Impact of charge state on 193 nm ultraviolet photodissociation of protein complexes

Abstract

As applications in mass spectrometry continue to expand into the field of structural biology, there have been an increasing number of studies on noncovalent biological assemblies. Ensuring that protein complexes maintain native-like conformations and architectures during the transition from solution to the gas phase is a key aim. Probing composition and arrangement of subunits of multi-charged complexes via tandem mass spectrometry (MS/MS) may lead to protein unfolding and the redistribution of charges on the constituent subunits, leading to asymmetric charge partitioning and ejection of a high-charged monomer. Additionally, the overall dissociation efficiency of many ion activation methods is often suppressed for low charge states, hindering the effectiveness of MS/MS for complexes that have low charge density. Ultraviolet photodissociation (UVPD) of proteins using 193 nm photons is a high-energy alternative to collisional activation and demonstrates little to no charge state dependence. Here the symmetry of charge partitioning upon UVPD is evaluated for an array of multimeric protein complexes as a function of initial charge state. The results demonstrate that high laser energies (3 mJ) for UVPD induces more symmetric charge partitioning and ejection of low-charged, presumably compact monomers than higher-energy collisional dissociation (HCD).

Figure 1.

ESI-MS of Cu/Zn superoxide dismutase (SOD) in a) 20 mM m-NBA, 80 mM ammonium acetate solution for supercharging, b) 100 mM ammonium acetate (standard native MS solution), and c) 20 mM triethylammonium acetate, 80 mM ammonium acetate for charge-reducing native solutions.

Figure 2.

MS/MS spectra of streptavidin (SA) in 15+ (top) and 10+ (bottom) charge states using a) a lab frame collision energy of 1 keV for HCD, b) 1 mJ UVPD, and c) 3 mJ UVPD, each using a single laser pulse. The precursor ion is labelled with a star.

Figure 3.

Percent sequence coverage of all of the charge states generated from supercharging, standard, and charge-reducing solutions for a) superoxide dismutase (SOD), b) streptavidin (SA), c) transthyretin (TTR), d) hemoglobin (Hb), e) C-reactive protein (CRP). A single pulse of 3 mJ was used for all UVPD measurements. HCD energy was optimized for each protein and ranged from 1400 to 2500 eV lab frame collision energy. Error bars equal to standard deviation of replicate data. Charge states with insufficient abundances for MS/MS analysis are not included.

Figure 4.

Weighted average charge (C_ES) of ejected monomers using varying UVPD laser pulse energies for the precursors of a) 10+ to 15+ streptavidin (SA), b) 11+ to 17+ transthyretin (TTR), c,d) 12+ to 17+ hemoglobin (Hb), and e) 17+ to 19+ and 23+ to 27+ C-reactive protein (CRP). Ejected monomers of Hb are shown separately as c) α-subunits and d) β-subunits. Error bars represent the standard deviation of replicate data. Values in parentheses next to each precursor charge indicate the theoretical charge state of each monomer following charge-symmetric partitioning.

Figure 5.

Asymmetric charge partitioning factor of a) 10+ to 15+ streptavidin (SA), b) 11+ to 17+ transthyretin (TTR), c) 12+ to 17+ hemoglobin (Hb), and d) 17+ to 19+ and 23+ to 27+ C-reactive protein (CRP) upon activation with HCD and low- and high-energy UVPD (1 pulse). The ACPF for Hb is shown as the average for α- and β-subunits. The lab frame collision energy was optimized for each precursor and is equal to 1000 eV for SA (a), 1200 eV for TTR and Hb (b and c), and 2000 eV for CRP (d). The ACPF for purely symmetric charge partitioning (1.0) is shown in each graph. Error bars are equal to standard deviation of replicate data.