Structural Mass Spectrometry of Protein Assemblies

Native MS and Ion Mobility-MS

In theory:

Shortly after the introduction of electrospray ionization (ESI) that revolutionized the MS analysis of biomolecules at the end of the 80s, several groups observed the presence of non-covalent complexes in the gas phase. First limited to small complexes, instrumental developments progressively enabled the analysis of increasingly larger complexes such as GroEL, the proteasome or even mega-Da assemblies such as ribosomes or viral capsids. Within the past 10 years, non-covalent -or native MS- has emerged and is now an integrative part of structural biology, providing a series of information on the structure and dynamics of protein complexes (Figure 1).

The toolbox of structural mass spectrometrists has also been further completed by ion mobility MS (IM-MS). This method enables the separation of ions in the gas phase, according to their size and conformation(s). Ionized proteins are introduced in a drift tube submitted to an electric field filled with an inert gas with which they collide. The larger the surface of the protein, the higher the number of collisions and the longer it will take to go through the ion mobility tube. By measuring the time taken to cross the tube (drift time), and after calibrating with proteins of known structure, averaged collisional cross section of the protein (CCS, in Å2) can be obtained. By acting like a “gas phase capillary electrophoresis”, ion mobility brings an additional dimension of separation, virtually increasing the peak capacity of the instrument allowing not only spectrum cleaning but also the detection of subtle conformational changes or differences in the unfolding pattern upon ligand binding. Moreover, CCSs constitute structural constraints that can be very useful for molecular modeling purposes.

Information obtained by native MS and ion mobility
Figure 1: Information obtained by native MS and ion mobility.

In practice:

Protein complexes (~ 10 µl at 10 µM i.e 100 pmol / 2.5 µg for a 25 kDa protein) are desalted (Biospin, Zeba, Vivaspin, dialysis) and directly infused on our SynaptG2Si (Waters) coupled to the Nanomate (Advion) (Figure 2, Left) or Q-Ex-HFX-Biopharma (Figure 2, Middle).

Figure 2. Our nanomate autosamplers (Advion) coupled to the SynaptG2Si (Waters, left) and Q-Ex-HFX-Biopharma (Thermo).The H-Class-Bio system can be used for SEC-Native MS.

Alternatively, protein complexes can be separated online by Size Exclusion Chromatography (BEH on a H-Class-Bio Acquity UPLC, Waters, Figure 2, Right) and the oligomers directly analysed by Native (Ion-mobility) MS.



Structural insights into chaperone addiction of toxin-antitoxin systems.
Guillet V, Bordes P, Bon C, Marcoux J, Gervais V, Sala AJ, Dos Reis S, Slama N, Mares-Mejía I, Cirinesi AM, Maveyraud L, Genevaux P, Mourey L.
Nat Commun. 2019 Mar 7;10(1):1187.

Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding.
Trcka F, Durech M, Vankova P, Chmelik J, Martinkova V, Hausner J, Kadek A, Marcoux J, Klumpler T, Vojtesek B, Muller P, Man P.
Mol Cell Proteomics. 2019 Feb;18(2):320-337.


Mangione PP, Verona G, Corazza A, Marcoux J, Canetti D, Giorgetti S, Raimondi S, Stoppini S, Esposito M, Relini A, Canale C, Valli M, Marchese L, Faravelli G, Obici L, Hawkins PN, Taylor GW, Gillmore JD, Pepys MB, Bellotti V. “Plasminogen activation triggers transthyretin amyloidogenesis” Journal of Biological Chemistry 293(37):14192-14199.


Ambrose S, Housden NG, Gupta K, Fan J, White P, Yen HS, Marcoux J, Kleanthous C, Hopper JTS, Robinson CV “Native desorption electrospray ionization liberates soluble and membrane protein complexes from surfaces” Andgewandte Chemie 56(46):14463-14468.

Raimondi S, Porcari R, Mangione PP, Verona G, Marcoux J, Giorgetti S, Ballico M, Zanini S, Taylor GW, Ellmerich S, Domanska K, Al-Shawi R, Simons JP, Corazza A, Fogolari F, Gillmore JD, Hawkins PN, Valli M, Stoppini M, Steyaert J, Robinson CV, Pepys MB, Esposito G, Bellotti V (2017) “A specific nanobody prevents amyloidogenesis of Asp76Asn β2-microglobulin in vitro and modify its tissue distribution in vivo” Scientific Reports 7: 46711.

Bourgeois G, Marcoux J, Saliou JM, Cianférani S, Graille M. “Activation mode of the eukaryotic m2G10 tRNA methyltransferase Trm11 by its partner protein Trm112” Nucleic Acids Research 45(4): 1971-1982.

Kadek A, Kavan D, Marcoux J, Stojko J, Felice AKG, Cianférani S, Ludwige R, Halada P, Man P (2017) “Interdomain electron transfer in cellobiose dehydrogenase is driven by surface electrostatics” BBA General Subjects 1861(2) : 157-167.


McDowell MA, Marcoux J, McVicker G, Johnson S, Fong YH, Stevens R, Degiacomi MT, Yan J, Wise A, Friede M, Benesch JLP, Deane JE, Tang CM, Robinson CV, Lea SM. “Characterisation of Shigella Spa33 and Thermotoga FliM/N reveals a new model for C-ring assembly in T3SS” Molecular Microbiology 99(4) : 749-766.

Mehmood S, Marcoux J, Gault J, Quigley A, Michaelis S, Young SG, Carpenter EP, Robinson CV. “Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24” Nature Chemistry 8(12) : 1152-1158


Marcoux J, Champion T, Colas O, Wagner-Rousset E, Corvaïa N, Van Dorsselaer A, Beck A, Sanglier-Cianférani S. “Native mass spectometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate” Protein Science 24(8) : 1210-1223.

Simonneau C, Leclercq B, Mougel A, Adriaenssens E, Paquet C, Raibaut, Ollivier N, Drobecq H, Marcoux J, Cianférani S, Tulasne D, De Jonge H, Melnyk O, Vicogne J. “Semi-synthesis of a HGF/SF kringle one (K1) domain scaffold generates a potent in vivo MET receptor agonist” Chemical Science 6(3) : 2110-2121.

 Marcoux J, Mangione PP, Porcari R, Degiacomi M, Verona G, Taylor GW, Giorgetti S, Raimondi S, Sanglier-Cianférani S, Benesch JL, Cecconi C, Naqvi MM, Gillmore JD, Hawkins PN, Stoppini M, Robinson CV, Pepys MB, Bellotti V. “A novel mechano-enzymatic cleavage mechanism underlies transthyretin amyloidogenesis” EMBO Molecular Medicine 7(10) : 1337-1349


Mangione PP, Porcari R, Gillmore JD, Pucci P, Monti M, Porcari M, Giorgetti S, Marchese L, Raimondi S, Serpell LC, Chen W, Relini A, Marcoux J, Clatworthy IR, Taylor GW, Tennent GA, Robinson CV, Hawkins PN, Stoppini M, Wood SP, Pepys MB, Bellotti V. “Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis” Proceedings of the National Academy of Sciences 111(4) : 1539-1544.

 Marcoux J, Politis A, Marshall D, Rinehart D, Wallace MI, Tamm LK, Robinson CV. “A new model for full length OmpA: native mass spectrometry reveals partial dimerization via C-ter domain” Structure 22(5) : 781-790.

Mehmood S, Marcoux J, Hopper JTS, Alison TM, Liko I, Borysik AJ and Robinson CV. “Charge reduction stabilizes intact membrane protein complexes for mass spectrometry” Journal of the American Chemical Society 136(49) : 17010-17012.


Rozbeský D, Sovová Z, Marcoux J, Man P, Ettrich R, Robinson CV, Novák P. “Structural model of lymphocyte receptor NKR-P1C revealed by mass spectrometry and molecular modelling” Analytical Chemistry 85(3) : 1597-604.

Lee SC, Bennett BC, Hong WX, Fu Y, Baker KA, Marcoux J, Robinson CV, Ward AB, Halpert JR,Stevens RC, Stout CD, Yeager M, Zhang Q. “Steroid-based Facial Amphiphiles for Stabilization and Crystallization of Membrane Proteins” Proceedings of the National Academy of Sciences 110(13) : E1203-1211.

 Marcoux J, Wang S, Politis A, Reading E, Ma J, Biggins P, Zhou M, Tao H, Zhang Q, Chang G, Morgner N, Robinson CV. “Mass spectrometry reveals synergistic binding of nucleotides, lipids and drugs to a multidrug resistance efflux pump” Proceedings of the National Academy of Sciences 110(24) : 9704-9709.


Deroo S, Hyung SJ, Marcoux J, Gordiyenko Y, Koripella RK, Sanyal S, Robinson CV. “Mechanism and rates of exchange of L7/L12 between ribosomes and the effects of binding EF-G” ACS Chemical Biology 7 (6) : 1120-1127.

Rollauer SE, Tarry MJ, Graham JE, Jääskeläinen M, Jäger F, Johnson S, Krehenbrink M, Liu SM, Lukey MJ, Marcoux J, McDowell MA, Roversi P, Stansfeld PJ, Robinson CV, Sansom MSP, Palmer T, Högbom M, Berks BC, Lea SM. “Structure of the twin arginine protein transport system” Nature 492 (7428) : 210-4.


Jaquet V, Marcoux J, Forest E, Leidal KG, McCormick S, Westermaier Y, Perozzo R, Plastre O, Fioraso-Cartier L, Diebold B, Scapozza L, Nauseef WM, Fieschi F, Krause KH and Bedard K. “NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action” British Journal of Pharmacology. 164 : 507–520.