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  • Secondary Ion Mass Spectrometry of Single Giant Unilamellar Vesicles Reveals Compositional Variability. Journal of the American Chemical Society Grusky, D. S., Bhattacharya, A., Boxer, S. G. 2023

    Abstract

    Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. It is generally assumed that the composition of individual GUVs is the same as the nominal stock composition; however, there may be significant compositional variability between individual GUVs. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified. To assess heterogeneity, we use secondary ion mass spectrometry (SIMS) to probe the composition of individual GUVs using non-perturbing isotopic labels. Both 13C- and 2H-labeled lipids are incorporated into a ternary mixture, which is then used to produce GUVs via gentle hydration or electroformation. Simultaneous detection of seven different ion species via SIMS allows for the concentration of 13C- and 2H-labeled lipids in single GUVs to be quantified using calibration curves, which correlate ion intensity to composition. Additionally, the relative concentration of 13C- and 2H-labeled lipids is assessed for each GUV via the ion ratio 2H-/13C-, which is highly sensitive to compositional differences between individual GUVs and circumvents the need for calibration by using standards. Both quantification methods suggest that gentle hydration produces GUVs with greater compositional variability than those formed by electroformation. However, both gentle hydration and electroformation display standard deviations in composition (n = 30 GUVs) on the order of 1-4 mol %, consistent with variability seen in previous indirect measurements.

    View details for DOI 10.1021/jacs.3c09039

    View details for PubMedID 38056605

  • Examining compositional variability of giant unilamellar vesicles via secondary ion mass spectrometry Grusky, D. S., Bhattacharya, A., Boxer, S. G. CELL PRESS. 2023: 81A

    View details for Web of Science ID 000989629700405

  • Examining compositional variability of giant unilamellar vesicles via secondary ion mass spectrometry. Biophysical journal Grusky, D. S., Bhattacharya, A., Boxer, S. G. 2023; 122 (3S1): 81a

    View details for DOI 10.1016/j.bpj.2022.11.642

    View details for PubMedID 36785033

  • Recombination between 13C and 2H to Form Acetylide (13C22H-) Probes' Nanoscale Interactions in Lipid Bilayers via Dynamic Secondary Ion Mass Spectrometry: Cholesterol and GM1 Clustering. Analytical chemistry Grusky, D. S., Moss, F. R., Boxer, S. G. 2022

    Abstract

    Although it is thought that there is lateral heterogeneity of lipid and protein components within biological membranes, probing this heterogeneity has proven challenging. The difficulty in such experiments is due to both the small length scale over which such heterogeneity can occur, and the significant perturbation resulting from fluorescent or spin labeling on the delicate interactions within bilayers. Atomic recombination during dynamic nanoscale secondary ion imaging mass spectrometry (NanoSIMS) is a non-perturbative method for examining nanoscale bilayer interactions. Atomic recombination is a variation on conventional NanoSIMS imaging, whereby an isotope on one molecule combines with a different isotope on another molecule during the ionization process, forming an isotopically enriched polyatomic ion in a distance-dependent manner. We show that the recombinant ion, 13C22H-, is formed in high yield from 13C- and 2H-labeled lipids. The low natural abundance of triply labeled acetylide also makes it an ideal ion to probe GM1 clusters in model membranes and the effects of cholesterol on lipid-lipid interactions. We find evidence supporting the cholesterol condensation effect as well as the presence of nanoscale GM1 clusters in model membranes.

    View details for DOI 10.1021/acs.analchem.2c01336

    View details for PubMedID 35759338

  • Recombination between C-13 and H-2 to form acetylide ((C2H-)-C-13-H-2) probes nanoscale interactions in lipid bilayers: cholesterol and GM1 clustering Grusky, D. S. CELL PRESS. 2022: 486A

    View details for Web of Science ID 000759523003180