Strategy for isolating and sequencing biologically derived MHC class I peptides.
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 are 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 3.10(9) Kb-derived EL-4 cells.
Tomlinson
A J
AJ
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.
Jameson
S
S
Naylor
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002457:Cell Extracts / Q000737:chemistry; D002793:Cholic Acids / Q000737:chemistry; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000737:chemistry; D019075:Electrophoresis, Capillary; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000302:isolation & purification*; D013058:Mass Spectrometry; D009862:Online Systems; D017421:Sequence Analysis / Q000379:methods; D013056:Spectrophotometry, Ultraviolet
D002457:Cell Extracts; D002793:Cholic Acids; D003902:Detergents; D015395:Histocompatibility Antigens Class I; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate