A comprehensive analysis of peptides presented by HLA‐A1

Abstract

Human leukocyte antigen (HLA)-A1 is one of the most common Caucasian HLA-A alleles. Here, we describe the comprehensive analysis of the HLA-A*01:01 ligand repertoire with the identification of 4735 naturally processed and presented peptides derived from 2477 source proteins. We found HLA-A*01:01 bound an equivalent number of ligands of 9 or 10 amino acids in length as well as being remarkably tolerant of even longer peptides. Indeed close to half of the HLA-A1 bound peptides identified ranged between 11 and 13 amino acids in length. These longer peptides contained the strong canonical motif of and acidic E/D residue at position 3 (P3) and Y at the C-terminus (CΩ), a motif that was still apparent in peptides of up to 18 amino acids in length. The identification of this large database of natural ligands will facilitate the refinement of predictive algorithms particularly with respect to longer peptide ligands.

Characterization of the Antigen Processing Machinery and Endogenous Peptide Presentation of a Bat MHC Class I Molecule

“…We also noted an increase in the use of small amino acids and a decrease in the frequency of bulky residues with increasing peptide length (Supplemental Fig. 3C) as we have previously reported for both murine and human class I molecules (28,29,33).…”

“…Some irrelevant GO terms (including plant-based terms) were manually removed. GO annotations for source proteins of Ptal-N*01:01 MHC peptides were compared with five previously published human and mouse MHC peptide datasets (28,29,33). Annotations were compiled for three human MHC class I alleles (HLA-A*01:01, HLA-B*27:05, HLA-Cw*04:01) and two murine class I alleles (H-2D…”

“…Cellular debris was removed by centrifugation at 3200 3 g for 10 min at 4˚C and lysates were precleared with unconjugated protein A-Sepharose (GE Healthcare). Bat MHC class Ipeptide complexes were immunoaffinity purified using anti-myc (9E10) mAb coupled to protein A-Sepharose as described (28)(29)(30)(31). Bound complexes were eluted with 10% (v/v) acetic acid, centrifuged at 16,000 3 g for 10 min to remove particulates, and neutralized with 1 M ammonium bicarbonate (pH 8.0).…”

“…Cell pellets (4 3 10 8 to 2 3 10 9 ) were ground using a Retsch mixer mill (MM400) and then lysed. MHC molecules were captured as above and peptides were isolated and analyzed as described (28)(29)(30)(31). Each experiment was performed in triplicate and the MHC-peptide mixtures were fractionated by reversed phase HPLC (RP-HPLC; Chromolith high-resolution RP-18e, Merck) using an Ettan HPLC system (GE Healthcare) as described in detail elsewhere (28)(29)(30)(31).…”

Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry*

“…We also highlight selected biological applications and discuss important current technical limitations that need to be solved to accelerate the development of this field. The immunopeptidome is referred to as the collection of peptides associated with and presented by major histocompatibility complex (MHC) molecules (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). MHC-associated peptides are recognized by T lymphocytes that are in turn activated to eliminate abnormal cells such as pathogen-infected and cancer cells.…”