Strategy for isolating and sequencing biologically derived MHC class I peptides

Andy J. Tomlinson, Stephen Jameson, Stephen Naylor

Research output: Contribution to journalArticlepeer-review

43 Scopus citations

Abstract

The presentation of MHC class I peptides at cell surfaces and the subsequent cytolytic T-lymphocyte response are critical components of the mammalian immune response. However, the identification and sequencing of such peptides present a considerable analytical challenge since >10,000 peptides at 10-15-10-18 M concentrations are often present in the mixture. We describe a two-dimensional chromatography approach in conjunction with tandem mass spectrometry to sequence and identify such peptides. After immunoaffinity concentration, and subsequent acetic acid release of MHC class I peptides from MHC protein complex, the peptides ate subjected to reversed phase HPLC, where they are separated based on their hydrophilic-hydrophobic character. These coarse fractions are then loaded onto a specially designed membrane preconcentration-capillary electrophoresis cartridge (mPC-CE) and subsequently subjected to on-line mPC-CE-MS analysis. The second dimension of chromatography by CE separation affords resolution of peptides based on their charge/mass (to a first approximation) ratio. Ultimately peptides are sequenced using mPC-CE-tandem mass spectrometry (mPC-CE-MS-MS). We describe the strategy for sequencing <60 femtomoles of a peptide obtained from 3-109 Kb-derived EL-4 cells.

Original languageEnglish (US)
Pages (from-to)273-278
Number of pages6
JournalJournal of Chromatography A
Volume744
Issue number1-2
DOIs
StatePublished - Sep 13 1996

Keywords

  • Peptides

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