Automated Ligand Purification Platform Accelerates Immunopeptidome Analysis by Mass Spectrometry

J Proteome Res. 2021 Jan 1;20(1):393-408. doi: 10.1021/acs.jproteome.0c00464. Epub 2020 Dec 17.


Major histocompatibility complex (MHC)-presented peptides (pMHC) give insight into T cell immune responses, a critical step toward developing a new generation of targeted immunotherapies. Recent instrumentation advances have propelled mass spectrometry to being arguably the most robust technology for discovering and quantifying naturally presented pMHC from cells and tissues. However, sample preparation has remained a major limitation due to time-consuming and labor-intensive workflows. We developed a high-throughput and automated platform with enhanced speed, sensitivity, and reproducibility relative to prior studies. This pipeline is capable of processing up to 96 samples in 6 h or less yielding high-quality pMHC mixtures ready for mass spectrometry. Here, we describe our efforts to optimize purification and mass spectrometer parameters, ultimately allowing us to identify as many as almost 5000 pMHC I and 7400 pMHC II from as little as 2.5 × 107 Raji cells each. We believe that this platform will facilitate and accelerate immunopeptidome profiling and benefit clinical research for immunotherapies.

Keywords: AssayMAP; MHC; automation; high throughput; immunopeptidome; immunoprecipitation; mass spectrometry; peptide; reproducibility; sensitivity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Immunotherapy*
  • Ligands
  • Mass Spectrometry
  • Peptides*
  • Reproducibility of Results


  • Ligands
  • Peptides