Navigating Critical Challenges Associated with Immunopeptidomics-Based Detection of Proteasomal Spliced Peptide Candidates

Abstract Within the tumor immunology community, the topic of proteasomal spliced peptides (PSP) has generated a great deal of controversy. In the earliest reports, careful biological validation led to the conclusion that proteasome-catalyzed peptide splicing was a rare event. To date, six PSPs have been validated biologically. However, the advent of algorithms to identify candidate PSPs in mass spectrometry data challenged this notion, with several studies concluding that the frequency of spliced peptides binding to MHC class I was quite high. Since this time, much debate has centered around the methodologies used in these studies. Several reanalyses of data from these studies have led to questions about the validity of the conclusions. Furthermore, the biological and technical validation that should be necessary for verifying PSP assignments was often lacking. It has been suggested therefore that the research community should unite around a common set of standards for validating candidate PSPs. In this review, we propose and highlight the necessary steps for validation of proteasomal splicing at both the mass spectrometry and biological levels. We hope that these guidelines will serve as a foundation for critical assessment of results from proteasomal splicing studies.