Surface-Induced Dissociation of Noncovalent Protein Complexes in an Extended Mass Range Orbitrap Mass Spectrometer

Abstract

Herein we demonstrate the first adaptation of surface-induced dissociation in a modified high-mass range, high-resolution Orbitrap mass spectrometer. The SID device was designed to be installed in the Q-Exactive series of Orbitrap mass spectrometers with minimal disruption of standard functions. The performance of the SID-Orbitrap instrument has been demonstrated with several protein complex and ligand-bound protein complex systems ranging from 53 to 336 kDa. We also address the effect of ion source temperature on native protein-ligand complex ions as assessed by SID. Results are consistent with previous findings on quadrupole time-of-flight instruments and suggest that SID coupled to high-resolution MS is well-suited to provide information on the interface interactions within protein complexes and ligand-bound protein complexes. <br>

Controlling protein assembly on inorganic crystals through designed protein interfaces

“…Sample purity and integrity was analyzed by on-line buffer exchange MS using an UltiMate™ 3000 RSLC (Thermo Fisher Scientific) coupled to an Exactive Plus EMR Orbitrap instrument (Thermo Fisher Scientific) modified to incorporate a quadrupole mass filter and allow for surface-induced dissociation 43 . 40 pmole protein (5 μL of 8 μM protein in TBS) were injected and on-line buffer exchanged to 200 mM ammonium acetate, pH 6.8 (AmAc) by a self-packed buffer exchange column 44 (P6 polyacrylamide gel, BioRad) at a flow-rate of 100 μL per min.…”

The Ims Paradox: A Perspective on Structural Ion Mobility‐mass Spectrometry

“…Instrument development for native‐MS is largely driven by needs to understand the structural details of large protein complexes, viz . solvent manipulation (Shi et al, 2014), effects of protein‐protein interactions (Cong et al, 2016), PTMs (Fornelli et al, 2020), stoichiometry (Kaltashov & Mohimen, 2005; Zhou & Wysocki, 2014; Shirzadeh et al, 2019; Stiving et al, 2019; VanAernum et al, 2019), and ligand binding events (Hyung, Robinson, & Ruotolo, 2009; Allison et al, 2015; Cong et al, 2016; Marchand et al, 2018), investigations of which are made possible by recent advances in MS instrumentation (Giles et al, 2019 Poltash et al, 2020). Advances in IM‐MS technology and experiments were accelerated by the readily adopted Waters SYNAPT platform.…”

“…Advances in IM‐MS technology and experiments were accelerated by the readily adopted Waters SYNAPT platform. The versatile SYNAPT platform allows for pre‐IM ion mass selection allowing for tandem MS experiments that can be further interrogated by the novel traveling‐wave IM with additional fragmentation, that is, CID (Mitchell Wells & McLuckey, 2005), SID (Zhou & Wysocki, 2014; Stiving et al, 2019; VanAernum et al, 2019), and electron transfer/capture dissociation (Zhurov et al, 2013). However, perturbations in the structure of native proteins are difficult to study, because the changes in structure may lead to small changes in CCS that are unresolvable on commercial instrumentation (Poltash et al, 2020).…”

Cellular mRNA triggers structural transformation of Ebola virus matrix protein VP40 to its essential regulatory form

“…The ring fraction of VP40-WT yielded a broad, unresolvable m/z spectrum (Figure 6B); this is as expected if the endogenous VP40 ring binds a variety of cellular RNA. To further investigate this ring sample, we partially disrupted it by tandem MS using surfaceinduced dissociation (SID) (VanAernum et al, 2019). This yielded .…”

“…For Figure 6C, the ring fraction was measured by direct infusion into a Q Exactive UHMR instrument modified with a surfaceinduced dissociation (SID) device (VanAernum et al, 2019). Sample ionization was accomplished using a Nanospray Flex ion source and in-house pulled nanospray emitters.…”