Dataset Information

Quantitative proteomics profiling of protein kinase A substrates - Jurkat clone E6.1

ABSTRACT: 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).

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Piero Giansanti

PROVIDER: PXD000148 | Pride | 2013-07-25

REPOSITORIES: Pride

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Publications

Interrogating cAMP-dependent kinase signaling in Jurkat T cells via a protein kinase A targeted immune-precipitation phosphoproteomics approach.

Giansanti Piero P Stokes Matthew P MP Silva Jeffrey C JC Scholten Arjen A Heck Albert J R AJ

Molecular & cellular proteomics : MCP 20130723 11

In the past decade, mass-spectrometry-based methods have emerged for the quantitative profiling of dynamic changes in protein phosphorylation, allowing the behavior of thousands of phosphorylation sites to be monitored in a single experiment. However, when one is interested in specific signaling pathways, such shotgun methodologies are not ideal because they lack selectivity and are not cost and time efficient with respect to instrument and data analysis time. Here we evaluate and explore a pept ...[more]

PMID: 23882029

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Project description:We combine high-resolution mass spectrometry with Ti4+-IMAC phosphopeptide enrichment and label-free quantification to monitor the phosphoproteome of Jurkat T-cells following stimulation by Prostaglandin E2. Jurkat T lymphoma cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (Lonza). For PGE2 stimulation, cells were centrifuged for 1 min at 1500g. The growth medium was removed and the cells were resuspended at a final concentration of 1-2 × 106 cells/ml in RMPI and supplemented with 0 (control) or 10 uM PGE2 and incubated for 5, 10, 20, 30 or 60 min. After cell lysis, reduction and alkylation, proteins were digested with Lys-C and Trypsin. The digests were desalted using Sep-Pak C18 cartridges, dried in vacuo and stored at −80 °C for further use. Phosphopeptide enrichment was performed as described in Zhou H, Ye M, Dong J, Corradini E, Cristobal A, Heck AJ, Zou H, Mohammed S. Nat Protoc. 2013 Mar;8(3):461-80. The enriched phosphopeptides were subjected to a reversed phase nano-LC–MS/MS analysis consisting of a Proxeon EASY-nLC 1000, an analytical column heater (40°C) and an LTQ-Orbitrap Elite. After the survey scans, the 20 most intense precursors were selected for subsequent CID or ETD-IT fragmentation. A programmed data-dependent decision tree determined the choice of the most appropriate technique for a selected precursor. In essence, doubly charged peptides were subjected to CID fragmentation and more highly charged peptides were fragmented using ETD. Raw data were processed with MaxQuant version 1.3.0.523, and the peptides were identified from the MS and MS/MS spectra searched against a concatenated forward-decoy Swissprot Homo sapiens database version 2012_09 (40,992 sequences) using the Andromeda search engine. The database search was performed with the following parameters: an initial mass tolerance of ±20 ppm for precursor masses; final mass tolerance of ±6 ppm: ±0.6 Da for CID and ETD-ion trap fragment ions, allowing two missed cleavages. Cysteine carbamidomethylation was used as a fixed modification and methionine oxidation, protein N-terminal acetylation and serine, threonine and tyrosine phosphorylation as variable modifications. For the identification, the false discovery rate was set to 0.01 for peptides, proteins and sites and the minimum peptide length allowed was six amino acids and a minimum peptide score of 60. The match between run feature was set on. A site localization probability of at least 0.75 and a score difference of at least 5 were used as threshold for the phosphoresidue localization. Normalization was performed by subtracting the median of log transformed intensities for each LC-MS/MS run.

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Dataset Information

Quantitative proteomics profiling of protein kinase A substrates - Jurkat clone E6.1

Publications

Interrogating cAMP-dependent kinase signaling in Jurkat T cells via a protein kinase A targeted immune-precipitation phosphoproteomics approach.

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