Targeted multiplex Imaging Mass Spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This manuscript describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet.
We have developed a method that combines the use of stable isotopes, MIMS and antibody. We began with using well-established antibodies, anti-actin and anti-synaptophysin, in mouse intestinal cells. We extended the method to an immunogold assay to specifically localize Ribeye, a major protein component of retina synaptic ribbons, or to localize a synaptic vesicle-containing protein, synaptophysin. Both are localized in presynaptic nerve terminal of photoreceptors cells in retina. Our results show that by MIMS analysis of the Au signal we can directly identify antibodies tagged with non amplified 1.4 nm gold nanoparticles. They also demonstrate that the gold nanoparticle-tagged antibodies do not dilute the 15N/14N signal used for measuring protein turnover. Thus we can simultaneously and directly use MIMS to measure protein turnover and to identify cell type or specific protein.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.