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Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity

ABSTRACT: Tandem mass tag spectrometry was performed on eluted peptides from 8 single cell clones derived from a DNA mismatch repair deficient (MMRd) lung cancer cell line, which was derived ex vivo from tumors induced autochthonously in the lungs of mice in the MMRd models described in Westcott, PMK, et al., Nature Genetics, 2023 (in press). Whole exome sequencing and neoantigen prediction was performed on all single cell clones, and predicted neoantigens associated with somatic mutations were provided in the inclusion list for searching spectra. Tandem mass spectra were searched with Sequest (Thermo Fisher Scientific; version IseNode in Proteome Discoverer 2.5.0.400). Sequest was set up to search a mouse uniprot database (v.July 3, 2020; 55650 entries containing common contaminants and the proteins GFP, Cas9, Puromycin, and P2A (present in the cell lines) assuming no digestion enzyme (unspecific). Sequest was searched with a fragment ion mass tolerance of 0.02 Da and a parent ion tolerance of 10.0 ppm. TMTpro was added as a fixed modification on the K and N terminus of peptides. Oxidation of methionine was specified in Sequest as a variable modification. Resulting peptides were filtered to exclude peptides with an isolation interference of greater than 30 percent and ppm error greater than plus/minus 3 ppm of the median ppm error of all peptide-spectrum matches (PSMs). Peptides were further prioritized based on concordance of relative abundance across the single cell clones with presence/absence of the associated somatic mutation.

INSTRUMENT(S): Orbitrap Exploris 480

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

SUBMITTER: Peter Westcott

PROVIDER: MSV000092096 | MassIVE | Sun Jun 04 19:47:00 BST 2023

REPOSITORIES: MassIVE

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