Quantitative analysis of supported membrane composition using the NanoSIMS

Kraft, Mary L.; Fishel, S F; Marxer, Carine Galli; Weber, Peter K.; Hutcheon, I. D.; Boxer, Steven G.

doi:10.1016/j.apsusc.2006.02.116

Cited by 34 publications

(33 citation statements)

References 35 publications

(47 reference statements)

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“…21, 23, 28–30 Because this instrument detects monoatomic and diatomic secondary ions, each species of interest must contain a distinct stable isotope so that the secondary ions generated during NanoSIMS analysis can be linked to the parent molecule. 21 The distinct stable isotopes that encode for component identity can be selectively incorporated into cholesterol and specific lipid species with established metabolic labeling techniques.…”

Section: Introductionmentioning

confidence: 99%

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

Wilson¹,

Frisz²,

Hanafin³

et al. 2012

Bioconjugate Chem.

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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.

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Section: Introductionmentioning

confidence: 99%

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

Wilson¹,

Frisz²,

Hanafin³

et al. 2012

Bioconjugate Chem.

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“…Ratio images ( 12 C 15 N − / 12 C 14 N − ) (10 × 10 μm, 256 × 256 pixels) were collected with a small primary aperture (D1 = 4) to match the pixel size in the images with the incident ion beam diameter (~70 nm). All data were collected without preliminary Cs + implantation to avoid sputtering away the thin samples [23], and the beam dwell time was set to 8,000 μs/pixel to collect information from surface layers only a few nanometres thick. The images were processed using an ImageJ plugin OpenMIMS software (MIMS, Harvard University; www.nrims.harvard.edu) and processed by a median filter with one-pixel radius.…”

Section: Methodsmentioning

confidence: 99%

Stable isotope imaging of biological samples with high resolution secondary ion mass spectrometry and complementary techniques

Jiang

Favaro

Goulbourne

et al. 2014

Methods

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Stable isotopes are ideal labels for studying biological processes because they have little or no effect on the biochemical properties of target molecules. The NanoSIMS is a tool that can image the distribution of stable isotope labels with up to 50 nm spatial resolution and with good quantitation. This combination of features has enabled several groups to undertake significant experiments on biological problems in the last decade. Combining the NanoSIMS with other imaging techniques also enables us to obtain not only chemical information but also the structural information needed to understand biological processes. This article describes the methodologies that we have developed to correlate atomic force microscopy and backscattered electron imaging with NanoSIMS experiments to illustrate the imaging of stable isotopes at molecular, cellular, and tissue scales. Our studies make it possible to address 3 biological problems: (1) the interaction of antimicrobial peptides with membranes; (2) glutamine metabolism in cancer cells; and (3) lipoprotein interactions in different tissues.

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“…5 Isotopically labeled molecules have previously been used to image molecules in biological structures with dynamic SIMS. 9, 10 In addition, the combination of deuterated lipids and Time of Flight Secondary Ion Mass Spectrometry (TOF-SIMS) has been used to quantify lipid transfer in artificial lipid membrane systems. 11 In TOF-SIMS only the top molecular layers of a surface are analyzed, which provides molecular information exclusively from the exposed surface of the sample.…”

Section: Introductionmentioning

confidence: 99%

Relative Quantification of Phospholipid Accumulation in the PC12 Cell Plasma Membrane Following Phospholipid Incubation Using TOF-SIMS Imaging

Lanekoff

Sjövall²,

Ewing

2011

Anal. Chem.

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Time of flight secondary ion mass spectrometry (TOF-SIMS) imaging has been used to investigate the incorporation of phospholipids into the plasma membrane of PC12 cells after incubation with phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The incubations were done at concentrations previously shown to change the rate of exocytosis in model cell lines. The use of TOF-SIMS in combination with an in situ freeze fracture device enables the acquisition of ion images from the plasma membrane in single PC12 cells. By incubating cells with deuterated phospholipids and acquiring ion images at high mass resolution, specific deuterated fragment ions were used to monitor the incorporation of lipids into the plasma membrane. The concentration of incorporated phospholipids relative to the original concentration of PC was thus determined. The observed relative amounts of phospholipid accumulation in the membrane ranges from 0.5 to 2 percent following 19 hours of incubation with PC at 100 to 300 μM and from 1 to 9 percent following incubation with PE at the same concentrations. Phospholipid accumulation is therefore shown to be dependent on the concentration in the surrounding media. In combination with previous exocytosis results, the present data suggests that very small changes in the plasma membrane phospholipid concentration are sufficient to produce significant effects on important cellular processes, such as exocytosis in PC12 cells.

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Quantitative analysis of supported membrane composition using the NanoSIMS

Cited by 34 publications

References 35 publications

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

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

Stable isotope imaging of biological samples with high resolution secondary ion mass spectrometry and complementary techniques

Relative Quantification of Phospholipid Accumulation in the PC12 Cell Plasma Membrane Following Phospholipid Incubation Using TOF-SIMS Imaging

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