1,2,3, MHC: a review of mass-spectrometry-based immunopeptidomics methods for relative and absolute quantification of pMHCs

Abstract

Quantitative mass-spectrometry-based methods to perform relative and absolute quantification of peptides in the immunopeptidome are growing in popularity as researchers aim to measure the dynamic nature of the peptide major histocompatibility complex repertoire and make copies-per-cell estimations of target antigens of interest. Multiple methods to carry out these experiments have been reported, each with unique advantages and limitations. This article describes existing methods and recent applications, offering guidance for improving quantitative accuracy and selecting an appropriate experimental set-up to maximize data quality and quantity.

Keywords: HLA; MHC; antigen presentation; immunopeptidomics; mass spectometry.

Figure 1

Schematics of immunopeptidomics workflows for standard discovery-based peptide major histocompatibility complex (pMHC) identifications (A), label-free quantification (LFQ) (B), stable isotope labeling (SIL) using amino acids (C), and multiplexed quantification with isobaric mass tags (D). MWCO, molecular weight cut-off; SPE, solid phase extraction; MS, mass spectrometry; TMT, tandem mass tag; RT, retention time; LC-MS/MS, liquid chromatography with tandem mass spectrometry.

Figure 2

(A) Absolute quantification of the light (L) endogenous peptide using single-point calibration with a heavy (H) isotopically-labeled peptide. (B) Schematic of immunopeptidomics workflow where isotopically-labeled standards may be added at points 1-3 for absolute quantification. (C) Fraction of peptide signal lost following peptide major histocompatibility complex (pMHC) enrichment and peptide isolation for n = 14 human leukocyte antigen-A∗02:01 peptides. (D) Relationship between fraction of signal lost and hydrophobicity (GRAVY score, left) and predicted affinity (right). (E) Absolute quantification using three embedded isotopologue heavy isotopically-labeled MHC peptide (hipMHC) calibrants and SureQuant targeted data acquisition. Light concentration [L] is determined by a linear fit of the three calibrants. (F) Schematic of absolute quantification strategy using titrated, tandem mass tag (TMT)-labeled exogenous peptide standards added to a single TMT-labeled sample. [L] is calculated using multipoint TMT-labeled calibrants. (G) Schematic of absolute quantification method using hipMHC standards titrated across replicate samples and labeled with TMT prior to liquid chromatography (LC)-mass spectrometry (LC-MS)/MS analysis. [L] is calculated using the isotopic, TMT-labeled calibrants. LFQ, label-free quantification; SIL, stable isotope labeling.