Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry

Abstract

The study of membrane protein structure and enzymology has traditionally been hampered by the inherent insolubility of membrane proteins in aqueous environments and experimental challenges in emulating an in vivo lipid environment. Phospholipid bilayer nanodiscs have recently been shown to be of great use for the study of membrane proteins since they offer a controllable, stable, and monodisperse model membrane with a nativelike lipid bilayer. Here we report the integration of nanodiscs with hydrogen exchange (HX) mass spectrometry (MS) experiments, thereby allowing for analysis of the native conformation of membrane proteins. gamma-Glutamyl carboxylase (GGCX), an approximately 94 kDa transmembrane protein, was inserted into nanodiscs and labeled with deuterium oxide under native conditions. Analytical parameters including sample-handling and chromatographic separation were optimized to measure the incorporation of deuterium into GGCX. Coupling nanodisc technology with HX MS offers an effective approach for investigating the conformation and dynamics of membrane proteins in their native environment and is therefore capable of providing much needed insight into the function of membrane proteins.

Figure 1

The integrated nanodisc-HX MS workflow. Loaded nanodiscs were assembled from a mixture of membrane scaffold protein (MSP), lipids, and the target membrane protein solubilized with detergent. Nanodiscs self-assembles as detergent was removed. Loaded nanodiscs were purified with SEC (see Supporting Information, Figure S1a) and exposed to deuterated buffer for various times before quenching the exchange reaction. Cholate was immediately added to the quenched reaction to begin disassembly of the nanodiscs. Protein was digested with pepsin for 5 minutes on ice. In the last minute of digestion, ZrO₂ resin was added to the digestion mixture to selectively remove phospholipid. Filtration removed immobilized-pepsin beads and the ZrO₂ resin. UPLC/ESIMS were used to measure the incorporation of deuterium.

Figure 2

Chromatographic traces of nanodisc peptic digests. (A) HPLC separation of 44 pmol of empty nanodisc (88 pmol MSP) with (red) and without (blue) addition of 400 μM sodium cholate. (B) UPLC separation of 50 pmol loaded nanodiscs (50 pmol GGCX, 100 pmol MSP) with (black) and without (green) addition of 480 μM sodium cholate. The more simple mixture of just empty nanodiscs was not analyzed with UPLC. Rather we went immediately to the much more complex loaded nanodiscs sample (MSP + GGCX mixture), which was the actual mixture that must be analysed for the final hydrogen exchange experiments. UPLC separation of loaded nanodiscs under HX MS analysis conditions included separation of peptic peptides from both MSP (24 kDa) and GGCX (94 kDa); a combined total mass of 118 kDa of unique sequence was digested. The average number of peptides identified in UPLC experiments with and without cholate is shown in the inset. HPLC and UPLC are shown on the same time scale to illustrate that reasonable HPLC performance required a longer gradient; even so, UPLC performance was superior for a more complex sample (loaded nanodisc) in a shorter time. In both chromatography systems, cholate was retained longer than the peptides and did not interfere with peptide ionization and identification (most runs, the UPLC was disconnected from the MS before the bulk of cholate eluted; cholate is shown in this trace for example purposes only.

Figure 3

Representative data for a nanodisc-HX MS experiment. (A) UPLC traces for empty (E) and loaded (L) nanodisc peptic digests at the deuterium exchange times indicated (UN – undeuterated). There were few differences in the empty and loaded traces as the most intense ions were always derived from the MSP (2:1 MSP:GGCX molar ratio). The bar at ~5.25 min corresponds to the scans combined to generate panel B. (B) Spectra for the m/z range 500–550. GGCX peptides (red) were not present in the empty nanodisc spectrum (top) and MSP peptides (black) are seen in both empty and loaded samples. Due to the high quality UPLC separations, very few peptides overlapped with one another, as shown here and found in most other spectra (data not shown). The bar corresponds to the zoomed region shown in panel C. (C) Magnification of the m/z range 532–545 showing typical mass spectral quality. (D) Deuterium uptake plots for the peptides shown in panel C. In these plots, the error of determining the deuterium level did not exceed ±0.3 daltons (see Supporting Information).

Figure 4

Sequence coverage plotted on the predicted topology map of gamma-glutamyl carboxylase. The protocol described in Figure 1 resulted in 71 unique, mostly non-overlapping GGCX peptic peptides that were identified and followed in duplicate nanodisc HX MS experiments. Residues within these peptides are shown in red and those not covered by these peptides are shown in gray. The five N-linked glycosylation sites are colored in blue. Recovery of peptic peptides containing post-translationally modified sites was not performed in the current HX-MS experiments..