Vacuum ultraviolet photofragmentation of peptide ions.

摘要

Tandem mass spectrometry is widely used to identify proteins based on the fragmentation of proteolytic peptides. The processes typically employed to activate ions in these experiments induce vibrational excitation. As a result, mass spectra are often dominated by the lowest energy fragmentation processes. Typically, incomplete fragment series are observed. The information contained in these spectra is not sufficient to determine peptide sequences directly. Instead, spectra are interpreted by comparing the experimental results with those obtained from experiments performed in silico using database sequences. Only peptide sequences found in the database will yield matches, so organisms without sequenced genomes cannot be investigated. Likewise, mutated or modified peptides are problematic. In this dissertation, a new peptide ion excitation technique using vacuum ultraviolet light is introduced. The resulting fragmentation spectra are significantly different than those observed with any other activation technique. In the presence of a favorable protonation site, the major fragment series correspond to cleavage of the peptide backbone between the alpha- and carbonyl-carbons with the charge sequestered at the basic site. Sequence coverage of over 90% is observed, with consistent intensities across each spectrum. Typical photodissociation spectra contain sufficient information to directly determine the sequence of a peptide without a database. By addressing the limitations of current techniques, photodissociation could enable more comprehensive peptide identification during proteomics studies.