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Lipidomics analysis of senescent SK-MEL103 cells

ABSTRACT: Lipidomics analysis of senescent SK-MEL103 cells and cells treated with GPX4 inhibitor RSL3 or SCLA2 (n=3). RAW datafiles were centroided and converted using MSConvert to mzXML format. Toza = tozasertib; Eto = etoposide; Fer = Ferrostatin; O17 = SCLA2

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Zoe Hall

PROVIDER: MSV000099167 | MassIVE |

REPOSITORIES: MassIVE

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Project description:Protein complexes and protein interaction networks are essential mediators of most biological functions. Complexes supporting transient functions such as signal transduction processes are frequently subject to dynamic remodeling. Currently, the majority of studies into the composition of protein complexes are carried out by affinity purification and mass spectrometry (AP-MS) and present a static view of the system. Therefore, to better understand inherently dynamic biological processes, methods to reliably quantify temporal changes of protein interaction networks are essential. In this study we used affinity purification combined with SWATH mass spectrometry (AP-SWATH) to study the dynamics of the 14-3-3B scaffold protein interactome after stimulation of the insulin/PI3K/AKT pathway.. The consistent and reproducible quantification of 1967 proteins across all stimulation time points provided new insights into the 14-3-3B interactome and its dynamic change following IGF1 stimulation. We therefore establish AP-SWATH as a tool to quantify dynamic changes in protein complex interaction networks. Data analysis: Profile mode wiff files from shotgun data acquisition were centroided and converted to mzML using the AB Sciex Data Converter v1.3, and further converted to mzXML using ProteoWizard67 MSConvert v3.04.238. Mass spectra were queried against the canonical UniProt complete proteome database for human (July 2011) appended with common contaminants and reversed sequence decoys (40,548 protein sequences including decoys) using XTandem with kscore plugin46, and additionally XTandem with native scoring47. Carbamidomethyl was set as fixed modification for cysteines, and methionine oxidation and phosphorylation on serine/threonine/tyrosine were set as variable modification. Semi-tryptic peptides and peptides with up to 2 missed cleavages were allowed, and mass error was set as 30 ppm and 75 ppm for precursor and product ions respectively. Results from both search engines were individually converted to the pepXML format using Tandem2XML v4.6.0, analysed with PeptideProphet48 v4.6.0, and combined using iProphet49 v4.6.0 including false identification rate calculation. Phosphosite localisation probabilities were estimated using PTMProphet v4.6.1 .

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Lipidomics analysis of senescent SK-MEL103 cells

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