Arginine (di)methylated Human Leukocyte Antigen class I peptides are favorably presented by HLA-B*07


Specific alterations in protein post-translational modification (PTMs) are recognized hallmarks of diseases. These modifications potentially provide a unique disease-related source of Human Leukocyte Antigen (HLA) class I-presented peptide antigens that can elicit specific immune responses. Although, phosphorylated HLA peptides have received already some attention, the frequency and characteristics of arginine methylated HLA class I peptide presentation have not been explored in detail. In a model human B-cell line we detected by mass spectrometry (MS) 149 HLA class I peptides harboring mono- and/or di-methylated arginine residues. The source proteins of these antigens play important roles in signal transduction, gene transcription and DNA repair. A striking preference was observed in presentation of arginine (di)methylated peptides predicted to bind HLA-B*07 molecules, most likely because the binding motifs of this allele resemble the substrates for arginine methyl-transferases. The HLA-B*07 peptides were preferentially di-methylated at the P3 position in the sequence, thus consecutively to the proline anchor residue at position P2. Such a proline-arginine sequnce has been associated with the arginine methyl-transferases CARM1 and PRMT5. Making use of the specific neutral losses in the MS/MS spectra we could further assign most of the peptides to be asymmetrically di-methylated, most likely by CARM1. The here presented data expand our knowledge of processing and presentation of arginine (di)methylated HLA class I peptides, indicating that this type of modification is frequently presented for recognition by T-cells and might thus present a potential target for immunotherapy.

Sample Processing Protocol

Cell culture conditions and isolation of HLA-peptides complexes was described previously.14 Briefly, two biological replicates of the HLA-A*01, A*03, B*07, -B*27, -C*02, and -C*07–positive B-lymphoblastoid cell line GR were grown in RPMI-1640 medium to a total number of 9 × 109 cells. HLA class I-peptide complexes were immunoprecipitated from lysed GR cells, using the HLA-A–, -B–, and -C–specific mouse monoclonal IgG2a antibody W6/32. HLA class I-peptides complexes were eluted using 10% (vol/vol) acetic acid and peptides were further purified by passage over a 10-kDa molecular weight cutoff membrane. HLA class I-eluted peptides were fractionated by SCX chromatography. The system comprises a Hypercarb trapping column (5 × 0.2 mm i.d., 7 µm particle size; Thermo Fisher) and SCX column (12 × 0.02 cm i.d. polysulfoethyl aspartamide, 5 μm; Poly LC). For each biological replicate a total number of nine SCX fractions were collected. The SCX fractions were analyzed directly by nanoscale LC-MS/MS using a Thermo Scientific EASY-nLC 1000 (Thermo Fisher Scientific) in combination with an ETD-enabled LTQ Orbitrap Elite (first biological replicate) or an Orbitrap Fusion (second biological replicate) mass spectrometer (Thermo Fisher Scientific). The LC system comprises a 20 × 0.1 mm i.d. trapping column (Reprosil C18, 3 μm; Dr. Maisch) and a 50 × 0.005 cm i.d. analytical column (Poroshell 120 EC-C18; 2.7 μm). For the Orbitrap Elite, full MS spectra were acquired in the Orbitrap at a resolution of 60,000 (FWHM) while fragment ions were detected in the Orbitrap at a resolution of 15,000 (FWHM). The 10 most abundant precursor ions were selected either for data-dependent EThcD, CID, ETD,or HCD as previously described 14. The maximum ion accumulation time for MS scans was set to 200 ms and for MS/MS scans to 2,500 ms. For the Orbitrap Fusion, both full MS and MS/MS spectra were acquired in an Orbitrap with a resolution of 60,000 (FWHM) and 15,000 (FWHM), respectively. The Top Speed method was enabled for fragmentation where all most abundant precursor ions in 3 seconds were selected for data-dependent EThcD. The maximum ion accumulation time for MS and MS/MS scans was set to 50 ms and 250 ms, respectively.

Data Processing Protocol

All raw data files were analyzed using Proteome Discoverer 1.4 software package (Thermo Fisher Scientific, Bremen, Germany). MS/MS scans were searched against the Swissprot human reviewed database (September 2015, 20203 entries) with no enzyme specificity using the SEQUEST HT mode. Precursor ion and MS/MS tolerances were set to 10 ppm and 0.05 Da. Methionine oxidation, arginine mono-methylation and arginine di-methylation were set as variable modifications. The peptides-to-spectrum matches were further filtered for precursor tolerance 5 ppm, < 1% FDR using Percolator,15 XCorr > 1.7 and peptide rank 1. Only peptides between 8 and 14 amino acid long were selected for further analysis. Precursor ion area detection node was added in order to obtain the area under the curve of the LC elution profiles of methylated, di-methylated and unmodified peptides. The areas under the curve of differently methylated peptides (none, mono-, di-) were only compared when detected in the same LC run. The NetMHC 3.4 algorithm16 was used to predict the HLA-peptide binding affinities for each of the identified peptide sequence. Predictions were enabled for HLA-A*01, -A*03, -B*07 and –B*27, and peptides were assigned to bind a particular allele when IC50 < 1000 nM. Sequence logo’s were generated by the IceLogo 17 algorithm with the Uniprot-Swissprot protein database as reference set and a p-value of 0.05. Gene Ontology (GO) analysis of the source proteins of the (di)methylated HLA class I peptides was performed by PANTHER.18


Fabio Marino, Utrecht University
Albert J.R. Heck, Biomolecular Mass Spectrometry and Proteomics ( lab head )

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    Mommen GP, Frese CK, Meiring HD, van Gaans-van den Brink J, de Jong AP, van Els CA, Heck AJ; Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD)., Proc Natl Acad Sci U S A, 2014 Mar 25, 111, 12, 4507-12, PubMed: 24616531

    Marino F, Mommen GP, Jeko A, Meiring HD, van Gaans-van den Brink JA, Scheltema RA, van Els CA, Heck AJ. Arginine (di)methylated Human Leukocyte Antigen class I peptides are favorably presented by HLA-B*07. J Proteome Res. 2016 Aug 8 PubMed: 27503676