Lian Ji Jin | PubMed Health

PubMed2003

Capillary electrophoresis with laser-induced fluorescence detection for laboratory diagnosis of galactosemia.

Galactosemia, a metabolic disorder associated with the intolerance to dietary galactose due to an inherited enzymatic deficiency, is indicated by heightened levels of galactose in urine (galactosuria). In this report, capillary electrophoresis (CE) with laser-induced fluorescence detection was evaluated for its ability to screen urinary carbohydrates, particularly galactose. Neonatal urine samples with normal and abnormal levels of galactose were analyzed with galactose concentrations quantitated relative to urinary creatinine concentrations to account for variable urinary dilution. Analysis of nine samples by CE in a single-blind manner defined four as negative (normal) and five as positive for galactosuria with galactose levels as high as 146.8 +/- 5.9 mM. Galactosuria was correlated with clinical galactosemia diagnoses for four of the positive samples, while the remaining positive was associated with a patient diagnosed with Hurler's syndrome.

Electrophoresis, Capillary↗

PubMed2003

A microchip-based proteolytic digestion system driven by electroosmotic pumping.

Microchip-based proteomic analysis requires proteolytic digestion of proteins in microdevices. Enzyme reactors in microdevices, fabricated in glass, silicon, and PDMS substrates, have recently been demonstrated for model protein digestions. The common approach used for these enzyme reactors is employment of a syringe pump(s) to generate hydrodynamic flow, driving the proteins through the reactors. Here we present a novel approach, using electroosmotic flow (EOF) to electrokinetically pump proteins through a proteolytic system. The existence of EOF in the proteolytic system packed with immobilized trypsin gel beads was proven by imaging the movement of a neutral fluorescent marker. Digestions of proteins were subsequently carried out for 12 min, and the tryptic peptides were analyzed independently using capillary electrophoresis (CE) and MALDI-TOF mass spectrometry (MS). The results from CE analysis of the tryptic peptides from the EOF-driven proteolytic system and a conventional water bath digestion were comparable. MALDI-TOF MS was used to identify the parent protein and the tryptic peptides using MS-Fit database searching. The potential utility of the EOF-driven proteolytic system was demonstrated by direct electro-elution of proteins from an acrylamide gel into the proteolytic system, with elution and tryptic digestion achieved in a single step. The EOF-driven proteolytic system, thus, provides a simple way to integrate protein digestion into an electrophoretic micro total analysis system for protein analysis and characterization.

Electrochemistry↗