An automated workflow to address proteome complexity and the large search space problem in proteomics and HLA-I immunopeptidomics.

Antigenic noncanonical epitope and novel protein discovery are research areas with therapeutical applications, predominantly done via mass spectrometry. The latter should rely on a well-characterized proteogenomic search space. Its size is barely known for antigenic noncanonical peptides and novel proteins, and this could impact on their identification. To address these issues, we here develop an automated workflow comprised of Sequoia for the creation of RNA sequencing informed and exhaustive sequence search spaces for various noncanonical peptide origins, and SPIsnake for pre-filtering and exploration of sequence search space prior to mass spectrometry searches. We apply our workflow to characterize the exact sizes of tryptic and nonspecific peptide sequence search spaces in a variety of definitions, their reduction when using RNA expression, their inflation by post-translational modifications, and the frequency of peptide sequence multimapping to different noncanonical origins. Furthermore, we explore the application of Sequoia and SPIsnake on HLA-I immunopeptidomes, thereby rescuing sensitivity in peptide identification when confronted with inflated search spaces. Taken together, Sequoia and SPIsnake pave the way for an educated development of methods addressing large-scale exhaustive proteogenomic discovery by exposing the consequences of database size inflation and ambiguity of peptide and protein sequence identification.