Speaker: Amina S. Woods, Johns Hopkins School of Medicine, Baltimore, MD

Topic: Antigen Discovery Using MALDI Mass Spectrometry

Place: Building 426, Conference Room, NCI-Frederick, Frederick, MD

Time: Wednesday, March 24, 1999, at 1:00 PM

Abstract: Recent technical breakthroughs in sequencing MHC class I peptides has allowed researchers to elucidate the structure of biomolecules that were thought to be beyond reach as they were available in infinitesimal amounts. A class Ib (Qa-1^b) restricted CTL clone has been isolated, which recognizes murine cells infected with Salmonella typhimirium as well as other gram-negative organisms. Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify this ubiquitous antigen from 5 X 10⁹ Qa-1^b transfected L-cells infected with S. typhirium. The mass spectrometer was equipped with a curved-field reflectron, which enables the recording of amino acid sequence mass spectra for peptides without stepping the reflectron voltage. A combination of exo- and endo-peptidase digests and post- and in-source decay were utilized to sequence the bioactive peptide. This approach was also used to characterize the structure of CD1d1 ligand from normal mouse, the ligand from a murine colorectal tumor, as well as other antigenic epitopes. The total amount of bioactive peptide purified was quantitated to be less than 100 femtomoles, and the amount used for structure elucidation was less than 20 femtomoles. This technological tour de force has implications in identifying T-cell antigens involved in autoimmunity, oncology and infectious diseases.