Extending the Comprehensiveness of Immunopeptidome Analyses Using Isobaric Peptide Labeling

Area of Science:

• Immunology
• Proteomics
• Cancer Research

Background:

• Identifying major histocompatibility complex class I (MHC I) peptides is crucial for cancer immunotherapy antigen discovery.
• Low abundance and mutational load in primary tumors present significant analytical challenges for antigen identification.

Purpose of the Study:

• To enhance the detection and identification of MHC I-presented peptides using isobaric peptide labeling.
• To improve the sensitivity for identifying low-abundance tumor-specific antigens (TSAs) and minor histocompatibility antigens (MiHAs).

Main Methods:

• Application of isobaric peptide labeling with tandem mass tag (TMT) for MHC I peptide analysis.
• Utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) with synchronous precursor selection (SPS) and high-field asymmetric waveform ion mobility spectrometry (FAIMS).

Main Results:

• TMT labeling promoted multiply charged ion formation and enhanced b-type fragment ions, improving MHC I peptide identification by 50%.
• The increased sensitivity allowed for the detection of low-abundance MHC I peptides, including TSAs and MiHAs.
• Quantification of MiHAs presented by B-cell lymphocytes was achieved, determining their expression levels.

Conclusions:

• Tandem mass tag (TMT) labeling is an effective strategy to enhance MHC I peptide identification sensitivity.
• This approach facilitates the discovery of critical low-abundance antigens for cancer immunotherapy development.
• The method enables precise quantification of minor histocompatibility antigens in relevant cell types.