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A novel and advanced Fc-binding probe – FcUni-RLuc namely – has been produced and functionally assayed for labelling IgGs. The Fc antibody binding sequence – HWRGWV – was fused to Renilla luciferase, and the purified probe was employed for bioluminescence enzyme-linked immunoabsorbance assay of Her2 positive cells. [ABSTRACT FROM AUTHOR]

A quantitative mass spectrometry imaging (QMSI) method for absolute quantification of glutathione (GSH) in healthy and cancerous hen ovarian tissues using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is presented. Using this technique, the ion abundance of GSH was normalized to that of a structural analogue, which was sprayed on the slide prior to mounting the tissue sections. This normalization strategy significantly improved the voxel-to-voxel variability; the variability is attributed to the overall ionization process. Subsequently, a series of calibration spots of stable isotope-labeled (SIL) GSH were pipetted on top of the tissue to construct a spatial calibration curve, and calculate the concentration of GSH in both tissue sections. The QMSI results were verified by LC-MS/MS quantification of GSH for the same tissues. GSH was extracted from tissue sections in a slightly acidic buffer and was then alkylated using N-ethylmaleimide to minimize autoxidation of GSH to glutathione disulfide. The alkylated GSH was separated from other contaminants using reversed phase liquid chromatography (RPLC) coupled to a triple quadrupole mass spectrometer, and the z-ion transition of NEM-GSH was used to quantify GSH in each tissue section. While the absolute values obtained using IR-MALDESI QMSI and LC-MS/MS were different, a ∼2-fold increase in the concentration of GSH in cancer tissue compared to the healthy tissue was observed using both techniques. Possible reasons for the difference between absolute concentration values obtained using IR-MALDESI QMSI and LC-MS/MS are also discussed.

Mass spectrometry imaging (MSI) is a rapidly evolving field for monitoring the spatial distribution and abundance of analytes in biological tissue sections. It allows for direct and simultaneous analysis of hundreds of different compounds in a label-free manner. In order to obtain a comprehensive metabolite and lipid data, a polarity switching MSI method using infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) was developed and optimized where the electrospray polarity was alternated from one voxel to the next. Healthy and cancerous ovarian hen tissue sections were analyzed using this method. Distribution and relative abundance of different metabolites and lipids within each tissue section were discerned, and differences between the two were revealed. Additionally, the utility of using mass spectrometry concepts such as spectral accuracy and sulfur counting for confident identification of analytes in an untargeted method are discussed.

The Ca(2+)-dependent binding of Annexin V to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this paper, we report a new class of Annexin V-based probes for apoptosis. Luciferase from Renilla reniformis (RLuc) was linked to Annexin V and expressed successfully in a soluble form in Escherichia coli BL21 (DE3). The new probe, Rluc/Annexin V, was purified and functionally assayed for detection of apoptosis in actinomycin D-induced apoptotic Jurkat cells. Moreover, the spontaneous apoptosis in neutrophils was shown using the new probe. The results indicate that Rluc/Annexin V can bind to the apoptotic cells, and the signal of Renilla luciferase can be detected by luminometric measurements. The availability of Rluc/Annexin V may be of potential commercial interest for improving current apoptosis assays.