InvitroSPI and a large database of proteasome-generated spliced and non-spliced peptides

Abstract

Noncanonical epitopes presented by Human Leucocyte Antigen class I (HLA-I) complexes to CD8+ T cells attracted the spotlight in the research of novel immunotherapies against cancer, infection and autoimmunity. Proteasomes, which are the main producers of HLA-I-bound antigenic peptides, can catalyze both peptide hydrolysis and peptide splicing. The prediction of proteasome-generated spliced peptides is an objective that still requires a reliable (and large) database of non-spliced and spliced peptides produced by these proteases. Here, we present an extended database of proteasome-generated spliced and non-spliced peptides, which was obtained by analyzing in vitro digestions of 80 unique synthetic polypeptide substrates, measured by different mass spectrometers. Peptides were identified through invitroSPI method, which was validated through in silico and in vitro strategies. The peptide product database contains 16,631 unique peptide products (5,493 non-spliced, 6,453 cis-spliced and 4,685 trans-spliced peptide products), and a substrate sequence variety that is a valuable source for predictors of proteasome-catalyzed peptide hydrolysis and splicing. Potential artefacts and skewed results due to different identification and analysis strategies are discussed.

Protein degradation by human 20S proteasomes elucidates the interplay between peptide hydrolysis and splicing

“…To note, in this analysis, the non-spliced and spliced peptide product database that was used to define the correct sequence assignment was derived from the invitroSPI-mediated peptide product identification in the in vitro polypeptide digestions. The invitroSPI precision was not 100% 60 . Therefore, we considered this a (pseudo) ground-truth dataset.…”

“…Although the digestion of full proteins might represent a remarkable progress in the understanding of PCPS as they should better mimic 20S proteasome activity in living cells, the size of the theoretically possible spliced peptide sequence database introduces statistical challenges in spliced peptide identification. This issue has been demonstrated for HLA-I immunopeptidomes 76 , whereas it was less relevant for studies focused on in vitro digestions of synthetic polypeptides shorter than 50 residues, which have been the main source of information to decipher PCPS dynamics so far 39 , 40 , 50 , 55 , 59 , 60 , 82 , 92 , 93 . For the analysis of peptide products of the latter dataset, we recently proposed and validated the invitroSPI method 60 .…”

“…This issue has been demonstrated for HLA-I immunopeptidomes 76 , whereas it was less relevant for studies focused on in vitro digestions of synthetic polypeptides shorter than 50 residues, which have been the main source of information to decipher PCPS dynamics so far 39 , 40 , 50 , 55 , 59 , 60 , 82 , 92 , 93 . For the analysis of peptide products of the latter dataset, we recently proposed and validated the invitroSPI method 60 . For the analysis of more complex systems such as HLA-I immunopeptidomes and tryptic proteome digestions, we recently proposed and benchmarked the inSPIRE method, which utilizes Prosit spectral and retention time prediction for enhanced peptide identification via Percolator 72 , 94 , 95 .…”

“…These issues are barely present in the MS-based analysis of in vitro digestions of synthetic polypeptides by purified proteasomes because the reference database size of spliced peptides is relatively small and cryptic peptide origin can be excluded since the substrate sequence is known. In this type of experiment, proteasomes produce a similar frequency of non-spliced, cis - and trans -spliced peptides 59 , 60 , although non-spliced peptides are on average produced in larger amount 50 , 77 . Correspondence between these in vitro experiments with synthetic polypeptides and in cellula and in vivo experiments has been demonstrated in various immunological studies 10 , 11 , 38 , 44 , 52 , 55 , 56 , 58 , 78 – 83 .…”

“…In contrast, the evidence supporting the biological relevance of trans -spliced peptides is scarcer. Homologous trans -spliced peptides have been identified in both in vitro experiments with purified proteasomes 14 , 39 , 40 , 50 , 59 , 60 as well as in cellula 54 . Heterologous trans -spliced peptides have been described so far only in a study on HLA-I immunopeptidomes 61 .…”