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IBA adipocyte phosphoproteomics

ABSTRACT: Library was constructed from 2 runs of Hph fractionated pooled sample after phosphopeptide enrichment. One run had FAIMS setting -40, -60, -80 V and the other run was -50, -70, -90 V. Data was collected on the samples using the -40,-60, -80 V settings.

INSTRUMENT(S): Orbitrap Eclipse (Thermo Scientific )

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Blaine Roberts

PROVIDER: MSV000086750 | MassIVE | Fri Jan 22 14:07:00 GMT 2021

SECONDARY ACCESSION(S): PXD023762

REPOSITORIES: MassIVE

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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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IBA adipocyte phosphoproteomics

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