For MS-based immunopeptidomics, major histocompatibility complex (MHC) peptides are typically isolated by immunoaffinity purification using specific anti-MHC antibodies. Subsequent separation of MHC peptides from proteins, including MHC and antibodies, can be achieved either by ultrafiltration using a molecular weight cutoff (MWCO) filter or by C18 solid-phase extraction (SPE). C18-SPE has been reported to have superior MHC peptide recovery relative to ultrafiltration and it combines desalting and protein separation in a single step. However, the upper limit of acetonitrile (ACN) concentration that can be applied for peptide elution is limited due to highly hydrophobic peptides, resulting in low recovery of highly hydrophobic peptides. In this work, restricted access material (RAM)-SPE, which combines size exclusion and reversed phase chromatography, was evaluated in sample preparation for MS-based immunopeptidomics. The coating of the porous C18 material with hydrophilic polymers and a cutoff of ~10 kDa enables the access of MHC peptides (1-3 kDa) to the pores and their interaction with the inner hydrophobic C18 stationary phase. Proteins (>10 kDa), on the other hand, are repelled from the outer hydrophilic stationary phase and have no access to the inner stationary phase, Therefore, subsequent elution of peptides with 80% ACN was achieved. Overall, RAM-SPE significantly increased the number of identified MHC peptides compared to C18-SPE. In particular, most hydrophobic peptides were exclusively detected using the RAM-SPE workflow. Additionally, the number of MHC peptides exclusively identified with RAM-SPE increased with the number of the hydrophobic amino acids. Altogether, RAM-SPE can be used to improve the overall MHC peptide recovery and extend MS-based immunopeptidome landscape towards hydrophobic peptides.
How was PEAKS used?
LC-MS/MS analysis was conducted on MHC peptides isolated by either C18-SPE or RAM-SPE workflow. A precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.02 Da were applied, and enzyme was set to none. The variable post-translational modifications (PTM) including oxidation (M) and pyro-Glu from Q (N-term Q) and a fixed PTM of carbamidomethylation (C) were added, and a maximum of 3 variable PTMs were allowed per peptide. For MHC-I peptides, de novo peptide sequencing was performed with PEAKS X+. Up to 10 de novo sequencing candidates reported for each identified MS2 spectrum were used for further analysis. MHC-II peptides were identified via database search with PEAKS X+. A fasta database generated by translating coding sequences (CDS) of ENSEMBL 90 (h.ens90.translated.fasta) was used for database searching. Results were filtered to 1% PSM FDR.
Bernhardt, Melissa, et al. “Extending the Mass Spectrometry-Detectable Landscape of MHC Peptides by Use of Restricted Access Material.” Analytical Chemistry 94.41 (2022): 14214-14222. https://doi.org/10.1021/acs.analchem.2c02198
Mass spectrometry-based immunopeptidomics enables the comprehensive identification of major histocompatibility complex (MHC) peptides from a cell culture as well as from tissue or tumor samples and is applied for the identification of tumor-specific and viral T-cell epitopes. Although mass spectrometry is generally considered an “unbiased” method for MHC peptide identification, the physicochemical properties of MHC peptides can greatly influence their detectability. Here, we demonstrate that highly hydrophobic peptides are lost during sample preparation when C18 solid-phase extraction (SPE) is used for separating MHC peptides from proteins. To overcome this limitation, we established an optimized protocol involving restricted access material (RAM). Compared to C18-SPE, RAM-SPE improved the overall MHC peptide recovery and extended the landscape of mass spectrometry-detectable MHC peptides toward more hydrophobic peptides.