Automated Ligand Purification Platform Accelerates Immunopeptidome Analysis by Mass Spectrometry

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.