Extending Proteome Coverage by Combining MS/MS Methods and a Modified Bioinformatics Platform Adapted for Database...

To extend proteome coverage obtained from bottom-up mass spectrometry approaches, three complementary ion activation methods, higher energy collision dissociation (HCD), ultraviolet photodissociation (UVPD), and negative mode UVPD (NUVPD), are used to interrogate the tryptic peptides in a human hepatocyte lysate using a high performance Orbitrap mass spectrometer. The utility of combining results from multiple activation techniques (HCD+UVPD+NUVPD) is analyzed for total depth and breadth of proteome coverage. This study also benchmarks a new version of the Byonic algorithm, which has been customized for database searches of UVPD and NUVPD data. Searches utilizing the customized algorithm resulted in over 50% more peptide identifications for UVPD and NUVPD tryptic peptide data sets compared to other search algorithms. Inclusion of UVPD and NUVPD spectra resulted in over 600 additional protein identifications relative to HCD alone.