STRATEGY FOR ISOLATING AND SEQUENCING BIOLOGICALLY DERIVED MHC CLASS-I PEPTIDES

The presentation of MHC class I peptides at cell surfaces and the subs equent 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 su ch peptides. After immunoaffinity concentration, and subsequent acetic acid release of MHC class I peptides from MHC protein complex, the pe ptides are subjected to reversed phase HPLC, where they are separated based on their hydrophilic-hydrophobic character. These coarse fractio ns are then loaded onto a specially designed membrane preconcentration -capillary electrophoresis cartridge (mPC-CE) and subsequently subject ed to on-line mPC-CE-MS analysis. The second dimension of chromatograp hy by CE separation affords resolution of peptides based on their char ge/mass (to a first approximation) ratio. Ultimately peptides are sequ enced using mPC-CE-tandem mass spectrometry (mPC-CE-MS-MS). We describ e the strategy for sequencing <60 femtomoles of a peptide obtained fro m 3 . 10(9) K-b-derived EL-4 cells.