One-dimensional protein analysis of an HT29 human colon adenocarcinoma cell.

A single HT29 human colon adenocarcinoma cell was introduced into a fused-silica capillary and lysed, and the protein content was fluorescently labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. The labeled proteins were separated by capillary electrophoresis in a submicellar buffer and detected by laser-induced fluorescence in a postcolumn sheath-flow cuvette. Several dozen components were resolved. A number of experiments were done to verify that these components were proteins. Most components of the single-cell electropherogram had the same mobility as components present in the 30-100 kDa fraction of a protein extract prepared from the cell culture. One component was identified as a approximately 100 kDa protein by co-injecting the sample with purified protein obtained from an SDS-PAGE gel. Protein expression varied significantly between cells, but the average expression was consistent with that observed from a protein extract prepared from 10(6) cells.

Labeling effects on the isoelectric point of green fluorescent protein.

We studied the effects of fluorescent labeling on the isoelectric points (pI values) of proteins using capillary isoelectric focusing with laser-induced fluorescence detection (cIEF-LIF). Specifically, we labeled green fluorescent protein (GFP) from the jellyfish Aequorea victoria with the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). cIEF-LIF was used to monitor the native fluorescence of GFP and showed pI changes in GFP's FQ-labeled products. Multiple labeling of GFP with FQ produced a series of products with pI values shifted towards a low pH. We verified cIEF-LIF results with traditional slab gel IEF. Our cIEF-LIF technique can routinely detect 10(-11) M of FQ-labeled protein, whereas traditional slab gel IEF with silver stain detection gives detection limits of 10(-7) M in the same samples.

Sodium dodecyl sulfate-capillary electrophoresis of proteins in a sieving matrix utilizing two-spectral channel laser-induced fluorescence detection.

We report a method for protein labeling, separation by capillary electrophoresis in a polymer sieving matrix, and detection by laser-induced fluorescence. Different dyes are used to label standard and sample proteins. A two-spectral channel detector resolves fluorescence from the sample and standards. Comparison of the migration time of the sample and standards permits the precise determination of molecular weight, irrespective of variations in run-to-run migration times.

General protease assay method coupling solid-phase substrate extraction and capillary electrophoresis.

Capillary electrophoresis with laser-induced fluorescence detection was used to develop a universal, highly specific protease assay. In this method, a peptide, biotinylated at the N-terminus, is labeled with fluorescein at a lysine residue near the C-terminus. Impurities are removed from the fluorescence labeling mixture by solid-phase extraction of the substrate on immobilized streptavidin, followed by extensive washing. The purified fluorescent substrate is dissociated from the streptavidin and incubated with the protease. The peptide sequence between the biotin and fluorescent label contains the cleavage sequence of the protease of interest. After cleavage, the fluorescent product does not contain a biotin group. A second solid-phase extraction is used to remove unreacted substrate to dramatically lower the background signal. The product is detected by capillary electrophoresis, which provides powerful discrimination against products generated by nonspecific proteases. With chymotrypsin as a test protease, product was detected with as little as 10 pg/mL (4.6 x 10(-13) M) chymotrypsin, or 5 amol of enzyme in the 10-microL sample volume.