Chemical modification of specific residues to improve peptide antigen sequencing by mass spectrometric fragmentation

Peptide sequencing via mass spectrometry is often accomplished by direct fragmentation of the peptide in the mass spectrometer. However, sequence determination with this method is often difficult, particularly in the case of peptides with internal basic residues, such as those which bind MHC molecules. The combination of peptide fragmentation and chemical modification can assist in the determination of peptide identity.; In this work, chemical modification of specific amino acid residues was accomplished through a variety of methods. First, guanidination of lysine was used to determine the peptide sequence GMKFDRGYI, a class IB MHC-associated antigen. Next, acetylation of the N-terminus to distinguish N-terminal product ions was utilized to sequence the class II peptide FAALNAQHVLA. Also, modification of arginine residues increased fragmentation and produced a pattern of product ions which can assist with de novo peptide sequencing.; A class-characteristic neutral loss from peptides containing an oxidized methionine residue was exploited to obtain sequence information in the MS/MS mode from peaks hidden in the chemical noise of a mass spectrum. Through this technique, the peptide GYGMPRQIL was sequenced. Finally, the use of a TOF/TOF instrument was found to increase the number of single amino acid immonium ions in a MS/MS spectrum. This process provided amino acid composition information which was utilized in conjunction with higher-mass product ions to yield peptide sequence.