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

Abstract

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.

Symmetry of 4-Oxalocrotonate Tautomerase Trimers Influences Unfolding and Fragmentation in the Gas Phase

“…38,39 Changes in abundances of sequence ions produced by UVPD as a function of unfolding from collisional heating may reveal changes in higher-order structures of proteins. 38,45 The loss of higher-order structure using a collision heating energy of at least −80 V results in a shift in the abundance of the various ion types as a function of the collision voltage (Figure S12), suggesting a change in the way energy is distributed throughout the protein during UVPD for the native-like versus collisionally unfolded trimers. Figure 3A−C displays the locations and magnitudes of significant changes (p value <0.02) in the backbone cleavage sites along the primary sequence of the 4-OT trimers based on the abundances of aand x-type ions produced by UVPD with and without collisional heating.…”

“…Cleavages of N-terminal to Pro residues such as this one are specific preferential cleavages also observed upon HCD, 46 often most prominently observed for b and y ions. 45 Here, the enhanced Pro cleavage is observed for the a 55 + 1 ion.…”

Mapping paratopes of nanobodies using native mass spectrometry and ultraviolet photodissociation

“…This correlation empowers characterization of conformational changes induced by point mutations, ligand binding, and protein complexation by UVPD. [26][27][28][29][30][31][32][33] Additionally, the high sequence coverage and rapid timescale of photodissociation enable detailed analyses of protein gas-phase structure, even informing proton sequestration with single residue resolution. 34 As native MS and UVPD increasingly gain broader utility for new protein applications, development and establishment of strategies to routinely study protein structure become imperative to cement these methodologies as cornerstones in the elds of structural biology and biotechnology that encompass development of new therapeutics, imaging agents, diagnostics and drug delivery agents.…”

“…The high diversity of fragment types characteristic of UVPD originates from competing pathways: direct dissociation from excited electronic states yields a/a + 1/x/x + 1 ions, and internal conversion to the ground state following intramolecular vibrational energy redistribution (IVR) produces b/y fragments. 32,33 IVR processes may preferentially sever weak non-covalent interactions, whereas direct dissociation from excited states occurs on a faster time-scale minimizing disruption of noncovalent interactions. 32,33 Consequently, the latter dissociation pathways and respective products, a/a + 1/x/x + 1 ions, are best suited to evaluate antigen-induced changes in nanobody topology.…”

“…32,33 IVR processes may preferentially sever weak non-covalent interactions, whereas direct dissociation from excited states occurs on a faster time-scale minimizing disruption of noncovalent interactions. 32,33 Consequently, the latter dissociation pathways and respective products, a/a + 1/x/x + 1 ions, are best suited to evaluate antigen-induced changes in nanobody topology. Variations in abundances of these four fragment types tended to be greater at the interface and CDRs, and inspection of the color-coded maps in Fig.…”