Secondary Ion Mass Spectrometry of Single Giant Unilamellar Vesicles Reveals Compositional Variability

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.

Figure 1.

Experimental design. Micron-sized GUVs formed by gentle hydration or electroformation are deposited over patterned Si/SiO₂ substrates. GUVs spontaneously rupture to form individual GUV-derived planar supported bilayer patches that are subsequently freeze-dried and analyzed via NanoSIMS. (A) Epifluorescence images of POPC GUVs containing 0.1% TR-DHPE. (B) GUVs are deposited over an oxidized silicon substrate with a chrome grid and are allowed to rupture. The patterning provides a visual guide for locating patches during NanoSIMS imaging. (C) Epifluorescence images of POPC GUV patches containing 0.1% TR-DHPE. Note that if small vesicles are present within the GUV these are lost or possibly deposited elsewhere upon bilayer patch formation.

Figure 2.

Isotopically labeled lipids used in this study. Color-coded circles represent the locations of isotopic labels.

Figure 3.

Concentration correlations of different labeling schemes. Correlations between the ²H⁻/(¹³C⁻ + ¹²C⁻) and ¹³C⁻/(¹³C⁻ + ¹²C⁻) ratios for each lipid mixture. The ²H⁻/(¹³C⁻ + ¹²C⁻) ratio tracks the amount of ²H-labeled lipid in the bilayer, while the ¹³C⁻/(¹³C⁻ + ¹²C⁻) ratio tracks the amount of ¹³C-labeled lipid in the bilayer. (A) Correlation between ²H_31-POPC and ¹³C₁₈-DSPC ratios in the ternary mixture DSPC:¹³C₁₈-DSPC:POPC:²H₃₁-POPC:CHOL 20:20:20:20:20. (B) Correlation between ²H₃₁-POPC and ¹³C₁₈-POPC ratios in the ternary mixture DSPC:¹³C₁₈-POPC:²H₃₁-POPC:CHOL 40:20:20:20. (C) Absence of correlation between ²H₃₁-POPC and ¹³C₁₈-POPC ratios in a pure POPC mixture with composition ¹³C₁₈-POPC:²H₃₁-POPC:POPC 20:20:60.

Figure 4.

Measured ²H⁻/¹³C⁻ ratios for different methods. GUV patches formed by gentle hydration and electroformation were compared to continuous bilayers formed from SUVs via their ²H⁻/¹³C⁻ ratios. All samples were produced from a master stock with nominal composition DSPC:¹³C₁₈-DSPC:POPC:²H₃₁-POPC:CHOL 20:20:20:20:20. (A) Displays the ²H⁻/¹³C⁻ ratio distributions for each preparation method. Significance was determined via an F-test. For this and subsequent plots, each point represents a measurement made on a single GUV patch or corral containing an SLB. Thirty bilayers were examined for each sample. For this and all subsequent plots, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. The displayed curves are normal distributions calculated by using the standard deviation and average from the GUV patch measurements. Note that these have been overlaid to guide the reader and that the area under the curve has not been normalized. A dot plot representation of the distributions can be seen in Figure S21. (B) Calculated standard deviations of the ²H⁻/¹³C⁻ ratio for each preparation method.

Figure 5.

Concentration quantification of GUV patches and monolayers. (A) Representative calibration curves for ¹³C₁₈-DSPC and ²H₃₁-POPC. (B) Calculated ¹³C₁₈-DSPC concentration distributions for GUV patches formed by gentle hydration and electroformation. All GUVs were formed using a ternary mixture with nominal composition DSPC:¹³C₁₈-DSPC:POPC:²H₃₁-POPC:CHOL 20:20:20:20:20. The gentle hydration ¹³C₁₈-DSPC concentration distribution is significantly different relative to the corresponding electroformation distribution, as determined by F-test. Red dashed lines indicate the nominal concentration of labeled lipids. (C) Displays the calculated ²H₃₁-POPC concentration distributions for GUV patches formed by gentle hydration and electroformation. These concentration distributions are compared to ²H₃₁-POPC concentrations measured in a monolayer composed of POPC with 20 mol % of ²H₃₁-POPC. Dot plot representations of these distributions can be seen in Figure S22. (D) Calculated standard deviations for each concentration distribution.

Figure 6.

Pure and ternary mixture concentration variability. ²H₃₁-POPC concentrations of individual GUV patches composed with pure POPC (¹³C₁₈-POPC:²H₃₁-POPC:POPC 20:20:60) or ternary (¹³C₁₈-DSPC:POPC:²H₃₁-POPC:CHOL 20:20:20:20:20) compositions were compared. Both pure POPC and ternary GUVs were formed by either electroformation (A) or gentle hydration (B). For both methods, the patches composed of pure POPC display significantly less ²H₃₁-POPC concentration variability than that of ternary patches formed using the same method. Dot plot representations of these distributions can be seen in Figure S23. (C) Displays the calculated standard deviations for each sample.

Figure 7.

Cholesterol concentration differences between electroformation and gentle hydration. GUVs were formed via electroformation or gentle hydration from a master stock with nominal composition DSPC:POPC:²H₃₁-POPC:¹³C₂₇-CHOL 40:20:20:20. (A) NanoSIMS image of a GUV patch formed via electroformation, which shows significant localization of ¹³C₂₇-CHOL to the edge of the GUV patch. (B) ¹³C₂₇-CHOL calibration curve. (C) Comparison of ¹³C₂₇-CHOL concentration in GUV patches formed via gentle hydration or electroformation. Error bars represent 95% confidence intervals. Comparisons were conducted either excluding the edge of the bilayer patch or including the edge of the bilayer patch. Regardless of the analysis method, electroformed GUVs contained less cholesterol on average. Replicate sample quantification along with internal controls (²H₃₁-POPC concentration quantification) can be found in Figures S19 and S20 of the Supporting Information.