Influence of Primary Structure on Fragmentation of Native-Like Proteins by Ultraviolet Photodissociation

Analysis of native-like
protein structures in the gas phase via
native mass spectrometry and auxiliary techniques has become a powerful
tool for structural biology applications. In combination with ultraviolet
photodissociation (UVPD), native top-down mass spectrometry informs
backbone flexibility, topology, hydrogen bonding networks, and conformational
changes in protein structure. Although it is known that the primary
structure affects dissociation of peptides and proteins in the gas
phase, its effect on the types and locations of backbone cleavages
promoted by UVPD and concomitant influence on structural characterization
of native-like proteins is not well understood. Here, trends in the
fragmentation of native-like proteins were evaluated by tracking the
propensity of 10 fragment types (a, a+1, b, c, x, x+1, y, y-1, Y, and z) in relation to primary structure in a native-top
down UVPD data set encompassing >9600 fragment ions. Differing
fragmentation
trends are reported for the production of distinct fragment types,
attributed to a combination of both direct dissociation pathways from
excited electronic states and those surmised to involve intramolecular
vibrational energy redistribution after internal conversion. The latter
pathways were systematically evaluated to evince the role of proton
mobility in the generation of “CID-like” fragments through
UVPD, providing pertinent insight into the characterization of native-like
proteins. Fragmentation trends presented here are envisioned to enhance
analysis of the protein higher-order structure or augment scoring
algorithms in the high-throughput analysis of intact proteins.