Individual mitochondrion characterization: a comparison of classical assays to capillary electrophoresis with laser-induced fluorescence detection.

A method has been developed that uses capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF) for measuring protein abundance in individual mitochondria collected from a discontinuous density gradient and labeled with Mitotracker Green. From these measurements we determined the distribution of protein content per mitochondrion and the relative abundance of mitochondrial proteins in density gradient fractions. In addition, this method is useful for counting mitochondria and, as a consequence, determining the number of mitochondria per unit volume or estimate mitochondria copy number per cell. It was determined that mitochondria accumulate in two interfaces defined by consecutive layers of 35% Metrizamide, 17% Metrizamide, and 6% Percoll. The presence of mitochondria in these interfaces was also confirmed using a modified Lowry assay that prevents interference from Metrizamide and Percoll and determines total protein content, and a succinate dehydrogenase assay that uses dichloroindophenol as an electron acceptor and that specifically indicates abundance of mitochondria. The CE-LIF analysis of mitochondrial properties, based on the individual mitochondrial determinations, has a wider scope than the average values determined by enzymatic or bulk protein assays.

Determination of properties of individual liposomes by capillary electrophoresis with postocolumn laser-induced fluorescence detection.

Individual liposome measurements by capillary electrophoresis with postcolumn laser-induced fluorescence detection facilitated the determination of liposome property distributions, two-dimensional plots, and an improved characterization of a liposomal preparation. This advancement in liposome analysis was feasible by using a high-sensitivity postcolumn laser-induced fluorescence detector wired for millisecond response. For each individual liposome containing fluorescein, peak height and migration time were determined. From these measurements the individual entrapped volumes and electrophoretic mobilities were determined. Distribution analysis of these properties facilitated comparison of various liposome dilutions and indicated that the method is reproducible and unaffected by the density of liposomes (10(7)-10(9) liposomes/mL) in the suspension. Furthermore, liposomes showed entrapped volumes that vary from 0.3 to 13 fL with apparent radius varying from 370 nm to 1.8 microns. Two-dimensional plots of reduced mobility versus kappa R (Debye parameter x liposome radius) revealed that the liposomes resuspended from a dried film of phospholipids are heterogeneous in regard to the surface charge density of individual liposomes. The described method has the potential of becoming a new tool for characterization of commercial liposomal preparations and theoretical studies.

Separation of free amino acids in human plasma by capillary electrophoresis with laser induced fluorescence: potential for emergency diagnosis of inborn errors of metabolism.

Free amino acids (AAs) in human plasma are derivatized with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) and analyzed by capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The labeling procedure is significantly improved over results reported previously. Derivatization can be completed in 40 min, with concentrations as low as 4 x 10(-8) M successfully labeled in favourable cases. Twenty-nine AAs (including 2 internal standards) are identified and can be reproducibly separated in 70 min. Migration time RSD values for 23 of these AAs were calculated and found in the range from 0.5 to 4%. The rapid derivatization procedure and the resolution obtained in the separation are sufficient for a semi-quantitative, emergency diagnosis of several inborn errors of metabolism (IEM). Amino acid profiles for both normal donor plasma samples and plasma samples of patients suffering from phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia, and citrullinemia are studied.

Instrumentation for chemical cytometry.

Capillary electrophoresis is ideally suited to chemical analysis of individual cells. Small mammalian somatic cells (approximately 15 microns in diameter) can be analyzed by injecting the intact cell into a capillary, lysing the cell, separating and detecting the cellular components, and reconditioning the capillary prior to the next injection. In this paper, we report on technical improvements to single-cell analysis. We designed an inexpensive multipurpose single-cell injector that facilitates the following: (i) monitoring of injection, (ii) reproducible pressure- or electrokinetic-driven injection of the cell, (iii) complete cell lysis by SDS within 30 s of injection, and (iv) pressure-driven capillary reconditioning. Furthermore, we report on the analysis of glycosylation and glycolysis in single human carcinoma cells (HT29 cell line). The reliability and quality of the analysis is confirmed by comparing electropherograms from single cells and those from purified cell extracts.

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.

Picomolar analysis of proteins using electrophoretically mediated microanalysis and capillary electrophoresis with laser-induced fluorescence detection.

We report a method for the analysis of picomolar concentration proteins using electrophoretically mediated microanalysis (EMMA) to label proteins on-column with a fluorogenic reagent. Labeling is followed by capillary zone electrophoresis separation and postcolumn detection based on laser-induced fluorescence. The method provides a concentration detection limit (3 sigma) of 3 x 10(-13) M for conalbumin. The method provides separation efficiency of 300,000 theoretical plates. Protein extract from a human colon adenocarcinoma cell line generated a dozen major components and many minor components in a 12-min separation; the protein extract from 2.5 cells was used for this analysis. When compared to UV absorbance detection, the EMMA method provides 7,000,000-fold improvement in detection limit.

Cationic and anionic polymeric additives for wall deactivation and selectivity control in the capillary electrophoretic separation of proteins in food samples.

Both cationic and anionic polymeric additives were used for the capillary electrophoretic separation of proteins in food samples. The cationic polyelectrolyte polydiallyldimethylammonium chloride was more effective in minimizing protein-wall interactions at pH 3 than at pH 7, presumably due to greater repulsion between the adsorbed polymer and proteins. Improved resolution was observed in the presence of the co-additive sodium octanesulphonate, presumably due to ion-pairing interactions with protein sample components. The anionic polymer dextran sulfate produced relatively high efficiencies, 120,000-180,000 theoretical plates, for protein separation, presumably because the polymer adsorbed to the capillary wall, rendering the surface more hydrophilic. In addition to reduced protein-wall interactions, improved resolution was observed, presumably due to analyte-polymer ion-exchange/ion-pairing interactions. When poly(vinyl sulphonic acid) was used instead of dextran sulfate, broader profiles were obtained and fewer components were resolved, presumably due to reduced wall deactivation that is related to the lower hydrophilicity of poly(vinyl sulphonic acid).

Picomolar assay of native proteins by capillary electrophoresis precolumn labeling, submicellar separation, and laser-induced fluorescence detection.

We report a method for the assay of proteins at concentrations lower than 10(-)(10) M with as little as 200 amol of protein. High sensitivity is accomplished by derivatizing the ε-amino group of the protein's lysine residues with the fluorogenic dye 5-furoylquinoline-3-carboxaldehyde and use of a sheath flow cuvette fluorescence detector. Most proteins have a large number of lysine residues; therefore, a large number of fluorescent molecules can be attached to each protein molecule. In general, precolumn labeling improves sensitivity but degrades resolution due to the inhomogeneity of the reaction products from multiple labeling. However, we demonstrate that, through careful manipulation of the separation and reaction conditions, high sensitivity can be obtained without excessive loss in separation efficiency. Over 190 000 theoretical plates are obtained for fluorescently labeled ovalbumin.

Solid-phase fluorescent labeling reaction of picomole amounts of insulin in very dilute solutions and their analysis by capillary electrophoresis.

The fluorescent labeling of peptides at concentrations as low as 10(-8) M can be achieved by using a solid-phase reactor. Using oxidized insulin chain B as a test peptide, we demonstrate the use of an Immobilon CD membrane to capture and preconcentrate peptides. Insulin chain B can then be labeled with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde, while it is still attached to the membrane. Unwanted fluorescent products (attributed to secondary reactions) can be washed away with methanol without significant removal of the labeled insulin chain B, which then can be extracted with a low pH buffer. The analysis by micellar electrokinetic capillary chromatography with post-column laser-induced fluorescence detection (mass limit of detection of 2.4 x 10(-21) moles insulin chain B) results in electropherograms that show great improvement in terms of unwanted peaks and high number of theoretical plates (up to 20 million). The use of the solid-phase reactor allows easy handling of as little as 5 picomoles of insulin chain B.

Fluorescence-based enzymatic assay by capillary electrophoresis laser-induced fluorescence detection for the determination of a few beta-galactosidase molecules.

beta-Galactosidase can be assayed by monitoring the generation of the fluorescent products, fluorescein-mono-beta-D-galactopyranoside and fluorescein, when the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside is used. We have used capillary electrophoresis with ultrasensitive laser-induced fluorescence detection to monitor the formation of the fluorescent products of off-column enzymatic reactions. By analyzing as little as 40 pl of the enzymatic mixture we obtain limits of detection of 6.5 x 10(-14) M beta-galactosidase or 1.6 molecules based on the detection of fluorescein-mono-beta-D-galactopyranoside.