Ultraviolet Photodissociation for Characterization of Whole Proteins on a Chromatographic Time Scale

Intact protein characterization using
mass spectrometry thus far
has been achieved at the cost of throughput. Presented here is the
application of 193 nm ultraviolet photodissociation (UVPD) for top
down identification and characterization of proteins in complex mixtures
in an online fashion. Liquid chromatographic separation at the intact
protein level coupled with fast UVPD and high-resolution detection
resulted in confident identification of 46 unique sequences compared
to 44 using HCD from prepared Escherichia coli ribosomes.
Importantly, nearly all proteins identified in both the UVPD and optimized
HCD analyses demonstrated a substantial increase in confidence in
identification (as defined by an average decrease in E value of ∼40 orders of magnitude) due to the higher number
of matched fragment ions. Also shown is the potential for high-throughput
characterization of intact proteins via liquid chromatography (LC)–UVPD-MS
of molecular weight-based fractions of a Saccharomyces cerevisiae lysate. In total, protein products from 215 genes were identified
and found in 292 distinct proteoforms, 168 of which contained some
type of post-translational modification.