Development of a fully automated lipid analyzer aimed at lipid metabolomics and disease analysis

开发针对脂质代谢组学和疾病分析的全自动脂质分析仪

• 批准号: 16390093
• 负责人: TOJO Hiromasa
• 金额: $ 9.66万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390093/
• 关键词: lipidomics; sphingolipids; phospholipids; HPLC; mass spectrometry; リピドミックス; 脂質; 順相クロマトグラフィー; エレクトロスプレー

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 … More 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

由于蛋白质组学和脂质代谢组学的并行分析有助于了解与脂质代谢相关的蛋白质功能，因此脂质组学受到了广泛关注。我们开发了一种自动正相微孔HPLC/电喷雾（ESI）离子阱质谱（MS）系统，用于分析更广泛的脂质类别，从中性脂质（例如，胆固醇酯、蜡、甘油酯、神经酰胺和鞘糖脂）到磷脂。该系统使用三元梯度和柱后添加溶剂，这有助于先前用非极性溶剂洗脱的中性脂质的ESI。开发了一种可以在低流速下稳定输送非极性溶剂梯度的无脉冲泵。为了提高离子化效率，使用了最近开发的氟聚合物涂层的ESI尖端，FortisTip，并且流出物通过T分流器分散，产生穿过尖端的10.5 μl/min的流速，并且用Finnigan LCQdeca-X监测 ...更多信息 P质谱仪设置为数据相关负或正串联MS ^2和MS ^3模式以及交替正负离子全扫描（交替极性切换）模式。用蒸发光散射光度计监测流速为1.50 μl/min的另一流出液，该方法可用于胆固醇的在线检测。胆固醇酯、三酰甘油、神经酰胺、胆固醇酯、磷脂酰乙醇胺、磷脂酰胆碱和具有非天然长度的酰基链的鞘磷脂用作内标。基于MS的色谱分析，包括中性脂质和磷脂，可用于检测和结构鉴定疾病状态下的新型脂质和脂质异常。该方法成功地应用于响应真实的分析需求，包括测定具有组织特异性破坏鞘脂代谢相关基因（如丝氨酸棕榈酰转移酶（SPT））的小鼠中脂质谱的变化，基因操作的酵母脂质谱变化，细菌SPT活性测定，脑中新的含葡萄糖磷脂（磷脂酰葡糖苷）的分析，和脂质分析在活性位点的酰基辅酶A氧化酶在单晶。少

项目成果

A cytosolic cytochrome b5-like protein in yeast cell accelerating the electron transfer from NADPH to cytochrome c catalyzed by Old Yellow Enzyme.

酵母细胞中的一种胞质细胞色素 b5 样蛋白，在老黄酶的催化下加速从 NADPH 到细胞色素 c 的电子转移。

• 发表时间: 2005
• 期刊: Biochem.Biophys.Res.Commun. 338
• 作者: Nakagawa;M.
• 通讯作者: M.

リパーゼファミリーに属するホスホリパーゼA2 触媒トライアッドの不思議

磷脂酶 A2，脂肪酶家族的一员：催化三联体的奥秘

• 发表时间: 2005
• 期刊: 化学と生物 43
• 作者: Kondo;G.;Tojo;H.;Nakatani;Y.;Komazawa;N.;Murata;C.;Yamagata;K.;Maeda;Y.;Kinoshita;T.;Okabe;M.;Taguchi;R.;Takeda;J.;東城 博雅
• 通讯作者: 東城 博雅

Analysis and determination of novel glycolipids in vertebrate brains by HPLC/mass spectrometry.

通过 HPLC/质谱法分析和测定脊椎动物脑中的新型糖脂。

• 期刊: Methods Enzymol. (印刷中)
• 作者: Nagatsuka;Y.
• 通讯作者: Y.

Three-dimensional structure of rat-liver acyl-CoA oxidase in complex with a fatty acid: insights into substrate-recognition and reactivity toward molecular oxygen.

• DOI: 10.1093/jb/mvj088
• 发表时间: 2006-04
• 期刊: Journal of biochemistry
• 影响因子: 2.7
• 作者: K. Tokuoka;Y. Nakajima;K. Hirotsu;I. Miyahara;Y. Nishina;K. Shiga;H. Tamaoki;C. Setoyama;H. Tojo;R. Miura

The Angiotensin-converting Enzyme (ACE) is a GPI-anchored Protein Releasing Factor Crucial for Fertilization.

血管紧张素转换酶 (ACE) 是一种对受精至关重要的 GPI 锚定蛋白释放因子。

• 发表时间: 2005
• 期刊: Nature Med. 11
• 作者: Kondo;G.;Tojo;H.;Nakatani;Y.;Komazawa;N.;Murata;C.;Yamagata;K.;Maeda;Y.;Kinoshita;T.;Okabe;M.;Taguchi;R.;Takeda;J.
• 通讯作者: J.