Protein Subunits Released by Surface Collisions of Noncovalent Complexes: Nativelike Compact Structures Revealed by Ion Mobility Mass Spectrometry

Abstract

The quaternary structure of proteins determines their biological function, and a majority of proteins exist as oligomers in vivo with miscellaneous architectures. [1] Mass spectrometry (MS) can be applied to study the stoichiometry and interactions of protein complexes by molecular weight measurements under gentle instrumental conditions where noncovalent interactions are preserved in the gas phase. [2] Recently, there have been extensive efforts to also utilize ion mobility (IM) techniques combined with MS for structural studies of noncovalent biological complexes, [3] including virus assembly pathways, [4] because IM provides conformational information, which is not accessible by MS, for these gas-phase ions. Experimentally measured collisional cross sections (CCSs) from IM can serve as constraints for architecture determination by molecular modeling. [5] Additionally, tandem MS can be used to dissociate gas-phase complexes. [2b, 6] A protein assembly would ideally dissociate into various noncovalent subcomplexes, and the topology of the original complex could be derived by piecing together all the subcomplex products. The common tandem MS method, collision induced dissociation (CID), involves activation of the complexes by collision with neutral gas atoms or molecules. Typically, CID results in an "asymmetric" dissociation into highly charged monomers and complementary (nÀ1)-mers [7] (although a few exceptions have been reported [8] ), and studies have suggested that unfolding of protein complexes occurs in CID. [9] It is therefore difficult to relate the CCS measurements of CID product ions to the complexes native structure.Tandem MS can alternatively be achieved by surface induced dissociation (SID) where the complexes collide with a surface target. Previous research in our group [7,10] has shown that several protein complexes dissociate in a more "symmetric" manner with SID than by CID, and have charge distributed more proportionally to the mass. We hypothesized that dissociation might occur in the absence of gradual monomer unfolding for SID because activation by SID is a single-step, higher-energy deposition, fast process that is different from the multistep, slower CID process. [7, 10b] SID has recently been applied to determining the quaternary structure of a heterohexameric protein with information from subunit product ions such as heterotrimers unique to SID. [10a] We present herein the first IM measurements on the SID products of several protein complexes, along with comparison to CID products, by using a modified quadrupole/IM/time-offlight (Q/IM/TOF) instrument. Briefly, the precursor ions are dissociated by CID or SID cells placed in front of the IM cell. The product ions are subsequently separated based on their size, shape, and charge under the influence of a continuous series of electrical pulses and friction with neutral gas in the IM cell. The drift times of the ions are recorded, with larger and lower-charged ions experiencing longer drift times. Experimental CCSs can be derived from th...

Salt Bridge Rearrangement (SaBRe) Explains the Dissociation Behavior of Noncovalent Complexes

“…Monomers (6+ and 7+) expelled from decomposing 18+ precursors were found to be compact with collision cross-sections (CCS) similar to calculated values and to all monomers (3+ - 6+) released by surface induced dissociation (SID) [74] (Fig. 3C).…”

“…Interestingly, two additional CID/SID comparisons yielded similar CCS values for identically charged monomer products from symmetric and asymmetric decompositions [74]. Transthyretin (TTR) tetramers (15+), subjected to SID and CID, yielded 3+-7+ and 5+-9+ products, respectively.…”

Human Defensins Facilitate Local Unfolding of Thermodynamically Unstable Regions of Bacterial Protein Toxins

“…3-molar excess of HNP1 was added to the protein, followed by addition of DDM. Nano-electrospray ionization mass spectrometry (nano-ESI/MS) analysis was conducted on a modified Quadrupole Ion Mobility Time of Flight (Q-IM-TOF) instrument (Synapt G2, Waters Corp., Manchester, U.K.) with a customized surface-induced dissociation (SID) device installed before the IM chamber as previously described (Zhou et al, 2012). See also Supplemental Experimental Procedures.…”

Surface-Induced Dissociation of Homotetramers with D2 Symmetry Yields their Assembly Pathways and Characterizes the Effect of Ligand Binding

“…Nanoelectrospray ionization mass spectrometry (ESI MS) analysis was conducted by utilizing a modified quadrupole ion mobility time-of-flight (Q-IM-TOF) instrument (Synapt G2-S, Waters) with a customized SID device installed before the IM chamber (SID-IM) as previously described (Zhou et al, 2012). The direct current (DC) voltages on the lenses of our customized SID device are tuned differently depending on the experiments being conducted.…”