Fluorinated Colloidal Gold Immunolabels for Imaging Select Proteins in Parallel with Lipids Using High-Resolution Secondary Ion Mass Spectrometry

Abstract

The local abundance of specific lipid species near a membrane protein is hypothesized to influence the protein’s activity. The ability to simultaneously image the distributions of specific protein and lipid species in the cell membrane would facilitate testing these hypotheses. Recent advances in imaging the distribution of cell membrane lipids with mass spectrometry have created the desire for membrane protein probes that can be simultaneously imaged with isotope labeled lipids. Such probes would enable conclusive tests of whether specific proteins co-localize with particular lipid species. Here, we describe the development of fluorine-functionalized colloidal gold immunolabels that facilitate the detection and imaging of specific proteins in parallel with lipids in the plasma membrane using high-resolution SIMS performed with a NanoSIMS. First, we developed a method to functionalize colloidal gold nanoparticles with a partially fluorinated mixed monolayer that permitted NanoSIMS detection and rendered the functionalized nanoparticles dispersible in aqueous buffer. Then, to allow for selective protein labeling, we attached the fluorinated colloidal gold nanoparticles to the nonbinding portion of antibodies. By combining these functionalized immunolabels with metabolic incorporation of stable isotopes, we demonstrate that influenza hemagglutinin and cellular lipids can be imaged in parallel using NanoSIMS. These labels enable a general approach to simultaneously imaging specific proteins and lipids with high sensitivity and lateral resolution, which may be used to evaluate predictions of protein co-localization with specific lipid species.

Hemagglutinin Clusters in the Plasma Membrane Are Not Enriched with Cholesterol and Sphingolipids

“…The 18 O-cholesterol was synthesized from i-cholesteryl methyl ether that was purchased from Sigma and 18 O-water that was purchased from Olinax (Hamilton, Ontario, Canada), as previously reported (49). The hemagglutinin was immunolabeled using a primary antibody (FC 125 antihemagglutinin) developed in mouse and fluorinated colloidal gold (average diameter ¼ 20 nm) immunolabels that were synthesized as previously reported (50).…”

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

“…This possibility was assessed by studying the stably expressed influenza hemagglutinin clusters in the membranes of uninfected Clone 15 cells instead of those in the membranes of influenza-infected cells to ensure that other viral proteins did not affect hemagglutinin localization within the plasma membrane. To permit visualization, the hemagglutinin on the metabolically labeled Clone 15 cells was labeled with a mouse anti-hemagglutinin antibody followed by an anti-mouse secondary antibody conjugated to a fluorinated colloidal gold particle (Wilson et al, 2012). High-resolution SIMS imaging of the 19 F − ions distinctive to the immunolabeled hemagglutinin in parallel with the 15 N-sphingolipids and 18 O-cholesterol revealed the fluorine-rich patches that located the hemagglutinin clusters were neither enriched with cholesterol nor well colocalized with 15 N-sphingolipid domains (Figures 3A–C; Wilson et al, 2015).…”

Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol

“…A high-resolution secondary ion mass spectrometry (SIMS) 2 technique that does not alter biomolecule distribution in membranes or cellular compartments (6–11) has enabled visualizing the organizations of rare isotope-labeled lipids in the plasma membrane by mapping their distinctive isotope enrichments (10–12). In a recent report, we used high-resolution SIMS, which was performed on a Cameca NanoSIMS 50, to image the distributions of metabolically incorporated 15 N-sphingolipids in the plasma membranes of intact cells.…”