Extended Data Fig. 3: Native MS of soluble and membrane protein assemblies performed using the Orbitrap Eclipse tribrid platform.

Soluble protein assemblies a, (17–386 kDa), and membrane protein assemblies b, (22–128 kDa) are ordered with increasing molecular mass and MS conditions were optimised to maximise ion intensity and retain the intact assembly. Charge state (CS) distributions are all approximately Gaussian (Note that DHODH has a slight LDAO effect29 and semiSWEET is mixture of monomer and dimer) and maximum CSs are as expected from native folded protein ions. The glycan profile of antibody Trastuzumab (Herceptin) is shown in the inset. In the cases of myoglobin and DHODH there is complete retention of the non-covalently bound co-factors haem and flavin mononucleotide (FMN) respectively. Most membrane proteins harbour residual detergent adducts - adduct peaks in the spectra of semiSWEET and AmtB, for example. All membrane proteins were electrosprayed from buffers containing ‘charge reducing’^29,34 detergents. This, together with the reduction in the number of ionisable residues and reduced exposed surface area for charging, means that CSs are shifted to lower m/z values compared to soluble protein assemblies of similar mass. (c) A plot of mass against expected m/z of the average charge state (Zave) based on previously observed empirical relationships29 indicates good agreement between the size of complexes predicted, and those achieved experimentally, up to the m/z 8,000 limit of the instrument. Arbitrary bounds of ±7% are included to represent the approximate width of the CS envelope. Curves are Zave = a MW^b where a = 0.0467 and b = 0.533 for soluble proteins and a = 0.0036, b = 0.71 for membrane proteins29. HSP16.5 oligomers usually appear at > m/z 8,000 but have been supercharged with meta nitro benzyl alcohol⁴⁷ to shift the CS envelope into a range below m/z 8,000.