Database search engines and target database features impinge upon the identification of post‐translationally cis‐spliced peptides in HLA class I immunopeptidomes

Abstract

Unconventional epitopes presented by HLA class I complexes are emerging targets for T cell targeted immunotherapies. Their identification by mass spectrometry (MS) required development of novel methods to cope with the large number of theoretical candidates. Methods to identify post‐translationally spliced peptides led to a broad range of outcomes. We here investigated the impact of three common database search engines – that is, Mascot, Mascot+Percolator, and PEAKS DB – as final identification step, as well as the features of target database on the ability to correctly identify non‐spliced and cis‐spliced peptides. We used ground truth datasets measured by MS to benchmark methods’ performance and extended the analysis to HLA class I immunopeptidomes. PEAKS DB showed better precision and recall of cis‐spliced peptides and larger number of identified peptides in HLA class I immunopeptidomes than the other search engine strategies. The better performance of PEAKS DB appears to result from better discrimination between target and decoy hits and hence a more robust FDR estimation, and seems independent to peptide and spectrum features here investigated.

inSPIRE: An Open-Source Tool for Increased Mass Spectrometry Identification Rates Using Prosit Spectral Prediction

“…K562-B∗07:02 and K562-A∗02:01 cell line clones express either the single HLA-B∗07:02 or HLA-A∗02:01 alleles. They derive from the leukemia K562 cell line (American Type Culture Collection CCL-243), which does not express endogenous HLA-I and HLA-II molecules, and its generation and growing conditions are described elsewhere ( 13 ).…”

“…HLA-I-bound peptides were isolated from 10 9 cells of K562-B∗07:02 and K562-A∗02:01 cell line clones, through HLA-I-peptide elution using W6/32 antibody, as described elsewhere ( 13 ). The MS files were already published ( 13 ).…”

“…MS data of HLA-I immunopeptidomes were collected using Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC nano pump (both from Thermo Fisher Scientific), as described elsewhere ( 13 ). The same method and instrument were used for the synthetic peptide library measurement.…”

“…Sequencing was performed using HiSeq 2x150 PE HO with a depth of 20 to 25 million reads per sample. Details about reads trimming, quantification, and data processing are described elsewhere ( 13 ).…”

“…Proteasomes are proteases that, in contrast to trypsin, can cleave after any amino acid, following complex dynamics ( 9 , 10 ), thereby impacting on the database search space. The size of the search space can be expanded even further with increasing interest in noncanonical peptides outside the commonly used reference proteomes ( 11 , 12 , 13 , 14 ). These enlarged search spaces can create a number of issues to traditional target-decoy search approaches, with a significant negative impact on both peptide yield and FDR estimation ( 15 , 16 ).…”