Application of capillary electrophoresis-electrospray ionization mass spectometry in the determination of molecular diversity.

By means of capillary electrophoresis coupled online to electrospray ionization MS, a library of theoretically 171 disubstituted xanthene derivatives was analyzed. The method allowed the purity and makeup of the library to be determined: 160 of the expected compounds were found to be present, and 12 side-products were also detected in the mixture. Due to the ability of capillary electrophoresis to separate analytes on the basis of charge, most of the xanthene derivatives could be resolved by simple capillary electrophoresis-MS procedures even though 124 of the 171 theoretical compounds were isobaric with at least one other molecule in the mixture. Any remaining unresolved peaks were resolved by MS/MS experiments. The method shows promise for the analysis of small combinatorial libraries with fewer than 1000 components.

DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.

A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method provides quantitative data in runs requiring < 20 min, from which dissociation constants are readily determined. As a test case we studied interactions of a developmentally important sea urchin embryo transcription factor, SpP3A2. As little as 2-10 x 10(6) molecules of specific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a single sea urchin egg lysed within the capillary column.

Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

Mass spectrometric and capillary electrophoretic investigation of the enzymatic degradation of heparin-like glycosaminoglycans

Difficulties in determining composition and sequence of glycosaminoglycans, such as those related to heparin, have limited the investigation of these biologically important molecules. Here, we report methodology, based on matrix-assisted laser desorption ionization MS and capillary electrophoresis, to follow the time course of the enzymatic degradation of heparin-like glycosaminoglycans through the intermediate stages to the end products. MS allows the determination of the molecular weights of the sulfated carbohydrate intermediates and their approximate relative abundances at different time points of the experiment. Capillary electrophoresis subsequently is used to follow more accurately the abundance of the components and also to measure sulfated disaccharides for which MS is not well applicable. For those substrates that produce identical or isomeric intermediates, the reducing end of the carbohydrate chain was converted to the semicarbazone. This conversion increases the molecular weight of all products retaining the reducing terminus by the “mass tag” (in this case 56 Da) and thus distinguishes them from other products. A few picomoles of heparin-derived, sulfated hexa- to decasaccharides of known structure were subjected to heparinase I digestion and analyzed. The results indicate that the enzyme acts primarily exolytically and in a processive mode. The methodology described should be equally useful for other enzymes, including those modified by site-directed mutagenesis, and may lead to the development of an approach to the sequencing of complex glycosaminoglycans.

The use of spreading depression waves for acute and long-term monitoring of the penumbra zone of focal ischemic damage in rats.

Slow potential recording was used for long-term monitoring of the penumbra zone surrounding an ischemic region produced by middle cerebral artery (MCA) occlusion in adult hooded rats (n = 32). Four capillary electrodes (El-E4) were chronically implanted at 2-mm intervals from AP -3, L 2 (El) to AP 0, L 5 (E4). Spontaneous or evoked slow potential waves of spreading depression (SD) were recorded during and 4 h after a 1-h MCA occlusion and at 2- to 3-day intervals afterward for 3 weeks. Duration of the initial focal ischemic depolarization was maximal at E4 and decreased with distance from the focus. SD waves in the penumbra zone were high at El and E2, low and prolonged at E3, and almost absent at E4. Amplitude of elicited SD waves was further reduced 3 days later and slowly increased in the following week. Cortical areas displaying marked reduction of SD waves in the first days after MCA occlusion either remained low or showed substantial (60%) recovery, the probability of which decreased with the duration of the initial focal ischemic depolarization and increased with the distance from the focus. It is concluded that the outcome of ischemia monitored by long-term SD recovery in the perifocal region can be partly predicted from the acute signs of MCA occlusion.