Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components and properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP–lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, and fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteolipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP–lipid organization. Applying this method, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.
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We acknowledge the late R. Kaback for several critical insights on making lipid bilayers. This work was partly supported by the National Institutes of Health grants R01GM141192 to K.G., R01DK027044 to J.E.R. and S.S.K., and R01GM122759 and R21NS105863 to L.G. A.J.W. was funded by a Pembroke College (University of Oxford) Rokos Award. A.L.D. was funded by the Biochemistry Department, University of Oxford, and Pembroke College Oxford.
The authors declare no competing interests.
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Detail flow chart of two alternate approaches that can yield native-MS ready proteo-liposomes with desired lipid composition and membrane biophysical properties. The formed proteo-liposomes can be isolated from the free protein and the empty liposomes through a floatation assay. For better visualization, we typically add 1% RhodPE in our lipid mix. Post-floatation, the quality of the liposomes is assessed via negative stain imaging and DLS. An optional step can be introduced to control the diameter, and in turn the curvature, of the liposomes by passing them through an extruder fitted with filters of desired dimension. The size is further assessed by negative stain or DLS. Between the two protocols, while float-up method is typically longer, for certain protein-detergent scenario, dialysis can be more effective way to reconstitute. Figure graphics were created using Biorender.com.
a–e - Negative stain TEM images of different lipid vesicles which mimics of different eukaryotic organellar membranes. f–h - Negative stain TEM images of different curvature synaptic vesicles. The desired curvature was achieved by passing the synaptic vesicles like lipid vesicles through different pore size membrane. All the sample was taken from a pool of vesicles that were subjected to nativeMS (Fig. 1), confirming the vesicles in the samples. All measurements were replicated over three independent measurements.
In the spectra of a, Golgi like lipid membrane, b, mitochondria like membrane, c, ER like lipid membrane, d, PM like lipid membrane, e, and f, bacterial inner plasma membrane like lipid membrane as observed in the Fig. 1. The identity of the lipids could be determined by directly comparing the adduct masses against the theoretical masses of the individual lipids used to make the bilayer (Supplementary Table 2). All measurements were replicated over three independent measurements.
Detailed lipidomics profile of the extracted E.coli membrane used to reconstitute LacY and AqpZ (Fig. 1). For a, PE, b, PG, c, CL, and d, LPE, the detected individual lipid components were divided as per the total chain length distribution and quantified. The data presented as mean ± s.e. (N = 2).
a, nMS analysis of IMP (VAMP2) from vesicles with increasing amount of cholesterol. All measurements were replicated over three independent measurements, b, The diffusion coefficient (µm2/s) plotted against the mol% of cholesterol in the bilayer. As can be seen from the plot, with increase in mol% of cholesterol in the bilayer the diffusion coefficient decreases. That means the fluidity of the bilayer decreases with increase in cholesterol in the bilayer. Each data point represents the average diffusion coefficient from three independent experiment. The data presented as mean ± s.e. (N = 3).
Extended Data Fig. 6 Isolation spectra of 4+ charge state lipid bound VAMP2 from synaptic vesicles mimicking vesicles. and Lipid bound/Apo-protein ratio.
a, Isolation spectra of 4+ charge state lipid bound VAMP2 from synaptic vesicles mimicking vesicles. b, Lipid bound/Apo-protein ratio calculated from area under the curve of apo-protein peaks and lipid bound protein peaks (and then total lipid bound area taken to get ratio) in nMS spectra of VAMP2 from synaptic vesicles mimicking vesicles.
Spectra of VAMP2 from SV-like lipid vesicles where all the PC were replaced by a, SM and b, EPC (lipid compositions shown in the respective inset tables). The arrows showing the absence of theoretically predicted SM/EPC bound m/z values. In both the cases, under the same MS conditions, no detectable amount of SM or EPC binding was observed. All measurements were replicated over three independent measurements.
Extended Data Fig. 8 Spectra of detergent solubilized VAMP2 incubated with the same mixture of lipids present in SV shows no specificity of lipid binding.
Spectra of detergent solubilized VAMP2 incubated with the same mixture of lipids present in SV-like liposomes. Taking the example of +4 charge state, the data clearly shows that VAMP2 binds to several phospholipids without any specificity to any specific lipids. Further no detectable amount of cholesterol binding was observed. The data was obtained in MS conditions identical to that used for detecting VAMP 2 from SV like liposomes. All measurements were replicated over three independent measurements.
a, % Fusion vs the time course data for the VAPM2 in SV like and EPC-SV vesicles. In both the cases, CDV (cytosolic domain of VAMP2) was used a negative control. In presence of excess CDV, CDV binds and occupies the VAMP2 binding domains of the t-SNAREs. Consequently, upon addition of VAMP2 reconstituted vesicles, these vesicular VAMP2s cannot bind to t-SNARE anymore and no fusion is observed. As seen, the EPC-SV vesicles, devoid of PC, fuses with the PM at a significantly lower rate than the PC containing native-like SV liposomes. b, SDS-Gel picture showing similar band intensity of VAMP2 in SV like and EPC-SV vesicles. c, Vesicle fusion assay performed between t-SNARE in PM like lipid composition vesicles (tSNARE-PM) and VAMP2 in SV like vesicles (VAMP2-PC). Subsequently, to assess the effect of EPC on fusion rate, PC was replaced with EPC in PM like vesicles containing t-SNARE. Similar rate of fusion was observed as observed in t-SNARE in PM like vesicles with PC. This confirms that the reduced fusion rate observed for SV-EPC vesicles (Fig. 2), is not because of replacing PC with EPC in the bilayer, but due to abrogation of VAMP2-PC binding. The data presented as mean ± s.e (N = 5).
Extended Data Fig. 10 NativeMS spectra of semisweet from a. detergent, b. bilayer with PE, PG without CL, c. bilayer with PE, PG, and 2.5% CL.
The individual peaks were plotted in the Origin software and then the area under the curve was obtained by fitting the peak with constant baseline mode. The 6+ monomer charge state and 9+ and 11+ dimer charge states were considered for oligomer % calculation. The peak areas of protein and lipid-bound protein from Origin software were then taken for percentage calculations and the final bar graph of this calculation is shown as an inset in a. The data clearly shows increase in CL% increases the dimeric population in the bilayer. The inset on the left side in each panel shows the mass plot obtained from UniDec analysis.
Mass spectrometry data in .xlsx format
Mass spectrometry data in .xlsx format and numerical data of bulk fusion assay in .xlsx format
Unprocessed TEM images of vesicles in .jpg and.tif format
Mass spectrometry data in .xlsx format
Lipidomics data in .xlsx format
Mass spectrometry data in .xlsx format and FRAP experimental data in .xlsx format
Mass spectrometry data in .xlsx format and lipid-bound and apoprotein calculation data in .xlsx format
Mass spectrometry data in .xlsx format
Mass spectrometry data in .xlsx format
Numerical data of bulk fusion assay in .xlsx format and unprocessed, uncropped gel image in .tiff format
Mass spectrometry data in .xlsx format
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Panda, A., Giska, F., Duncan, A.L. et al. Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer. Nat Methods 20, 891–897 (2023). https://doi.org/10.1038/s41592-023-01864-5