| Budget Amount *help |
¥15,100,000 (Direct Cost: ¥15,100,000)
Fiscal Year 2005: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 2004: ¥10,100,000 (Direct Cost: ¥10,100,000)
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| Research Abstract |
Lipidomics has attracted much attention because a side-by-side analysis of proteome and lipid metabolome could greatly facilitate to understand protein functions relevant to lipid metabolism. We developed an automated normal-phase microbore HPLC/electrospray (ESI) ion trap mass spectrometry (MS) system for analyzing a wider range of lipid classes, from neutral lipids (e.g., cholesterol esters, waxes, glycerides, ceramides, and glycosphingolipids) to phospholipids, on a single chromatographic run. The system uses a ternary gradient and post-column addition of solvent that aids ESI of neutral lipids eluted earlier with non-polar solvents. A pulse-less pump that can stably deliver gradients of non-polar solvents at low flow rates was developed. To increase ionization efficiency, the recently developed fluoropolymer-coated ESI tip, FortisTip, was used, and the effluent was dispersed via a T splitter, yielding a flow rate of 〜0.5μl/min across the tip, and monitored with a Finnigan LCQdeca-X
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P spectrometer set to either data dependent negative or positive tandem MS^2 and MS^3 mode as well as to alternate positive and negative ion full scan (alternate polarity switching) mode. The other effluent at 〜50μl/min was monitored with an evaporative light scattering photometer, which is useful for detecting cholesterol on-line. Choresterol ester, triacylglycerol, ceramide, cerebroside, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin with acyl chain(s) of unnatural length were used as internal standards. The MS-based chromatographic profiling, encompassing neutral lipids and phospholipids, is useful for detecting and structurally identifying a novel lipid and lipid anomaly in diseased states. This method was successfully applied in response to real analytical needs, including determination of changes in lipid profile in mice with tissue-specific disruption of sphingolipid metabolism-related gene, such as serine palmitoyl transferase (SPT), yeast lipid profile changes on gene manipulation, bacterial SPT activity assay, analysis of a novel glucose-containing phospholipid, phosphatidylglucoside, in brain, and lipid analysis in the active site of acyl CoA oxidase in single crystals. Less
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