Isolation of HLA Bound Peptides by Immunoaffinity Capture and Identification by Mass Spectrometry
Kirti Pandey, Sri H. Ramarathinam, Anthony W. Purcell
Abstract
This article describes the purification of HLA-bound peptides and their subsequent sequencing by mass spectrometry. These methods can be used for both HLA class I and class II molecules and can be adapted to different species depending on the availability of specific antibodies. Peptides can be successfully isolated from a variety of sample types, including in vitro cultured cells and primary tissues. The method involves the affinity capture of HLA-peptide complexes and separation of peptides from HLA heavy chains, followed by tailored interrogation by mass spectrometry to take into account the non-tryptic nature of endogenously derived HLA-bound peptides. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of immunoaffinity column Alternate Protocol 1: Preparation of microscale immunoaffinity column Basic Protocol 2: Generation of cell lysate and HLA immunoaffinity purification Alternate Protocol 2: Microscale immunoaffinity purification Basic Protocol 3: Separation of HLA peptides by reverse-phase high-performance liquid chromatography (RP-HPLC) Alternate Protocol 3: Isolation of HLA peptides using molecular weight cutoff (MWCO) filter Basic Protocol 4: Mass spectrometry and data analysis.