Rapid, parallel separations of d1S80 alleles in a plastic microchannel chip.

We have performed fast, parallel separations of alleles of the D1S80 locus in a plastic, multi-channel chip, replicated from a microfabricated master and laminated with a plastic film. The array of 16 channels was filled with a replaceable sieving polymer, and a size-dependent, electrophoretic separation of the DNA fragments was performed in all channels in less than 10 min, representing a 30-fold increase in throughput compared to that on a single-capillary instrument. To detect the fragments in all 16 channels in parallel during the run, we designed and built a scanning, confocal, laser-induced fluorescence system. The electropherograms were then used to determine the sample genotype. To demonstrate the use of multiplexed, microchannel arrays for real-life samples, we amplified D1S80 alleles from genomic DNA extracted from whole blood and separated these alleles by electrophoresis in the plastic chip. Evaluation of the electrophoretic data showed that, using a 300- and a 1,000-base pair fragment as internal mobility markers, 83% of the alleles were assigned correctly, using the allele identification from a single capillary instrument as a reference. This work demonstrates that, with improvements in the microchannel electrophoresis system, it is feasible to perform rapid, parallel genotyping in mass-produced, inexpensive, disposable plastic devices for large-scale applications in medicine and the life sciences.

Mass spectral study of polymorphism of the apolipoproteins of very low density lipoprotein.

New isoforms of apolipoprotein (apo)C-I and apoC-III have been detected in delipidated fractions from very low density lipoprotein (VLDL) using matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) mass spectrometry (MS). The cleavage sites of truncated apoC-III isoforms have also been identified. The VLDL fractions were isolated by fixed-angle single-spin ultracentrifugation using a self-generating sucrose density gradient and delipidated using a newly developed C18 solid phase extraction protocol. Fifteen apoC isoforms and apoE were identified in the MALDI spectra and the existence of the more abundant species was verified by ESI-MS. The relative intensities of the apoCs are closely correlated in three normolipidemic subjects. A fourth subject with type V hyperlipidemia exhibited an elevated apoC-III level and a suppressed level of the newly discovered truncated apoC-I isoform. ApoC-II was found to be particularly sensitive to in vitro oxidation. The dynamic range and specificity of the MALDI assay shows that the complete apoC isoform profile and apoE phenotype can be obtained in a single measurement from the delipidated VLDL fraction.

Characterization and quantitation of apolipoprotein B-100 by capillary electrophoresis.

The interaction of low density lipoproteins (LDL) with different surfactants was studied by capillary electrophoresis (CE) and sucrose density gradient ultracentrifugation as part of developing a method for quantitation of apoB-100 in serum. A mixture of surfactants consisting of 70% sodium dodecyl sulfate (SDS), 25% sodium myristyl sulfate, and 5% sodium cetyl sulfate was found to delipidate LDL particles more effectively than pure SDS or sodium decyl sulfate. The delipidation products of LDL [apolipoprotein B-100 (apoB-100) and lipids] were resolved as two distinct peaks by CE when using a 3.5 mM 70% SDS mixture, 20% (v/v) aceto nitrile, 50 mM sodium borate, pH 9.1 buffer. This CE method was also used to characterize apoB-100 derived from samples of lipoprotein [a] and very low density lipoproteins (VLDL). A CE-based quantitation method for apoB-100 was developed utilizing the observed linear relationship between apoB-100 concentration and its corrected 214 nm absorbance peak area measured on-line by CE. Concentration values of apoB-100 in LDL and VLDL samples were determined by CE and found to be accurate when compared to values obtained by immunoturbidimetric analysis and the Lowry method. Capillary electrophoresis can be used as a precise, accurate, and specific on-line method for the qualitative and quantitative analysis of the apoB-100 component of VLDL and LDL-related lipoproteins.

Development of a lipoprotein profile using capillary electrophoresis and mass spectrometry.

A new program for lipoprotein characterization is outlined where capillary electrophoresis (CE) plays a central role in the analysis of intact lipoprotein serum components and the apoprotein domains. The first characterization step involves separation and particle density analysis of very low-, low-, and high-density lipoprotein fractions (VLDL, LDL, HDL) by ultracentrifugation and image analysis. VLDL, HDL, and LDL fractions are analyzed by capillary electrophoresis. Sodium dodecyl sulfate (SDS) at low concentrations in the background electrolyte used in the CE analysis is incorporated into the lipoprotein particle without appreciable delipidation, as determined by ultracentrifuge particle density analysis. Increasing the concentration of SDS results in extensive delipidation, resulting in the release of apoproteins (apo) which are detected as components of the electropherogram. Apo B-100 is detected in the delipidated VLDL and LDL fractions along with micelles of the lipids. Micelles from LDL delipidation have uniform charge densities. Apo A-I and A-II are detected in the HDL fraction. A new method for lipoprotein delipidation is introduced where the lipoprotein fraction is adsorbed on a reversed-phase hydrophobic cartridge. Delipidation and recovery of the apoprotein fractions is made by serial elutions with acetonitrile. CE of the lipid-free apoprotein mixture shows the presence of apoC-I,II,III and apoE in the VLDL fraction, and apoA-I,II apoC-I and apoE in the HDL fraction. Electrospray ionization mass spectrometry analysis gives the isoform distribution for each apoprotein. The identification of the apoproteins in the electropherograms is the first step in developing a CE-based quantitation method for measuring serum levels of these apoproteins and their distribution between the lipoprotein fractions. The assay described in this paper is being used as a level 2 and 3 cardiac risk profile analysis for individuals with normal lipid profiles who have a documented or family history of cardiovascular disease.

Characterization and quantitation of the apoproteins of high-density lipoprotein by capillary electrophoresis.

A method has been developed using capillary electrophoresis (CE) to quantitate plasma levels of apoprotein A-I (apoA-I) and apoprotein A-II (apoA-II) in high-density lipoprotein (HDL) samples. ApoA-I and apoA-II are resolved by CE in delipidated and non-delipidated HDL samples. Concentrations of apoA-I and apoA-II were calculated from their peak areas in the electropherogram. Results of the analysis of Sigma plasma standards (Controls 1 and 2) using CE are in good agreement with values obtained by Sigma using immunoturbidimetric assay. CE and reverse-phase high-performance liquid chromatography (RP-HPLC) were found to be complementary in the study of apoA-I and apoA-II. RP-HPLC resolves the isoforms of purified apoA-I and apoA-II, but it cannot resolve mixtures of them because the retention times of the isoforms overlap. CE separates apoA-I from apoA-II, but it does not resolve the isoforms. Matrix-assisted laser desorption/ ionization mass spectrometry was used to identify the isoforms of apoA-I and apoA-II by their molecular weight (M(r)) in fractions collected from RP-HPLC.

Influence of dodecyl sulfate ions on the electrophoretic mobilities of lipoprotein particles measured by HPCE.

The authors have developed a method for profiling plasma lipoproteins based on differences in their surface chemical properties using HPCE. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were used as model compounds representing components of the lipoprotein population with high surface polarity (HDL) and low surface polarity (LDL). Using an uncoated fused-silica capillary in the HPCE measurement, the effective electrophoretic mobilities (mu eff) of HDL and LDL at high pH were found to be nearly identical in different buffer systems and ionic strengths. Both HDL and LDL particles have a high affinity for dodecyl sulfate anions (DS-), increasing the mu eff for both particles by almost a factor of two but not giving significantly different mu eff values. Decreasing the polarity of the solution by the addition of acetonitrile resulted in a partitioning of the DS- ions between the solvent and lipoprotein particles. The more hydrophobic LDL particles retain a larger fraction of DS- ions than do HDL particles. Under these conditions, the LDL particles have a significantly higher charge/volume ratio than do HDL particles and a large difference in mu eff values is achieved. The conditions for maximizing the mu eff difference were found by studying the influence of the concentration of the DS- ions and percent acetonitrile of the mu eff of LDL and HDL at high pH.

Characterization of lipoprotein a by capillary zone electrophoresis.

Lipoprotein a [Lp(a)] has been recognized as a significant marker for premature coronary heart disease (CHD). In this paper, we present the results of Lp(a) analysis based on capillary zone electrophoresis (CZE). CZE separation of Lp(a) and its reduced species, lipoprotein a- [Lp(a-)] and apolipoprotein a [apo(a)], was accomplished using 50 mM borate buffer containing 3.5 mM sodium dodecyl sulfate (SDS) and 20% (v/v) acetonitrile (ACN). Low density lipoprotein (LDL) and high density lipoprotein (HDL) were separated under the same buffer conditions. The electrophoretic mobilities of both Lp(a) and Lp(a-) were found to be different from that of LDL. Benzyl alcohol (BA) and methanol (MeOH) were used as electroosmotic flow markers. BA molecules associated with Lp(a-) and LDL to enhance their UV absorbance, but did not change their effective electrophoretic mobilities. Our results show that CE is a very efficient and effective technique for lipoprotein analysis.