Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry

Abstract

The myriad of peptides presented at the cell surface by class I and class II major histocompatibility complex (MHC) molecules are referred to as the immunopeptidome and are of great importance for basic and translational science. For basic science, the immunopeptidome is a critical component for understanding the immune system; for translational science, exact knowledge of the immunopeptidome can directly fuel and guide the development of next-generation vaccines and immunotherapies against autoimmunity, infectious diseases, and cancers. In this mini-review, we summarize established isolation techniques as well as emerging mass spectrometry-based platforms (i.e. SWATH-MS) to identify and quantify MHC-associated peptides. We also highlight selected biological applications and discuss important current technical limitations that need to be solved to accelerate the development of this field.

Fig. 1.

Performance profiles of general resources for the analysis of MHC immunopeptidomes. In the radar charts, variables are represented on axes by a spoke. The highest value on the scale (10) indicates top performance. The lowest value on the scale (0) indicates bad performance. (Upper panel) Cell lines, primary mouse and human tissues represent best-established model systems for the analysis of immunopeptidomes. “Transfectants” refers to cell lines expressing low levels of endogenous MHC molecules but high levels of transfected MHC molecules. (Middle panel) IP and MAE are two established methods for the isolation of MHC-associated peptides. Strong acid elution from whole cell lysis is represented by the dotted green spoke as the performances are hypothetical. (Lower panel) Application of DDA, targeted and DIA MS for the analysis of immunopeptidomes. MS2 spectra (DDA) and chromatographic ion traces (targeted and DIA) are illustrated.

Fig. 2.

Mass spectrometer configurations used for the analysis of MHC-associated peptides. All settings comprise isolation and fragmentation of precursor ions and recording of the mass to charge ratio of MS2 fragment ions in a mass analyzer. (Upper panel) In DDA MS, only the most abundant precursor ions (TopN) per MS1 scan are selected for consecutive isolation and fragmentation. A linear ion trap, an Orbitrap, or a TOF analyzer is used for the high-throughput acquisition of MS1 and MS2 spectra. (Middle panel) In targeted MS, S/MRM exploits the capability of a triple quadrupole to screen a list of transitions (tr), i.e. precursor-fragment ion pairs. The transitions are monitored over time to yield ion traces corresponding to the peptides of interest. PRM experiments are conducted using an Orbitrap-type high-resolution and accurate mass spectrometer (e.g. Q-Exactive). All MS2 product ions derived from predefined peptides (MS1 precursors) are recorded over time to generate the ion chromatographic traces. (Lower panel) HRM and SWATH MS are two fundamentally similar data-independent acquisition methods employing an Orbitrap-type and a quadrupole TOF high-resolution accurate mass spectrometer, respectively. In DIA mode, multiplexed ion traces are acquired by repeatedly cycling through predefined consecutive precursor isolation windows (originally 32 × 25 Th) and by monitoring all fragment ions.