LC-MSMS of 3 DLBCL cell lines (class I immunopeptidome and whole proteome)

Accumulating evidence indicates that alternative translation events occur in all cell types, suggesting that the proteome is more complex than previously anticipated. We elected to broaden the repertoire of human cryptic proteins by designing an approach combining ribosome profiling, which gives a precise view of truly active translation events, and mass spectrometry, which is so far the best way to screen with confidence the cellular proteome. This allowed building complete but noiseless customized protein databases suitable for MS-based protein identification. The analysis of the immunopeptidome and whole proteome of DLBCL cell lines led to the identification of 710 novel protein isoforms along with 1856 new cryptic proteins, from which 72% derived from allegedly non-coding regions of the genome. Globally, cryptic proteins were very distinct from canonical proteins, as they were shorter, less stable, initiated at a diversified set of start codons and were at least 5-fold more efficient for MHC-I peptide generation. The translation efficiency of novel isoforms and cryptic proteins was at least as good as for canonical proteins and we propose that some of them could regulate the translation of canonical proteins involved in cellular stress and cancer cell growth.