Charge site assignment in native proteins by ultraviolet photodissociation (UVPD) mass spectrometry

Abstract

Characterization of all gas-phase charge sites of natively sprayed proteins and peptides is demonstrated using 193 nm UVPD. The high sequence coverage offered by UVPD is exploited for the accurate determination of charge sites in protein systems up to 18 kDa, allowing charge site to be studied as a function of protein conformation and the presence of disulfide bonds. Charging protons are found on both basic sidechains and on the amide backbone of less basic amino acids such as serine, glutamine, and proline. UVPD analysis was performed on the 3+ charge state of melittin, the 5+ to 8+ charge states of ubiquitin, and the 8+ charge state of reduced and oxidized β-lactoglobulin. The location of charges in gas-phase proteins is known to impact structure; molecular modeling of different charge site motifs of 3+ melittin demonstrates how placement of protons in simulations can dramatically impact the predicted structure of the molecule. The location of positive charge sites in ubiquitin and β-lactoglobulin are additionally found to depend on the presence or absence of salt-bridges, columbic repulsion across the length of the peptide, and protein conformation. Charge site isomers are demonstrated for ubiquitin and β-lactoglobulin but found to be much less numerous than previously predicted.

Characterization of hydrogen bonding motifs in proteins: hydrogen elimination monitoring by ultraviolet photodissociation mass spectrometry

“…We have recently used 193 nm UVPD to localize the three charge sites of melittin (3+) and have used molecular dynamics simulations to identify a structure consistent with the charge sites determined by UVPD and with the collisional cross section obtained from ion mobility measurements. 49 The resulting structure was the lowest energy one for the protonation scheme derived from UVPD analysis and was within 1.5% of the experimental CCS, thus validating the assignment. UVPD was used to generate fragment ions from the 3+ charge state of melittin.…”

“…We have previously shown that 193 nm UVPD can be used to localize charge sites in gas-phase proteins. 49 In brief, UVPD of native proteins was shown to generate a and x fragment ions with charge states that were consistent with the protonation sites of the intact protein. Based on this strategy, the 5+ charge state of PA was evaluated.…”

“…In Figure 8, the fractional abundances of a and x fragment ion charge states are shown as function of sequence and used to infer the charge sites in a manner described previously. 49 Two charging schemes are evident from the splitting in observed charge states between Lys11 and His68 and are denoted A and B, with A referring to the more abundant population. For example, the bar at Gln49 in Figure 8 reflects the a 49 ion population for which ~60% is detected in the 7+ charge state (thus representative of A) and 40% is detected in the 8+ charge state (representing B).…”

193 nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology

“…We have previously demonstrated that it is possible to assign the charge sites of native-like protein ions by tabulating the relative abundances of the charge states of the a and x ion product ions. 40 Using this strategy it was determined that 1) the charge state of the fragmenting protein can be deduced by identifying and tallying all charge sites, and 2) that charge site heterogeneity correlates with protein unfolding. 40 Thus, we postulated that a similar charge site analysis of the products arising from backbone dissociation of tetrameric SA and TTR could be used to assess the fragmentation pathway(s) by which these products are generated from 193 nm UVPD of multi-protein complexes.…”

“…Thus, mapping the charge states of the complementary a and x ions offers a convenient way to estimate the locations of charge sites of the protein. 40 …”