Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Abstract

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHCassociated peptides from these cell lines, including the steps to verify the beadsbinding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra-and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Induction of senescence renders cancer cells highly immunogenic

“…Bead coupling and immunopurification of MHC class I peptides were performed as previously described (53). Briefly, 80 mg of sepharose CNBr activated beads was coupled with 2 mg of anti-mouse H2 antibody.…”

“…An equivalent volume of cell lysis buffer (1% CHAPS in PBS containing protease inhibitors, 1 pellet/10 mL) was added to the cell suspension (final concentration of the lysis buffer of 0.5% Chaps), followed by an incubation for 60 min at 4 °C using a rotator device (10RPM) and centrifugation at 18,000 × g for 20 min at 4 °C. The cell lysis supernatant containing the MHC-peptide complexes was transferred to a new 2 mL microcentrifuge tube and kept on ice until use for immunopurification.Bead coupling and immunopurification of MHC class I peptides were performed as previously described(53). Briefly, 80 mg of sepharose CNBr activated beads was coupled with 2 mg of anti-mouse H2 antibody.…”

Integrating Machine Learning-Enhanced Immunopeptidomics and SARS-CoV-2 Population-Scale Analyses Unveils Novel Antigenic Features for Next-Generation COVID-19 Vaccines

“…Immunopurification of HLA-class I peptides was performed as previously described 32,63 . To isolate MHC class I peptides, a frozen pellet of 1 x 10 8 cells was resuspended in 500 µL of PBS by pipetting up and down until homogenization.…”

SpectiCal: m/z Calibration of MS2 Peptide Spectra Using Known Low Mass Ions

“…In order to test and demonstrate the performance of the tool, the following public ProteomeXchange data sets were downloaded and processed: PXD008369, PXD015162, PXD019252, PXD023650, and PXD028633 . These five data sets were selected as being (1) publicly accessible in the ProteomeXchange repository, (2) conveniently on disk locally already for local reprocessing, (3) from the most commonly used instruments, and (4) relatively small (less than 50 MS runs).…”

“…In order to test and demonstrate the performance of the tool, the following public ProteomeXchange 20 data sets were downloaded and processed: PXD008369, 21 PXD015162, 22 PXD019252, 23 PXD023650, 24 and PXD028633. 25 These five data sets were selected as being (1) normalized intensity. The intensity of an a{II})/a{LL} ion, which is one of the most common high-intensity ions appearing in every MS run, is normalized at 100, and all other peak intensities are normalized to this ion.…”