Glycosphingolipids (GSL) on the surface of cells are important receptors in antigen/microbial recognition and cell adhesion. However, their functional characterization is often challenging. We have developed a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for the identification of specific interactions between water-soluble proteins or protein complexes with GSL incorporated into nanodiscs. The specificity and sensitivity of the assay is demonstrated for interactions involving cholera toxin and Shiga toxin, with their natural GSL receptors, the ganglioside GM1, and the globotriaosylceramide Gb3, respectively. The detection of binding between cholera toxin and GM1 within a mixture of lipids extracted from cell membranes highlights the potential of this assay for the discovery of biologically relevant protein-GSL interactions.

The selective ligation of hydrazine and amino-oxy compounds with carbonyls has gained popularity as a detection strategy with the recognition of aniline catalysis as a way to accelerate the labeling reaction in water. Aldehydes are a convenient functional group choice since there are few native aldehydes found at the cell surface. Aldehydes can be selectively introduced into sialic acid containing glycoproteins by treatment with dilute sodium periodate. Thus, the combination of periodate oxidation with aniline-catalyzed ligation (PAL) has become a viable method for detection of glycoconjugates on live cells. Herein we examine two fluorescent nitrobenzoxadiazole dyes for labeling of glycoproteins and cell surface glycoconjugates. We introduce a novel 4-aminooxy-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDAO) (5) fluorophore, and offer a comparison to commercial dyes including the known 4-hydrazino-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDH) (2) and Bodipy FL hydrazide. We confirm specificity for sialic acid moieties and that both dyes are suitable for in vitro and in vivo labeling studies using PAL and fluorescence spectroscopy. The dyes examined here are attractive labeling agents for microscopy, as they can be excited by a 488 nm laser line and can be made in a few synthetic steps. These carbonyl-reactive chromophores provide a one step alternative to avidin-biotin labeling strategies and simplify the detection of sialic acid in cells and glycoproteins.

Fast, high-yielding, and selective bioorthogonal "click" reactions employing nontoxic reagents are in high demand for their great utility in the conjugation of biomolecules in live cells. Although a number of click reactions were developed for this purpose, many are associated with drawbacks and limitations that justify the development of alternative systems for both single- or dual-labeling applications. Recent reports have highlighted the potential of boronic ester formation as a bioorthogonal click reaction between abiotic boronic acids and diols. Boronic ester formation is a fast dehydrative process; however it is intrinsically reversible in aqueous medium. We designed and optimized a synergic system based on two bifunctional reagents, a thiosemicarbazide-functionalized nopoldiol and an ortho-acetyl arylboronic acid. Both reagents were shown to be chemically stable and nontoxic to HEK293T cells at concentrations as high as 50 μM. The resulting boronate/thiosemicarbazone adduct is a medium-sized ring that forms rapidly and irreversibly without any catalyst at low μM concentrations, in neutral buffer, with a rate constant of 9 M s as measured by NMR spectroscopy. Control experiments in the presence of competing boronic acids showed no crossover side-products and confirmed the stability and lack of reversibility of the boronate/thiosemicarbazone conjugates. Formation of the conjugates is not affected by the presence of biological diols such as fructose, glucose, and catechol, and the thiosemicarbazide-functionalized nopoldiol is inert to aldehyde electrophiles of the sort found on protein-bound glyoxylyl units. The suitability of this system in the cell-surface labeling of live cells was demonstrated using a SNAP-tag approach to install the boronic acid reagent onto the extracellular domain of the Beta-2 adrenergic receptor in HEK293T cells, followed by incubation with the optimal thiosemicarbazide-functionalized nopoldiol reagent labeled with fluorescein dye. Successful visualization by fluorescence microscopy was possible with a reagent concentration as low as 10 μM, thus confirming the potential of this system in biological applications.