Surface-induced Dissociation Mass Spectrometry as a Structural Biology Tool

Abstract

Native mass spectrometry (nMS) is evolving into a workhorse for structural biology. The plethora of online and offline preparation, separation, and purification methods as well as numerous ionization techniques combined with powerful new hybrid ion mobility and mass spectrometry systems has illustrated the great potential of nMS for structural biology. Fundamental to the progression of nMS has been the development of novel activation methods for dissociating proteins and protein complexes to deduce primary, secondary, tertiary, and quaternary structure through the combined use of multiple MS/MS technologies. This review highlights the key features and advantages of surface collisions (surface-induced dissociation, SID) for probing the connectivity of subunits within protein and nucleoprotein complexes and, in particular, for solving protein structure in conjunction with complementary techniques such as cryo-EM and computational modeling. Several case studies highlight the significant role SID, and more generally nMS, will play in structural elucidation of biological assemblies in the future as the technology becomes more widely adopted. Cases are presented where SID agrees with solved crystal or cryoEM structures or provides connectivity maps that are otherwise inaccessible by "gold standard" structural biology techniques.

Figure 1:

Schematic representations of CID and SID of noncovalent protein complexes with corresponding simplified potential energy diagrams shown at the bottom. In CID (left) protein complexes undergo multiple collisions with the collision gas, which can result in rearrangement/unfolding and ejection of elongated, highly charged monomer and complementary (N-1)mer (A). In SID (right) the high, rapid energy jump can favor a faster, more direct dissociation pathway (B) into folded subunits carrying charge proportional to their mass (surface area), referred to as ‘symmetric charge partitioning’. Reproduced with permission from ref . Copyright 2014 American Chemical Society.

Figure 2:

Charge-reduced species have more native-like SID fragmentation patterns than their normal-charge counterparts. (a) SID spectrum of (charge-reduced) 18+ C-reactive protein (CRP) at 1 keV, (b) CID spectrum of CRP 18+ at 3.6 keV, (c) SID spectrum of CRP 24+ (no charge reduction) at 1 keV, and (d) CID spectrum of CRP 24+ at 3.6 keV. Adapted from ref. with permission from the Royal Society of Chemistry.

Figure 3:

SID can distinguish between tetramers of different arrangements. A) PISA interfacial analysis for C4 tetramer aquaporinZ, and D2 tetramers streptavidin, neutravidin and transthyretin. B) Low energy SID for 13+ aquaporin Z, adapted with permission from ref with permission from the Royal Society of Chemistry. C) low energy SID of 11+ streptavidin, adapted with permission from ref. .

Figure 4:

Predicted AE, based on the initial optimized model as shown in equation 1, shows good correlation to experimental AE. Reproduced with permission from ref .

Figure 5:

SID can distinguish between different arrangements of subunits. A) SID from a sample at 4 °C of a TTR UU/TT tetramer yielding an MS spectrum with equal signal intensity for UU and TT dimers. (B) SID-MS spectrum of an equimolar solution of UU/TT, UT/UT, and UT/TU. Reproduced with permission from Ref . Copyright 2019 American Chemical Society.

Figure 6:

Workflow for characterizing the TNH structure by complementary mass spectrometric tools, reproduced with permission from ref . Copyright 2015 American Chemical Society.

Figure 7:

Using data from nMS and SID experiments, along with homology and ab initio models, a structural model for Mnx could be proposed. Reproduced with permission from ref .

Figure 8:

Comparison of (a) CID and (b) SID spectra of 19+ toyocamycin nitrile hydratase heterohexamer (αβγ)₂ on a Micromass/Water QTOF II mass spectrometer retrofitted with a Gen 1 SID device. Reproduced from ref. . Copyright 2011 American Chemical Society.

Figure 9:

SID cells for Q-IM-TOF platforms. Schematic diagram of (a) Waters Synapt G2 platform with three integrated SID cells with locations noted. Three generations of SID cells have been installed in the G2, (a) Gen 1,^, (b) Gen 2, (c), Gen 3. Note that the Gen 1 & 2 devices can be located before and after the IM cell, whereas the Gen 3 install location is in the quadrupole chamber prior to the Trap.

Figure 10:

SID-IM reveals the connectivity within a heterohexamer. SID-IM of 14+ TNH on a Waters G2-S fitted with a Gen 1 SID device prior to the IM cell. Shown are low- and high-energy spectra at (a) 700 eV and (b) 1680 eV, respectively. Reproduced from ref. . Copyright 2015 American Chemical Society.

Figure 11:

SID-IM-TOF of the 18+ charge state of holoTRAP 11mer (in 200 mM EDDA with 14 equiv of trp) on a Synapt G2. All oligomeric fragments are observed in mobility space, consistent with the cyclic arrangement of the subunits. Reproduced with permission from Ref . Copyright 2020 American Chemical Society.

Figure 12:

SID-IM of the 43+ and 44+ rabbit 20s proteasome at 150 V. A) full MS (top) and isolation (bottom), B and C) IM-MS analysis of SID products, D-J) extracted spectra for the different regions underlined in panel B. Reproduced with permission from ref . Copyright 2020 American Chemical Society.

Figure 13:

IM-SID can be used to individually probe different conformations of in-source activated CRP (cone 200 V). Right hand panels show extracted SID spectra (1260 eV) from the highlighted regions in the left hand panel. Cartoon representations of the structure are also shown as inserts on the CCS panel. Reproduced with permission from ref . Copyright 2015 American Chemical Society.

Figure 14:

SID cells for Orbitrap platforms. (a) Schematic of a Thermo Scientific Extended Mass Range (EMR) Orbitrap Exactive platform with SID cell taking the place of a transport multipole, (b) the Gen 1 SID cell design and (c) Gen 3 SID cell design, (d) SID spectrum of GroEL 14mer (stacked 7mer rings) 65+ to 74+ (165 V) obtained on a UHMR equipped with a Gen 1 SID cell, and (e) Unidec deconvoluted mass spectrum with 7mer as a prominent fragment. Numbers indicate the oligomeric state that corresponds to each peak in the mass domain. Panel (a) reproduced from ref. . Copyright 2019 American Chemical Society. Panels (b) and (c) adapted from ref. . Copyright 2020 American Chemical Society. Panels (d) and (e) reproduced from ref. with permission from The Royal Society of Chemistry.

Figure 15:

SID distinguishes ligand binding locations in pentamers CRP (with phosphocholine, PC) and CTB (with GM1s). CID spectra of (a) 18+ CRP at 2700 eV and (c) 18+ CTB at 2200 eV and corresponding SID spectra of (b) 18+ CRP at 630 eV and (d) 18+ CTB at 605 eV. Reproduced from ref. . Copyright 2018 American Chemical Society.

Figure 16:

(a) Deconvoluted mass spectrum of DMPC nanodiscs. Waterfall plots showing (b) CID and (c) SID spectra of DMPC nanodiscs with increasing collision energy. Reproduced from ref. with permission from the Royal Society of Chemistry.

Figure 17:

SID cells for FT-ICR platforms. (a) Schematic of the solariX FT-ICR platform, (b) CAD renderings of three generations of hybrid SID-CID cells (which replace the red collision cell), (c) illustration of transmission vs. SID modes, (d) schematic of the ‘Gen 3’ split lens SID design in the front endcap of the Bruker collision cell, (e) mass spectrum of TNH charge-reduced with EDDA, (f) SID spectrum using acceleration voltage of 45 V on the Gen 1 SID-CID cell, (g) zoom-in of the 6+ charge state of the α subunit showing isotopic resolution, and (h) Gen 3 SID spectrum of the mass selected 16+ charge state of TNH, showing an increase in S/N compared to the Gen 1 configuration. Panels (a, adapted), (c), and (e-g) reproduced from ref. . Copyright 2017 American Chemical Society. Panels (b) and (d) reproduced from ref. . Copyright 2020 American Chemical Society.

Figure 18:

Surface-induced dissociation of 211 kDa multicopper oxidase Mnx on an ultrahigh resolution 15 T FT-ICR platform. (a) SID spectrum of Mnx 26+ through 29+ charge states (inset shows precursor ion population), (b) experimental isotopic distribution of peaks near m/z 3087 and comparison to theoretical isotope distributions for (c) MnxE + 2Cu + Fe and (d) MnxE + C₆H₁₀O, and (e) experimental isotope distributions observed near m/z 3813 and theoretical isotope distributions for (f) MnxF + Cu + C₆H₁₀O₆, (f) MnxF + Cu + C₈H₉NO₂S, and (f) MnxF + 4Cu. Adapted from ref. . Copyright 2018 American Chemical Society.

Figure 19:

FT-ICR offers unparalleled resolution for quantifying oligomer abundances from SID. (a) Mass spectrum of cholera toxin B charge-reduced with EDDA and (b) SID fragmentation pattern at collision energy 715 eV. The ultrahigh resolution is demonstrated for the overlap peak at m/z 5803 using (c) an 18s transient or (d) a 36 s transient. Reproduced from ref. . Copyright 2020 American Chemical Society.

Figure 20:

(a) Illustration of SID in an ELIT, (b) native mass spectrum of triose phosphate isomerase, (c) isolation of the 14+ charge state by mirror switching, and (d) SID spectrum of the 14+ charge state to produce symmetrically charged monomers. Panel (a) adapted from . Copyright 2014 American Chemical Society. Panels (b-d) adapted from ref. with written permission from the author.

Figure 21:

Illustration of SID-IM-SID. (a) SID spectrum of 19+ tryptophan synthase at a collision energy of 570 eV, (b) SID-IM at a higher energy of 1330 eV, (c) SID-IM-SID at 2280 eV (second stage) of 12+ αββ trimer, and (d) 1330 eV SID of the 8+ ββ dimer. The dissociation pathways are illustrated in the insets. Reproduced from ref. with permission from the Royal Society of Chemistry.

Figure 22:

Illustration of SID-IM-SID for native TNH heterohexamer. SID-IM-SID spectra of heterotrimer αβγ (a) 8+ and (b) 9+ produced from a first stage of SID of the heterohexamer (αβγ)₂ on a Synapt G2-S platform equipped with SID cells prior to and after the TWIM cell. Reproduced from ref. . Copyright 2015 American Chemical Society.

Figure 23:

Illustration of SID-Q-SID on an ultrahigh resolution 15 T FT-ICR. (a) native mass spectrum of HFQ65 homohexamer charge reduced with TEAA, (b) single stage SID spectrum of the entire charge state distribution in the entrance lens of the FT-ICR quadrupole, and SID-Q-SID of the isolated (c) dimer 3+, (d) trimer 4+, (e) tetramer 5+, and (f) pentamer 7+. The utility of isotopic resolution is evident when comparing the fragment ion peak at m/z 7186 for each subcomplex. Adapted from ref. with permission from Elsevier.

Figure 24:

Comparison of CIU and SIU plots for bovine serum albumin (BSA) 15+ and the N-terminal domain of anthrax lethal factor (LF_N¹⁰⁺). (A) CIU OF BSA15+, (b) SIU of BSA15+, (c) CIU of LF_N¹⁰⁺, and (d) SIU of LF_N¹⁰⁺. Adapted from ref. with permission from The Royal Society of Chemistry.

Figure 25:

(a) CID and (b) SID spectra of (Cyt c)₂¹¹⁺ Reproduced from ref. . Copyright 2006 American Chemical Society.

Figure 26:

SID spectra of protein complexes exhibit symmetric charge partitioning. SID spectra of (a) phosphorylase B dimer 29+ at 110 V, (b) phosphorylase B dimer 21+ at 150 V, (c) glutamate dehydrogenase hexamer 39+ at 130 V, and (d) glutamate dehydrogenase hexamer 27+ at 190 V. Adapted from ref. with kind permission from Springer Science & Business Media.

Figure 27:

Monomer orientations during asymmetric dissociation of cytochrome c dimer 10+ at a center-of-mass distance of (a) 6 nm, (b) 9 nm, and (c) 11 nm. The yellow monomer has 8 charges, and the green monomer has 2 charges. Reproduced from ref. . Copyright 2010 American Chemical Society.

Figure 28:

Comparison of CCSs of subcomplexes produced by (a,c) CID and (b,d) SID of pentamer C-reactive protein and tetramer concanavalin A. While monomer CCSs agree well with crystal structures, the CCSs of larger oligomers suggest collapse/restructuring in both CID and SID. Open symbols are product ions from normal-charge precursors (24+ CRP and 19+ ConA) while filled circles originate from charge-reduced precursors (18+ CRP and 13+ ConA). Reproduced from ref. with permission from The Royal Society of Chemistry.