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J Proteomics. 2014 Sep 23;109:240-4. doi: 10.1016/j.jprot.2014.07.009. Epub 2014 Jul 19.

Accurate quantitation of MHC-bound peptides by application of isotopically labeled peptide MHC complexes.

Author information

1
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.
2
Department of Hematology, Laboratory of Experimental hematology, Leiden University Medical Center, Leiden, The Netherlands.
3
OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
4
Department of Cell Biology II, The Netherlands Cancer Institute (NKI-AVL), Amsterdam, The Netherlands.
5
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: p.a.van_veelen@lumc.nl.

Abstract

Knowledge of the accurate copy number of HLA class I presented ligands is important in fundamental and clinical immunology. Currently, the best copy number determinations are based on mass spectrometry, employing single reaction monitoring (SRM) in combination with a known amount of isotopically labeled peptide. The major drawback of this approach is that the losses during sample pretreatment, i.e. immunopurification and filtration steps, are not well defined and must, therefore, be estimated. In addition, such losses can vary for individual peptides. Therefore, we developed a new approach in which isotopically labeled peptide-MHC monomers (hpMHC) are prepared and added directly after cell lysis, i.e. before the usual sample processing. Using this approach, all losses during sample processing can be accounted for and allows accurate determination of specific MHC class I-presented ligands. Our study pinpoints the immunopurification step as the origin of the rather extreme losses during sample pretreatment and offers a solution to account for these losses. Obviously, this has important implications for accurate HLA-ligand quantitation. The strategy presented here can be used to obtain a reliable view of epitope copy number and thus allows improvement of vaccine design and strategies for immunotherapy.

KEYWORDS:

Epitopes; MS; Quantitation; SRM; hpMHC

PMID:
25050860
DOI:
10.1016/j.jprot.2014.07.009
[Indexed for MEDLINE]

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