Enhancing Top-Down
Analysis of Proteins by Combining
Ultraviolet Photodissociation (UVPD), Proton-Transfer Charge Reduction
(PTCR), and Gas-Phase Fractionation to Alleviate the Impact of Nondissociated
Precursor Ions
posted on 2023-12-27, 18:37authored bySean D. Dunham, Jennifer S. Brodbelt
Recent advances in
top-down mass spectrometry strategies
continue
to improve the analysis of intact proteins. 193 nm ultraviolet photodissociation
(UVPD) is one method well-suited for top-down analysis. UVPD is often
performed using relatively low photon flux in order to limit multiple-generation
dissociation of fragment ions and maximize sequence coverage. Consequently,
a large portion of the precursor ion survives the UVPD process, dominates
the spectrum, and may impede identification of fragment ions. Here,
we explore the isolation of subpopulations of fragment ions lower
and higher than the precursor ion after UVPD as a means to eliminate
the impact of the surviving precursor ion on the detection of low
abundance fragment ions. This gas-phase fractionation method improved
sequence coverage harvested from fragment ions found in the m/z regions lower and higher than the precursor
by an average factor of 1.3 and 2.3, respectively. Combining this
gas-phase fractionation method with proton transfer charge reduction
(PTCR) further increased the sequence coverage obtained from these m/z regions by another factor of 1.3 and
1.4, respectively. Implementing a post-UVPD fractionation + PTCR strategy
with six fractionation events resulted in a sequence coverage of 75%
for enolase, the highest reported for 193 nm UVPD.