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Native mass spectrometry (MS) of proteins and protein assemblies reveals size and binding stoichiometry, but elucidating structures to understand their function is more challenging. Native top-down MS (nTDMS), i.e., fragmentation of the gas-phase protein, is conventionally used to derive sequence information, locate post-translational modifications (PTMs), and pinpoint ligand binding sites. nTDMS also endeavors to dissociate covalent bonds in a conformation-sensitive manner, such that information about higher-order structure can be inferred from the fragmentation pattern. However, the activation/dissociation method used can greatly affect the resulting information on protein higher-order structure. Methods such as electron capture/transfer dissociation (ECD and ETD, or ExD) and ultraviolet photodissociation (UVPD) can produce product ions that are sensitive to structural features of protein complexes. For multi-subunit complexes, a long-held belief is that collisionally activated dissociation (CAD) induces unfolding and release of a subunit, and thus is not useful for higher-order structure characterization. Here we show not only that sequence information can be obtained directly from CAD of native protein complexes but that the fragmentation pattern can deliver higher-order structural information about their gas- and solution-phase structures. Moreover, CAD-generated internal fragments (i.e., fragments containing neither N-/C-termini) reveal structural aspects of protein complexes.

Native mass spectrometry (MS) was used to detect the membrane protein, bacteriorhodopsin (bR), in its 27 kDa monomeric form and trimeric assemblies directly from lipid-containing purple membranes (PMs) from the halophilic archaeon, Halobacteriumsalinarum. Trimer bR ion populations bound to lipid molecules were detected with n-octyl β-d-glucopyranoside as the solubilizing detergent; the use of octyl tetraethylene glycol monooctyl ether or n-dodecyl-β-d-maltopyranoside resulted in only detection of monomeric bR. The archaeal lipids phosphotidylglycerolphosphate methyl ester and 3-HSO3–Galp-β1,6-Manp-α1,2-Glcp-α1,1-sn-2,3-diphytanylglycerol were the only lipids in the PMs found to bind to bR, consistent with previous high-resolution structural studies. Removal of the lipids from the sample resulted in the detection of only the bR monomer, highlighting the importance of specific lipids for stabilizing the bR trimer. To the best of our knowledge, this is the first report of the detection of the bR trimer with resolved lipid-bound species by MS. [ABSTRACT FROM AUTHOR]