Mild Acid Elution and MHC Immunoaffinity Chromatography Reveal Similar Albeit Not Identical Profiles of the HLA Class I Immunopeptidome

Abstract

To understand and treat immunology-related diseases, a comprehensive, unbiased characterization of major histocompatibility complex (MHC) peptide ligands is of key importance. Preceding the analysis by mass spectrometry, MHC class I peptide ligands are typically isolated by MHC immunoaffinity chromatography (MHC-IAC) and less often by mild acid elution (MAE). MAE may provide a cheap alternative to MHC-IAC for suspension cells but has been hampered by the high number of contaminating, MHC-unrelated peptides. Here, we optimized MAE, yielding MHC peptide ligand purities of more than 80%. When compared with MHC-IAC, obtained peptides were similar in numbers, identities, and to a large extent intensities, while the percentage of cysteinylated peptides was 5 times higher in MAE. The latter benefitted the discovery of MHC-allotype-specific, distinct cysteinylation frequencies at individual positions of MHC peptide ligands. MAE revealed many MHC ligands with unmodified, N-terminal cysteine residues which get lost in MHC-IAC workflows. The results support the idea that MAE might be particularly valuable for the high-confidence analysis of post-translational modifications by avoiding the exposure of the investigated peptides to enzymes and reactive molecules in the cell lysate. Our improved and carefully documented MAE workflow represents a high-quality, cost-effective alternative to MHC-IAC for suspension cells.

Keywords: HLA ligandome; MHC bound peptides; MHC immunoaffinity chromatography; cysteinylation; mild acid elution; post-translational modification.

Figure 1

MAE and MHC-IAC identify a large overlapping set of peptides with correlating MS1 intensities. Data are derived from exactly the same cell culture using 5 × 10⁸ JY cells each for MAE and MHC-IAC. (A) Most of the peptides obtained by MAE are also obtained by MHC-IAC and vice versa. The numbers in italics indicate absolute numbers of peptides. (B) Quantitative MS1 information derived from MAE versus MHC-IAC is similar for most peptides. The mean area of MS1 intensities was calculated for all peptides shared between MAE and MHC-IAC based on the three LC-MS replicates per extraction method. Predicted MHC non-binders (NetMHC IC₅₀ ≥ 500 nM) are highlighted with red circles in the dot plot.

Figure 2

Peptides isolated by MAE and MHC-IAC show a similar distribution of predicted MHC binding affinities. All displayed data originate from the same cell culture harvesting using 5 × 10⁸ JY cells each for MAE and MHC-IAC. Each data point is derived from the mean area of MS1 intensities of a single peptide as measured in triplicate LC-MS injections. The gray bars represent the median of the NetMHC-IC₅₀ and the median of the corresponding peptide intensities, respectively. Medians were calculated separately for predicted MHC binders (IC₅₀ < 500 nM) and non-binders (IC₅₀ ≥ 500 nM) and are indicated with gray numbers. (A) Peptides obtained by MAE. (B) Peptides obtained by MHC-IAC.

Figure 3

The modestly higher peptide yield of MHC-IAC as compared to MAE was mainly due to a better performance for HLA-B allotypes. Each peptide was assigned to that MHC allotype of the respective cell line yielding the highest NetMHC binding affinity. Peptides with a NetMHC IC₅₀ ≥ 500 nM were considered as non-binders. Absolute peptide numbers are indicated in italics. (A) Peptides obtained from exactly the same cell culture using 5 × 10⁸ JY cells each for MAE and MHC-IAC. (B) Peptides obtained from exactly the same cell culture using 1.25 × 10⁸ THP-1 cells each for MAE and MHC-IAC.

Figure 4

Cysteinylated peptides isolated by MAE are enriched in HLA-A*02:01 binders and diminished in HLA-B*07:02 binders. Data for JY cells are derived from five MAEs of JY cells that were analyzed employing EThcD. Data from THP-1 cells represent four MAEs performed in parallel and measured using CID. Identified peptides of each sample were grouped into two bins: non-cysteinylated (pale blue) and cysteinylated (dark blue) peptides. The mean number of non-cysteinylated peptides per MAE was 4640 for JY cells and 969 for THP-1 cells. The mean number of cysteinylated peptides per MAE amounted to 105 for JY cells and 32 for THP-1 cells. Peptides with a NetMHC IC₅₀ of <500 nM were considered as binders. Columns represent means, and error bars indicate standard deviation. Depicted P values are derived from two-tailed, heteroskedastic Student’s t-tests with Bonferroni correction for three comparisons. (A) Proportion of predicted HLA-A*02:01 binders among all identified peptides. (B) Proportion of predicted HLA-B*07:02 binders among all identified peptides.

Figure 5

Cysteinylated cysteine residues are preferentially located at position C-1 of both HLA-A2 and HLA-B7 peptide ligands, whereas a preference for position C-4 occurs in the context of HLA-A2 but not HLA-B7. The columns represent means from five MAEs of JY cells that were analyzed employing EThcD. To adjust for different peptide lengths, more C-terminal amino acid positions were also counted relative to the C-terminus, so, e.g., “C-4” refers to the position four amino acids N-terminal of the C-terminus, whereas “C-1” represents the position adjacent to the C-terminus. The error bars indicate the standard deviation. Accompanying P values are given in Supplementary Tables S3 and S4. (A) Frequency of modified cysteine residues in cysteine-containing peptides with a NetMHC IC₅₀ of <500 nM for HLA-A*02:01; numbers of HLA-A*02:01 motif peptides containing modified cysteine (n) = 114, 114, 64, 102, and 117, respectively, in total 173 non-redundant peptides. (B) Frequency of modified cysteine residues in cysteine-containing peptides with a NetMHC IC₅₀ of <500 nM for HLA-B*07:02; numbers of HLA-B*07:02 motif peptides containing modified cysteine (n) = 32, 31, 27, 35, and 40, respectively, in total 66 non-redundant peptides.