Delivering Transmembrane Peptide Complexes to the Gas Phase Using Nanodiscs and Electrospray Ionization

Abstract

The gas-phase conformations of dimers of the channel-forming membrane peptide gramicidin A (GA), produced from isobutanol or aqueous solutions of GA-containing nanodiscs (NDs), are investigated using electrospray ionization-ion mobility separation-mass spectrometry (ESI-IMS-MS) and molecular dynamics (MD) simulations. The IMS arrival times measured for (2GA + 2Na) ions from isobutanol reveal three different conformations, with collision cross-sections (Ω) of 683 Å (conformation 1, C1), 708 Å (C2), and 737 Å (C3). The addition of NHCHCO produced (2GA + 2Na) and (2GA + H + Na) ions, with Ω similar to those of C1, C2, and C3, as well as (2GA + 2H), (2GA + 2NH), and (2GA + H + NH) ions, which adopt a single conformation with a Ω similar to that of C2. These results suggest that the nature of the charging agents, imparted by the ESI process, can influence dimer conformation in the gas phase. Notably, the POPC NDs produced exclusively (2GA + 2NH) dimer ions; the DMPC NDs produced both (2GA + 2H) and (2GA + 2NH) dimer ions. While the Ω of (2GA + 2H) is similar to that of C2, the (2GA + 2NH) ions from NDs adopt a more compact structure, with a Ω of 656 Å. It is proposed that this compact structure corresponds to the ion conducting single stranded head-to-head helical GA dimer. These findings highlight the potential of NDs, combined with ESI, for transferring transmembrane peptide complexes directly from lipid bilayers to the gas phase. Graphical Abstract ᅟ.

Engineering Nanodisc Scaffold Proteins for Native Mass Spectrometry

“…Nanodiscs are a promising platform for native MS of membrane proteins due to their size, homogeneity, and relative monodispersity. − To complement conventional detergent-based methods, nanodiscs allow membrane proteins to be delivered for native MS in a lipid bilayer environment, which may be a better model of the physiological environment of membrane proteins. Nanodiscs are nanoscale lipoprotein particles consisting of a lipid bilayer surrounded by two membrane scaffold protein (MSP) belts.…”

“…Nanodiscs are nanoscale lipoprotein particles consisting of a lipid bilayer surrounded by two membrane scaffold protein (MSP) belts. There are a number of different MSP constructs available, but they are all derived from ApoAI, the primary component of high-density lipoprotein particles. − Klassen and co-workers have employed nanodiscs with catch-and-release ESI to investigate glycolipid interactions with soluble proteins. ,,,,,− , We showed that nanodisc complexes can be preserved intact for native MS, , and Robinson and co-workers later showed that nanodiscs can be used to deliver membrane protein complexes into the gas phase. , …”

“…20−24 Klassen and co-workers have employed nanodiscs with catch-and-release ESI to investigate glycolipid interactions with soluble proteins. 1,3,5,10,11,[13][14][15]17 We showed that nanodisc complexes can be preserved intact for native MS, 12,18 and Robinson and co-workers later showed that nanodiscs can be used to deliver membrane protein complexes into the gas phase. 4,16 Despite their utility, native MS analysis of nanodiscs is limited by potential challenges with isobaric species that can complicate data analysis and interpretation.…”

Scratching the surface: native mass spectrometry of peripheral membrane protein complexes

“…Marty et al [35] showed further that large numbers of lipids bound to the ejected proteins could still be detected, providing vital information about interactions within the lipid environment. In these and other studies, integral membrane proteins were co-assembled into nanodiscs and then released in the mass spectrometer, a 'build-and-release' approach [50,[55][56][57].…”

Expanding the Types of Lipids Amenable to Native Mass Spectrometry of Lipoprotein Complexes

“…Due to their relatively low polydispersity and high homogeneity, lipoprotein nanodiscs have emerged as a promising membrane mimetic for native MS. Nanodiscs have been used to deliver integral membrane proteins [8,11,12], peripheral membrane proteins [13,14], and transmembrane peptides [15] for native MS and to characterize large numbers of lipids bound to membrane protein complexes [16]. Recent work found that charge manipulation reagents enable a wide range of dissociation pathways, allowing the membrane protein to be either ejected from the nanodisc or characterized within a nearly intact nanodisc complex [17].…”