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 (MH C) class I molecules on cell surfaces. Recent work indicates that tumo r antigens are processed and presented in a manner similar to viral an tigens. Identification of the peptides recognized by tumor-specific CT L would provide valuable information about their parent proteins, as w ell as allowing for the development of recombinant antigen-specific tu mor vaccines. While highly represented MHC-bound peptides have been ro utinely purified by reversed-phase HPLC for Edman degradation sequenci ng, identification and sequencing of infrequent peptides that represen t 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 th is technique (femtomolar range) allows for the sequencing of specific MHC-bound peptides derived from as few as 1 X 10(9) cells. (C) 1995 Ac ademic Press, Inc.