Simplified high-sensitivity sequencing of a major histocompatibility complex class I-associated immunoreactive peptide using matrix-assisted laser desorption/ionization mass spectrometry.
Cytotoxic T cells (CTL) are known to recognize small peptide fragments of cytoplasmic proteins bound to major histocompatibility complex (MHC) class I molecules on cell surfaces. Recent work indicates that tumor antigens are processed and presented in a manner similar to viral antigens. Identification of the peptides recognized by tumor-specific CTL would provide valuable information about their parent proteins, as well as allowing for the development of recombinant antigen-specific tumor vaccines. While highly represented MHC-bound peptides have been routinely purified by reversed-phase HPLC for Edman degradation sequencing, identification and sequencing of infrequent peptides that represent the biologically relevant targets of tumor-specific CTL have proved elusive. We have combined matrix-assisted laser desorption/ionization mass spectrometry with on-slide exopeptidase digestion to successfully identify and directly sequence a model tumor-specific peptide antigen derived from an integrated viral gene. The enhanced sensitivity of this technique (femtomolar range) allows for the sequencing of specific MHC-bound peptides derived from as few as 1 x 10(9) cells.
Woods
A S
AS
Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Huang
A Y
AY
Cotter
R J
RJ
Pasternack
G R
GR
Pardoll
D M
DM
Jaffee
E M
EM
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000276:immunology; D002268:Carboxypeptidases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D007931:Leucyl Aminopeptidase / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D012680:Sensitivity and Specificity; D017421:Sequence Analysis / Q000379:methods*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D015395:Histocompatibility Antigens Class I; D010455:Peptides; C025427:carboxypeptidase P; D002268:Carboxypeptidases; D007931:Leucyl Aminopeptidase; D000645:Ammonium Sulfate