Browsing by Author "Wu, June-Tai"

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Automated system for extraction and instantaneous analysis of millimeter-sized samples
(2014)
Hu, Jie-Bi
;
Chen, Ssu-Ying
;
Wu, June-Tai
;
Chen, Yu-Chie
;
Urban, Pawel L.
Adequate sample treatment is critical when employing mass spectrometry (MS) in the analyses of complex biological matrices. Despite various improvements, it is generally difficult to automate the process of preparing solid biological samples for MS analysis. Here we demonstrate a facile approach for automation of the whole analysis (from an untreated sample to the final result). The proposed platform enables disruption and extraction of relatively small samples (individual fruit flies, fragments of tea leaves, powdered drug sampled with a cotton bud), and almost simultaneous analysis of the obtained extract within less than 10 min, and with very little intervention of the analyst. The operation is straightforward: once a sample is placed in the sample chamber, the analyst only needs to press a button on the touch screen of the user interface. The programmed open-source electronic device triggers addition of a small amount of solvent, and subsequent mechanical disruption/extraction of the specimen under controlled conditions (thermostated chamber). A small volume of the extract is directed to the ion source of the mass spectrometer incorporating a Venturi pump. During the operation of the instrument, fluorescence intensities (excitation wavelength windows: 320-380 and 460-500 nm) as well as MS extracted ion currents are recorded simultaneously. In the case of fruit fly samples, similar to 70 signals were recorded in both modes while the analysis of green tea leaves yielded similar to 30 signals. The resulting data reveal time-resolved extraction profiles characterizing every sample.
Isotope Label-Aided Mass Spectrometry Reveals the Influence of Environmental Factors on Metabolism in Single Eggs of Fruit Fly
(2012-11-21)
Tseng, Te-Wei
;
Wu, June-Tai
;
Chen, Yu-Chie
;
Urban, Pawel L.
;
應用化學系
In order to investigate the influence of light/dark cycle on the biosynthesis of metabolites during oogenesis, here we demonstrate a simple experimental protocol which combines in-vivo isotopic labeling of primary metabolites with mass spectrometric analysis of single eggs of fruit fly (Drosophila melanogaster). First, fruit flies were adapted to light/dark cycle using artificial white light. Second, female flies were incubated with an isotopically labeled sugar (C-13(6)-glucose) for 12 h - either during the circadian day or the circadian night, at light or at dark. Third, eggs were obtained from the incubated female flies, and analyzed individually by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS): this yielded information about the extent of labeling with carbon-13. Since the incorporation of carbon-13 to uridine diphosphate glucose (UDP-glucose) in fruit fly eggs is very fast, the labeling of this metabolite was used as an indicator of the biosynthesis of metabolites flies/eggs during 12-h periods, which correspond to circadian day or circadian night. The results reveal that once the flies adapted to the 12-h-light/12-h-dark cycle, the incorporation of carbon-13 to UDP-glucose present in fruit fly eggs was not markedly altered by an acute perturbation to this cycle. This effect may be due to a relationship between biosynthesis of primary metabolites in developing eggs and an alteration to the intake of the labeled substrate - possibly related to the change of the feeding habit. Overall, the study shows the possibility of using MALDI-MS in conjunction with isotopic labeling of small metazoans to unravel the influence of environmental cues on primary metabolism.
Microscale MALDI Imaging of Outer-Layer Lipids in Intact Egg Chambers from Drosophila melanogaster
(2011-05-15)
Urban, Pawel L.
;
Chang, Chia-Hsien
;
Wu, June-Tai
;
Chen, Yu-Chie
;
應用化學系
Fruit fly (Drosophila melanogaster) is a standard model organism used in genetics and molecular biology. Phospholipids are building blocks of cellular membranes, and components of a complex signaling network Here, we present a facile method, based on matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), for molecular imaging of phospholipid distributions in submillimeter-sized components of the fruit fly reproductive system. Individual egg chambers were deposited on a specially prepared MALDI target comprising an aluminum slide with a rough surface created by ablation with a microsecond-laser: this helped to immobilize biological specimens, remove excess of saline solution by adhesive forces, carry out microscopic observations, and facilitated distribution of the MALDI matrix. A continuous-flow ultrasound-assisted spray was used for the deposition of MALDI matrix (9-aminoacridine) onto the sample. The upper surface of the specimen was then scanned with a 355-nm solid-state laser with a preset beam focus of 10 mu m to obtain negative-ion mode MALDI-MS images. Overall, this provided sufficient spatial resolution to reveal micrometer-scale gradient-like patterns of phospholipids along the anterior/posterior axis of egg chambers. Several phosphatidylinositols are seen to be segregated according to the number of unsaturated bonds, with an elevated abundance of polyunsaturated phosphatidylinositols within the oocyte compartment.