Innovations Toward Immunopeptidomics

Abstract

Over the past 30 years, immunopeptidomics has grown alongside improvements in mass spectrometry technology, genomics, transcriptomics, T cell receptor sequencing, and immunological assays to identify and characterize the targets of activated T cells. Together, multiple research groups with expertise in immunology, biochemistry, chemistry, and peptide mass spectrometry have come together to enable the isolation and sequence identification of endogenous major histocompatibility complex (MHC)-bound peptides. The idea to apply highly sensitive mass spectrometry techniques to study the landscape of peptide antigens presented by cell surface MHCs was innovative and continues to be successfully used and improved upon to deepen our understanding of how peptide antigens are processed and presented to T cells. Multiple research groups were involved in this bringing immunopeptidomics to the forefront of translational research, and we will highlight the contributions of one of the earliest developers, Professor Donald F. Hunt, and his research group at the University of Virginia. The Hunt laboratory applied cutting edge mass spectroscopy-based immunopeptidomics to study cancer, autoimmunity, transplant rejection, and infectious diseases. Across these diverse research areas, the Hunt laboratory and collaborators would characterize previously unknown MHC peptide-binding motifs and identify immunologically active antigens using ultra sensitive mass spectrometry techniques. Amazingly, many of the MHC-bound peptide antigens discovered in collaborations with the Hunt laboratory were sequenced by mass spectrometry before the completion of the human genome using manual de novo sequencing. In this perspective article, we will chronicle the work of the Hunt laboratory and their many collaborators that would be a major part of the foundation for mass spectrometry-based immunopeptidomics and its application to immunology research.

Keywords: HLA; MHC; antigen; immunopeptidomics; peptidomics.

Graphical abstract

Fig. 1

Overview of the splitter workflow that enabled simultaneous immunopeptidomics and immunogenicity testing of HLA peptide fractions. A, microcapillary LC-MS/MS system with splitter to direct the HLA peptide effluent simultaneously to the mass spectrometer and to wells of a microtiter plate for downstream T cell reactivity assays. B, to create a zero dead volume splitter, a PRP-1 (Hamilton) microcapillary HPLC column (100 um by 22 cm) was connected to two small capillaries of different lengths and interior diameters (25 um and 40 um ID, Polymicro Technologies). The HPLC column was eluted into the union and 1/6 of the material was deposited into 50 uL of culture media in microtiter plate wells. The larger of the two capillaries directed the remaining 5/6 of the material was used for LC-MS/MS analysis on a Finnigan-MAT triple quadrupole mass spectrometer. C, example MS1 spectra of a peptide fraction shown in (D) that activated patient T cells. HLA, HLA, human leukocyte antigen; MS, mass spectrometry.