Construction of capillary electrophoresis/mass spectrometry interface

毛细管电泳/质谱接口的构建

• 批准号: 07558219
• 负责人: TITANI Koiti
• 金额: $ 2.37万
• 依托单位: Fujita Health University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558219/
• 关键词: mass spectrometry; capillary electrophoresis; protein; nucleic acid; structural analysis; 電気泳動; 蛋白質

In the first fiscal year, we made a coaxial type capillary electrophoresis/mass spectrometry (CE/MS) interface for the connection between the existing electrospray mass spectrometer (Sciex API III) and the newly purchased capillary electroresis (CE) apparatus (Beckman P/ACE 5000). During the second year, the efforts were concentrated in the optimization of the electrophoresis conditions that are compatible with the electrospray ionization. The results obtained were very close to our initial expectations ; the resolution and the sensitivity observed with the new interface were superior to those obtained with the existing liquid-junction type interface. Furthermore, the amounts of samples needed for the analysis are minimal. The amounts are less than one percent of those used for the previous LC/MS analysis. This is a clear advantage in the protein analysis, since the rest of the samples can be used to other analyzes such as amino acid sequencing. In the third (final) year, we introduced the so-called nanospray ionization method to achieve higher sensitivities. For this purpose, we established the existing nanospray method using glass microinjection capillary as well as the new microspray method using pulled-fused silica capillaries. The latter method allowed us to get stable electrospray ionization at a flow rate around 20-50 nl/min. This method, therefore, can be well combined with the capillary electrophoresis.

在第一个会计年度，我们为现有的电喷雾质谱仪（SCIEX API III）与新购买的新购买的毛细管电动机（CE）（Beckman P/ACE 5000）之间的连接进行了同轴类型的毛细管电泳/质谱（CE/MS）接口。在第二年，努力集中在与电喷雾电离兼容的电泳条件的优化中。获得的结果非常接近我们的最初期望。新界面观察到的分辨率和灵敏度优于现有液体结型界面获得的分辨率和敏感性。此外，分析所需的样本量很小。这些量少于用于先前的LC/MS分析的量。这在蛋白质分析中是一个明显的优势，因为其余样品可以用于其他分析，例如氨基酸测序。在第三年（最后），我们引入了所谓的纳米喷雾电离方法，以达到更高的敏感性。为此，我们使用玻璃微注射毛细管以及使用拉式融合的二氧化硅毛细管的新微喷雾方法建立了现有的纳米喷雾方法。后一种方法使我们能够在20-50 nl/min的流速下获得稳定的电喷雾电离。因此，该方法可以与毛细管电泳充分结合。

项目成果

谷口寿章: "キャピラリーLC/MS法による蛋白質・核酸の修飾の解析" J.Mass Spectron.Soc.Japan. 44. 443-457 (1996)

Toshiaki Taniguchi：“通过毛细管 LC/MS 方法分析蛋白质和核酸的修饰”J.Mass Spectron.Soc.Japan 44. 443-457 (1996)。

Yamauchi,E.,et al.: "The C-terminal conserved domain of marcks is phosphorylated in vivo by proline-directed protein linase: application of ion trap mass spectrometry to the determination of protein phosphorylation sites." J.Biol.Chem.273. 4367-4371 (1998

Yamauchi,E.,et al.：“marcks 的 C 端保守结构域在体内被脯氨酸导向的蛋白连接酶磷酸化：应用离子阱质谱法测定蛋白磷酸化位点。”

Hayashi,N.,et al.: "Circular dichroism and ^1H nuclear magnetic resonance studies on the solution and membrane structures of Gap-43 calmodulin-binding domain." J.Biol.Chem.272. 7639-7645 (1997)

Hayashi,N.,et al.：“Gap-43 钙调蛋白结合域的溶液和膜结构的圆二色性和 ^1H 核磁共振研究。”

Yamauchi, E., Kiyonami, R., Kanai, M., and Taniguchi, H.: "Presence of conserved domains in the C-terminus of MARCKS,a major in vivo substrate of protein kinase C : application of ion trap mass spectrometry to the elucidation of protein structures" J.Bioc

Yamauchi, E.、Kiyonami, R.、Kanai, M. 和 Taniguchi, H.：“蛋白激酶 C 的主要体内底物 MARCKS C 末端存在保守结构域：离子阱质谱的应用

Usami, Y., et al.: "Primary Structure of Alboagggregin-B Purified from the Venom of Trimeresurus albolabris." Biochem. Biophys. Res. Commun.(in press). (1996)

Usami, Y. 等人：“从 Trimeresurus albolabris 毒液中纯化的 Alboagggregin-B 的主要结构”。