Identification of Natural MHC Class II Presented Phosphopeptides and Tumor-Derived MHC Class I Phospholigands

Meyer, Verena S.; Drews, Oliver; Günder, Marc; Hennenlotter, Jörg; Rammensee, Hans‐Georg; Stevanović, Stefan

doi:10.1021/pr800937k

Cited by 50 publications

(59 citation statements)

References 83 publications

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“…S9). Comparison with previous studies revealed that 16 HLA-B7-associated phosphopeptides had been reported previously (16,24,26), whereas a total of 24 phosphorylation sites are annotated in the Uniprot protein database (www.uniprot.org/). The correct identification of selected phosphopeptides could be nicely confirmed by the spectra we generated from their synthetic peptide counterparts (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 59%

Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD)

Mommen

Frese

Meiring

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

174

205

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The identification of peptides presented by human leukocyte antigen (HLA) class I is tremendously important for the understanding of antigen presentation mechanisms under healthy or diseased conditions. Currently, mass spectrometry-based methods represent the best methodology for the identification of HLA class I-associated peptides. However, the HLA class I peptide repertoire remains largely unexplored because the variable nature of endogenous peptides represents difficulties in conventional peptide fragmentation technology. Here, we substantially enhanced (about threefold) the identification success rate of peptides presented by HLA class I using combined electron-transfer/higher-energy collision dissociation (EThcD), reporting over 12,000 high-confident (false discovery rate <1%) peptides from a single human B-cell line. The direct importance of such an unprecedented large dataset is highlighted by the discovery of unique features in antigen presentation. The observation that a substantial part of proteins is sampled across different HLA alleles, and the common occurrence of HLA class I nested sets, suggest that the constraints of HLA class I to comprehensively present the health states of cells are not as tight as previously thought. Our dataset contains a substantial set of peptides bearing a variety of posttranslational modifications presented with marked allele-specific differences. We propose that EThcD should become the method of choice in analyzing HLA class I-presented peptides.human leukocyte antigen class I | electron-transfer dissociation | major histocompatibility complex | phosphorylation | binding motif C lass I molecules of the human leukocyte antigen (HLA) complex present short peptides, typically 8-11 aa in length at the cell surface, for scrutiny by the immune system (1). These peptide fragments are generated in the cytoplasm by proteasomal degradation of source proteins, translocated into the endoplasmic reticulum (ER) and subjected to N-terminal trimming to a size that is suitable for loading onto the HLA (2). Loading is governed by physicochemical binding motifs typical for each HLA class I allele (3). Depending on the motif required for the HLA class I allele(s) expressed, an ER-residing peptide may become presented or not. Recognition of specific HLA class I peptide complexes by CD8 T lymphocytes on pathogen infected or cancerous cells leads to the activation of a cytotoxic response and the clearance of the diseased cell. The identification of these HLA class I-associated peptides has important consequences for understanding the biology of cells, vaccine design, and tumor immunotherapy (4, 5).Today mass spectrometry (MS) is the core technology for the analysis of HLA class I-presented peptides. These peptides are typically enriched from cell lysates through the affinity purification of HLA class I peptide complexes, released from the HLA by acid elution, and separated by liquid chromatography (LC) before introduction into the mass spectrometer. Identification is commonly accomplished by...

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Section: Resultsmentioning

confidence: 59%

Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD)

Mommen

Frese

Meiring

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

174

205

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“…The identification of phosphorylated species associated with HLA class I molecules has gained considerable attention since they were proposed as putative targets for cancer immunotherapy (12,13). Nevertheless, the identification of phosphopeptides via mass spectrometry is challenging because of several analytical limitations, such as their low stoichiometry, their inefficient ionization, or the poorly informative MS2 spectra obtained upon their fragmentation by collision-induced dissociation (18,19).…”

Section: To Test This Hypothesis the Natural Ligands S[ps]ygnirav Anmentioning

confidence: 99%

“…In the same way, identification of HLA class I-associated phospholigands has benefited from the implementation of these approaches (11)(12)(13). We combined IMAC and TiO 2 affinity chromatography before LC-MS analysis, resulting in the identification of 85 B40-bound phosphopeptides.…”

Section: To Test This Hypothesis the Natural Ligands S[ps]ygnirav Anmentioning

confidence: 99%

“…Among other modifications, N-terminal acetylation (10), phosphorylation (11)(12)(13), methylation (14), and glycosylation (15) have been described in MHC class I-bound peptidomes. In this context, phosphorylated ligands have raised much interest owing to their potential as targets in T-cellbased cancer immunotherapy (12,13), given that aberrant phosphorylation is a hallmark of malignant transformation (16,17) and phosphorylated epitopes can be specifically recognized by CTLs (11). Therefore, the characterization of the phosphopeptidome associated with MHC class I molecules and the identification of tumor-derived phosphopeptides are necessary in order for such immunotherapeutic approaches to be implemented.…”

mentioning

confidence: 99%

“…Several such strategies rely on the enrichment of the phosphorylated species prior to LC-MS analysis, typically by means of IMAC or TiO 2 affinity chromatography (20). These techniques have also been successfully applied to the characterization of the phosphopeptidomes of several class I molecules (11)(12)(13).…”

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Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue

Marcilla

Alpízar

Lombardía

et al. 2014

Molecular & Cellular Proteomics

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Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with highresolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO 2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy. Molecular & Cellular Proteomics 13: 10.1074/mcp.M113.034314, 462-474, 2014. Major histocompatibility complex (MHC)1 class I molecules are cell surface glycoproteins that are expressed on almost every nucleated cell in vertebrates. They result from the noncovalent interaction of a polymorphic heavy chain, a constant light chain (␤-2-microglobulin (␤2m)), and a peptide ligand (1). The extracellular region of the heavy chain encompasses three domains, ␣ 1 , ␣ 2 , and ␣ 3 , with ␣ 1 and ␣ 2 forming a groove that accommodates a peptide ligand of, typically, 8 to 11 amino acid residues. The binding of the ligand to the groove is governed by the interaction of the side chains of certain peptide residues, called anchor positions, with several pockets of the heavy chain named A to F (1, 2). The size and chemical nature of these pockets impose restrictions on the peptide repertoire that can be associated with a particular class I antigen. It is reckoned that the ligandome of a given class I allotype may comprise up to 10,000 different peptides (3), although recent reports suggest that this number may be underestimated (4).Peptides displayed by MHC class I molecules derive from the intracellular degradation of endogenous proteins in the nucleus and cytosol and reach the lumen of the endoplasmic reticulum by means of the transporter associated with antigen processing. Inside the endoplasmic reticulum, peptides bind to the heavy chain and ␤2m in a multistep process involving several c...

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Surfaceome: a new era in the discovery of immune evasion mechanisms of circulating tumor cells

Masmoudi,

Vialaret,

Hirtz

et al. 2024

Molecular Oncology

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Circulating tumor cells (CTCs) are cancer cells that detach from the original site and reach the bloodstream. The most aggressive CTCs survive various immune system attacks and initiate metastasis formation. Importantly, CTCs are not specifically targeted by the current immunotherapies due to the limited knowledge on specific targets. Proteomic profiling can be a powerful tool for understanding some of the immune evasion mechanisms used by cancer cells and particularly CTCs. These mechanisms are generally linked to the expression of specific surface proteins/peptides (i.e. the surfaceome). The study of the peptides that bind to class I molecules of the major histocompatibility complex (MHC‐I) and of the various glycoproteins expressed on CTC surface may open a completely new avenue for the discovery of novel mechanisms of immune evasion. In this review, we discuss how immunopeptidomic and glycoproteomic studies of CTCs that interact with immune cells could help to better understand how metastasis‐initiator CTCs escape the host immune response. We also describe how immunopeptidomic and glycoproteomic studies are carried out.

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Identification of Natural MHC Class II Presented Phosphopeptides and Tumor-Derived MHC Class I Phospholigands

Cited by 50 publications

References 83 publications

Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD)

Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD)

Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue

Surfaceome: a new era in the discovery of immune evasion mechanisms of circulating tumor cells

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