The very strong association of human leukocyte antigen (HLA)-B27 with spondyloarthritis might be related to its peptide-presenting properties. The natural polymorphism of this molecule influences both peptide specificity and disease susceptibility. In this study, we present a comprehensive compilation of known natural ligands of HLA-B27 arising from endogenous proteins of human cells, together with a statistical assessment of residue usage among constitutive peptide repertoires of multiple HLA-B27 subtypes. This analysis provides evidence that every peptide position, including "non-anchor" ones, may be subjected to selection on the basis of its contribution to HLA-B27 binding and also allows a quantization of residue preferences at known anchor positions. The present registry is intended as a basis on which to build up reliable criteria to assess the effect of HLA-B27 polymorphism on peptide presentation, for T-cell epitope predictions, and for molecular mimicry studies.

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and β2-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

Phosphorylation is one of the most relevant and ubiquitous post-translational modifications. Despite its relevance, the analysis of protein phosphorylation has been revealed as one of the most challenging tasks due to its highly dynamic nature and low stoichiometry. However, the development and introduction of new analytical methods are modifying rapidly and substantially this field. Especially important has been the introduction of more sensitive and specific methods for phosphoprotein and phosphopeptide purification as well as the use of more sensitive and accurate MS-based analytical methods. The integration of both approaches has enabled large-scale phosphoproteome studies to be performed, an unimaginable task few years ago. Additionally, methods originally developed for differential proteomics have been adapted making the study of the highly dynamic nature of protein phosphorylation feasible. This review aims at offering an overview on the most frequently used methods in phosphoprotein and phosphopeptide enrichment as well as on the most recent MS-based analysis strategies. Current strategies for quantitative phosphoproteomics and the study of the dynamics of protein phosphorylation are highlighted.

In contrast to HLA-B*2705, B*2709 is weakly or not associated to ankylosing spondylitis. Both allotypes differ by a single D116H change. We compared the B*2705-and B*2709-bound peptide repertoires by mass spectrometry to quantify the effect of B*2709 polymorphism on peptide specificity. In addition, shared and differentially bound ligands were sequenced to define the structural features of the various peptide subsets. B*2705 shared 79% of its peptide repertoire with B*2709. Shared ligands accounted for 88% of the B*2709-bound repertoire. All B*2705 ligands not bound to B*2709 had Cterminal basic or Tyr residues. Most B*2709-bound peptides had C-terminal aliphatic and Phe residues, but two showed C-terminal Arg or Tyr. The B*2709-bound repertoire included 12% of peptides not found in B*2705. These had aliphatic C-terminal residues, which are also favored in B*2705. However, these peptides bound weakly B*2705 in vitro, indicating distinct contribution of secondary anchor residues in both subtypes. Differences in peptide binding did not affect the ratio of native to ␤ 2 -microglobulin-free HLA-B27 heavy chain at the cell surface. Our results suggest that weaker association of B*2709 with ankylosing spondylitis is based on differential binding of a limited subset of natural ligands by this allotype.

The emergence of proteomics has placed great interest in the understanding of the mechanisms of MS/MS fragmentation of peptides under low-energy collision-induced dissociation. In this work, we describe the presence of anomalous fragments, which correspond to neutral loss elimination of internal amino acids from ions of the b series in quadrupole ion trap MS/MS spectra from naturally occurring peptides. Internal amino acid elimination occurred preferentially with aliphatic amino acids. The phenomenon was more apparent when doubly charged precursors were fragmented and was inhibited when peptides were N-acetylated at the N-terminus. Fragmentation of isomeric peptides where some internal amino acids were relocated in N-terminal position produced MSn spectra indistinguishable from those of the original peptides, indicating that some b ions underwent a structural rearrangement process. Formation of anomalous fragments required a minimum activation time. Our data are consistent with a nucleophile attack of the N-terminal nitrogen over the electrophilic carbonyl carbon at one peptide bond, forming a cyclic b ion intermediate that, by reopening at preferential sites, exposes internal amino acids to the C-terminal side.

Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts.

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The influence of various factors along the processing-loading pathway in limiting the diversity of HLA-B27-bound peptides around a core protein sequence was analyzed. The C5 proteasome subunit-derived RRFFPYYV and RRFFPYYVY peptides are natural B*2705 ligands. The octamer is an allospecific CTL epitope. Digestion of a 27-mer fragment of C5 revealed that both ligands are generated from this precursor substrate with the 20S proteasome in vitro in a ratio comparable to that in the B*2705-bound peptide pool. The C5 sequence allowed to derive a nested set of six additional peptides with 8–11 residues containing the core octamer sequence and the Arg2 motif of HLA-B27, none of which was found in the B27-bound pool. Together, low proteasomal yield, disfavored TAP-binding motifs, and low affinity for B*2705 accounted for the absence of four of the six peptides. The two remaining differed from the natural octamer or nonamer ligands only by an additional N-terminal Ser residue. Their stability in complex with B*2705 was lower than the respective natural ligands, raising the possibility that N-terminal trimming might have favored a shift toward the more stable peptides. The results suggest that the B*2705-bound peptide repertoire has a highly restricted diversity around a core alloantigenic sequence. This is not explained by a single bottleneck feature, but by multiple factors, including proteasomal generation, TAP-binding motifs, MHC-binding efficiency, and perhaps optimized stability through N-terminal trimming. Tapasin-dependent restrictions, although not excluded, were not required to explain the absence in vivo of the particular peptide set in this study.