Extending Proteome Coverage by Combining MS/MS Methods and a Modified Bioinformatics Platform Adapted for Database Searching of Positive and Negative Polarity 193 nm Ultraviolet Photodissociation Mass Spectra

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.