SAPrIm, a semi-automated protocol for mid-throughput immunopeptidomics.

Area of Science:

• Immunology
• Proteomics
• Cancer Research

Background:

• Human leukocyte antigen (HLA) molecules present peptides, defining the immunopeptidome and guiding adaptive immunity.
• Understanding the immunopeptidome is vital for developing effective cancer immunotherapies like vaccines and T-cell therapies.
• Personalized cancer treatments necessitate comprehensive immunopeptidome profiling.

Purpose of the Study:

• To introduce SAPrIm, a novel semi-automated immunopeptidomics workflow.
• To enable mid-throughput analysis of the immunopeptidome.
• To facilitate the identification and quantification of HLA-presented peptides.

Main Methods:

• Utilized a semi-automated workflow with the KingFisher platform for immunopeptidome isolation.
• Employed anti-HLA antibodies coupled to magnetic microbeads for efficient peptide capture.
• Implemented a variable window data independent acquisition (DIA) mass spectrometry method.
• Enabled parallel processing of up to 12 samples.

Main Results:

• Successfully identified and quantified approximately 400 to 13,000 unique peptides from 5e5 to 5e7 cells, respectively.
• Demonstrated concordant peptide identification and quantification across samples.
• Validated the workflow's capability for mid-throughput immunopeptidome analysis.

Conclusions:

• SAPrIm provides an efficient tool for immunopeptidome profiling in the mid-throughput era.
• This workflow is crucial for future immunopeptidomics studies, particularly for mid-size cohorts.
• SAPrIm supports comparative immunopeptidomics and the advancement of personalized cancer therapies.