here we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the cAMP-dependent protein kinase A (PKA) consensus motif. This targeted phospho-proteomic strategy is used to profile temporal quantitative changes of protein PKA substrates in Jurkat T-lymphocytes upon prostaglandin E2 stimulation, which increases intracellular cAMP activating PKA. Our method combines ultra-high specificity PKA-motif-based immunoaffinity purification with cost-efficient stable isotope dimethyl labeling. The data set coprises of 4 raw files, 3 of them (IP, IP_2 and IP_3) are the LC-MSMS technical replicates of the eluate of the immunoprecipitation, while the MIX raw files correpsond to the LC-MSMS analysis of the pre-IP cell lysate digest.Each raw data file was processed and quantified with Proteome Discoverer (version 1.3, Thermo Scientific). Peak lists containing HCD and ETD fragmentation were generated with a signal-to-noise threshold of 1.5. The ETD non-fragment filter was also taken into account with the following settings: the precursor peak was removed within a 4 Da window, charged reduced precursors were removed within a 2 Da window, and neutral losses from charged reduced precursors were removed within a 2 Da window and the maximum neutral loss mass was set to 120 Da. All generated peak lists of the IP were searched against a concatenated forward-decoy Swissprot human database (version 2012_09, 40,992 sequences) while the MIX file was searched against a Swissprot database version 2012_09 with taxonomy Homo sapiens (20,235 sequences) by the use of Mascot software (version 2.3.02 Matrix Science). The database search was performed with the following parameters: a mass tolerance of ±50 ppm for precursor masses; ±0.6 Da for ETD-ion trap fragment ions; ±0.05 Da for HCD and ETD-Orbi trap fragment ions, allowing two missed cleavages, cysteine carbamidomethylation as fixed modification. Light, intermediate and heavy dimethylation of peptide N-termini and lysine residues; methionine oxidation; phosphorylation on serine and threonine (only for the IP) were set as variable modifications. The enzyme was specified as Lys-C and the fragment ion type was specified as electrospray ionization FTMS-ECD, ETD-TRAP, and ESI-QUAD-TOF for the corresponding mass spectra. The phosphorylation site localization of the identified phosphopeptides was performed by the phosphoRS algorithm 2.0. The dimethyl-based quantitation method was chosen in Proteome Discoverer, with mass precision requirement of 2 ppm for consecutive precursor mass measurements. We applied 0.5 min of retention time tolerance for isotope pattern multiplets and allowed spectra with maximum 1 missing channels to be quantified. After identification and quantification, we combined all results originating from the same biological replica and filtered them with the following criteria: (i) mass deviations of ±10 ppm, (ii) Mascot Ion Score of at least 20, (iii) a minimum of 6 amino-acid residues per peptide and (iv) position rank 1. Mascot results of the MIX analysis were filtered with the same parameters and with the integrated Percolator based filter using an FDR <1% (based on PSMs).

Dataset Information

Developing Soybean Seed Lysine-N-epsilon-acetylome

Publications

Initial description of the developing soybean seed protein Lys-N(ε)-acetylome.

Similar Datasets

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