RESUMEN
An analysis of the glycan processing event is of particular importance to understand the nontemplate dependent synthetic mechanism of the multiple glycosylation reactions taking place in the Golgi apparatus in connection with the post-translational modification of biomolecules. In our efforts to address the issue, we constructed an analysis platform using nano-liquid chromatography (LC), which also worked as a spray tip, with an optical-fiber-based blue (470 nm) light emitting diode (LED)-induced fluorescence (520 nm) detector coupled with a microelectrospray ionization (ESI)-quadrupole ion trap (QIT)-time of flight (TOF) mass spectrometer (MS). This system was designed to enable both quantitative and qualitative analyses of fluorescently tagged molecules such as BODIPY-tagged lactosylceramide. Owing to the zero dead volume after LC separation, an extremely high sensitivity was achieved for the quantitative analysis (260 amol). It was also shown that a simultaneous online structural analysis based on MS could be achieved for the same quantity of analyte. To further demonstrate its potential, an enzymatic reaction of fluorescently tagged lactosylceramide using sialyltransferase was carried out, and the conversion yield was obtained on the basis of fluorescence detection. In addition, the structural details of a product, sialyl lactosylceramide, were obtained by MS and MS/MS analyses.
Asunto(s)
Cromatografía Liquida/métodos , Fluorescencia , Glicoesfingolípidos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Antígenos CD/química , Gangliósido G(M3)/análisis , Gangliósido G(M3)/química , Glicoesfingolípidos/química , Lactosilceramidos/química , Microquímica/métodos , Sensibilidad y Especificidad , Sialiltransferasas/química , Sialiltransferasas/metabolismo , Factores de TiempoRESUMEN
A microfluidic chip carrying three reaction chambers was designed and constructed to examine sequential multiple enzymatic reactions. The synthesis of oligosaccharides in living cells is carried out in the Golgi apparatus where multiple enzymes such as glycosidase and glycosyltransferases act on a variety of substrates to generate glycoconjugates that include glycolipids and glycoproteins. The regulatory mechanism of the process however remains unknown. A microchip-based analysis platform may provide a valuable tool with which to address the issue by mimicking the Golgi function. We thus examined 3 sequential glycosyltransfer reactions on a chip, and succeeded in the synthesis of a tetrasaccharide using immobilized enzymes. Also, the kinetic parameters for a recently identified glycosyltransferase, proteoglycan GalT-I, were obtained for the first time.
Asunto(s)
Enzimas Inmovilizadas , Glicosaminoglicanos/química , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/estadística & datos numéricos , Oligosacáridos/síntesis química , Secuencia de Carbohidratos , Cromatografía de Gases y Espectrometría de Masas , Glicosaminoglicanos/metabolismo , Glicosilación , Datos de Secuencia Molecular , Oligosacáridos/química , Sefarosa/químicaRESUMEN
A microchip-based method was developed for on-line determination of trace sulfur dioxide (SO(2)) in air. Gaseous SO(2), which diffused through the porous glass materials on the microchip, was absorbed into an absorption solution of triethanolamine (TEA) as sulfite ions and reacted with N-(9-acridinyl)maleimide (NAM), which was used as a fluorescent reagent. The fluorescence of NAM-sulfite in micro-fluidic channel was detected. The calibration curve of sulfite ions in the range of 1.5-30mumol/L (SO(2) 3-60ppbv) showed a linear relation R(2)=0.995, and the relative standard deviation (R.S.D.) was 1.9% for 10mumol/L sulfite ions in five measurements. The entire measurement procedure was achieved by the integrated microchip, and the consumption of reagents was drastically reduced. It was satisfactory to apply this method to determine on-line the SO(2) level in the air.