TY - JOUR
T1 - Strategy for isolating and sequencing biologically derived MHC class I peptides
AU - Tomlinson, Andy J.
AU - Jameson, Stephen
AU - Naylor, Stephen
PY - 1996/9/13
Y1 - 1996/9/13
N2 - 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.
AB - 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.
KW - Peptides
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UR - http://www.scopus.com/inward/citedby.url?scp=0030582353&partnerID=8YFLogxK
U2 - 10.1016/0021-9673(96)00333-0
DO - 10.1016/0021-9673(96)00333-0
M3 - Article
C2 - 8843675
AN - SCOPUS:0030582353
SN - 0021-9673
VL - 744
SP - 273
EP - 278
JO - Journal of Chromatography A
JF - Journal of Chromatography A
IS - 1-2
ER -