LC-MS data of peptides from p53 isolated by custom immunoaffinity chromatography

ABSTRACT: We exploited a primary human fibroblast system in which the p53 tumor suppressor protein accumulates to high concentrations due to infection by an adenovirus type 5 mutant that lacks the E1B 55kDa E3 ubiquitin ligase, which targets p53 for degradation, to examine select post-translational modifications (PTMs) present on this transcriptionally inactivated endogenous human p53, as well as on p53 activated in response to etoposide treatment. These forms of p53 were isolated from whole cell lysates by immunoaffinity chromatography using a cocktail of monoclonal antibodies, followed by SDS-PAGE, and peptides derived from these species were subjected to nano-ultra-high performance liquid chromatography-MS and MS/MS analyses on high resolution Orbitrap platforms. Proteomics informatics: Raw LC-MS/MS data were processed into mgf-format peaklist files and searched using Mascot (v. 2.2.7) against the human p53 sequence determined by direct sequencing of the p53 gene from cDNA derived from the specific batch of HFFs used in this study (identical to the consensus sequence for UniProt P04637), employing a window of 8 ppm for the precursor and 1.2 Da for the fragment ion species and allowing for <= 3 missed trypsin and semi-trypsin cleavages, carbamidomethylation of cysteines as a fixed modification, with methionine oxidation, N-terminal protein acetylation, phosphorylation of Ser and Thr, acetylation of Lys, methylation, dimethylation, and trimethylation of Lys and Arg, diglycine modification of Lys (ubiquitinylation), and deamidation of Asn and Gln, iteratively as variable modifications. Aggregate search results for each sample were collated and consolidated using Scaffold (vs. 4.0.7) according to the PeptideProphet and ProteinProphet parsimony algorithms implemented therein, and filtered to the 90% peptide confidence level and to a precursor mass accuracy within 2-3 ppm.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Cercopithecus Aethiops (green Monkey) (grivet)

TISSUE(S): Kidney

SUBMITTER: David Perlman  

LAB HEAD: David Perlman

PROVIDER: PXD000464 | Pride | 2013-10-17

REPOSITORIES: Pride