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Data‐Independent Acquisition of HLA Class I Peptidomes on the Q Exactive Mass Spectrometer Platform
Author(s) -
Ritz Danilo,
Kinzi Jonny,
Neri Dario,
Fugmann Tim
Publication year - 2017
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201700177
Subject(s) - human leukocyte antigen , mass spectrometry , computational biology , biomarker discovery , tandem mass spectrometry , identification (biology) , proteomics , chemistry , computer science , biology , antigen , chromatography , immunology , biochemistry , gene , botany
The characterization of peptides presented by human leukocyte antigen (HLA) class I molecules is crucial for understanding immune processes, biomarker discovery, and the development of novel immunotherapies or vaccines. Mass spectrometry allows the direct identification of thousands of HLA‐bound peptides from cell lines, blood, or tissue. In recent years, data‐independent acquisition (DIA) mass spectrometry methods have evolved, promising to increase reproducibility and sensitivity over classical data‐dependent acquisition (DDA) workflows. Here, we describe a DIA setup on the Q Exactive mass spectrometer, optimized regarding the unique properties of HLA class I peptides. The methodology enables sensitive and highly reproducible characterization of HLA peptidomes from individual cell lines. From up to 16 DDA analyses of 100 million human cells, more than 10 000 peptides could be confidently identified, serving as basis for the generation of spectral libraries. This knowledge enabled the subsequent interrogation of DIA data, leading to the identification of peptide sets with >90% overlap between replicate samples, a prerequisite for the comparative study of closely related specimens. Furthermore, >3000 peptides could be identified from just one million cells after DIA analysis using a library generated from 300 million cells. The reduction in sample quantity and the high reproducibility of DIA‐based HLA peptidome analysis should facilitate personalized medicine applications.