Tube-gel digestion - A novel proteomic approach for high throughput analysis of membrane proteins
Tube-gel digestion - A novel proteomic approach for high throughput analysis of membrane proteins
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DOI：
10.1074/mcp.m500138-mcp200
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发表时间：
2005-12-01
影响因子：
7
通讯作者：
Zhu, HN
中科院分区：
文献类型：
--
作者：
Lu, XN;Zhu, HN
这项研究描述了一种新的蛋白质消化方案，其中可以使用多种去污剂来溶解膜蛋白并以更高的效率促进胰蛋白酶消化。在此方案中，蛋白质溶解在含有各种去污剂的溶液中，并直接掺入聚丙烯酰胺凝胶基质中，无需电泳。随后，洗涤剂从凝胶基质中去除，而蛋白质仍然固定在凝胶基质中。蛋白质凝胶内消化后，使用 LC-MS/MS 分析提取的肽以进行蛋白质鉴定。该方案的独特之处在于它允许在起始溶液中使用各种去垢剂，而不会干扰 LC-MS/MS 分析。我们在此证明，不同的去污剂，包括离子 SDS、非离子 Triton X-100 和正辛基 β-D-吡喃葡萄糖苷以及两性离子 CHAPS，可用于实现膜蛋白的最大溶解，同时对 LC-MS/MS 分析的干扰最小。增强的消化，即检测到的肽的数量和强度的提高，也被证明适用于耐消化的蛋白质，例如肌红蛋白、泛素和细菌视紫红质。 Tube-Gel 消化方案的另一个优点是，即使没有电泳分离，它也可以与 LC-MS/MS 结合使用，对复杂的蛋白质混合物进行高通量分析。该方案用于分析由前列腺癌细胞制备的复杂膜蛋白混合物。该方案仅涉及一次消化和 2.5 小时的 LC-MS/MS 分析，并鉴定出 178 种膜蛋白。相比之下，通过 SDS-PAGE 解析相同的膜部分，并切下 20 个凝胶切片并通过 LC-MS/MS 单独消化和分析。更精细的工作需要超过 50 小时的 LC-MS/MS 分析并鉴定出 268 种蛋白质。新的管凝胶消化方案是膜蛋白高通量分析的替代方法。
This study describes a new protein digestion protocol in which a variety of detergents can be used to solubilize membrane proteins and facilitate trypsin digestion with higher efficiency. In this protocol, proteins are dissolved in solutions containing various detergents and directly incorporated into a polyacrylamide gel matrix without electrophoresis. Detergents are subsequently eliminated from the gel matrix while proteins are still immobilized in the gel matrix. After in-gel digestion of proteins, LC-MS/MS is used to analyze the extracted peptides for protein identification. The uniqueness of the protocol is that it allows usage of a variety of detergents in the starting solution without interfering with LC-MS/MS analysis. We hereby demonstrate that different detergents, including ionic SDS, non-ionic Triton X-100 and n-octyl beta-D-glucopyranoside, and zwitterionic CHAPS, can be used to achieve maximum solubilization of membrane proteins with minimal interference with LC-MS/MS analysis. Enhanced digestions, i.e. improved number and intensity of detected peptides, are also demonstrated for digestion-resistant proteins such as myoglobin, ubiquitin, and bacteriorhodopsin. An additional advantage of the Tube-Gel digestion protocol is that, even without electrophoresis separation, it allows high throughput analysis of complex protein mixtures when coupled with LC-MS/MS. The protocol was used to analyze a complex membrane protein mixture prepared from prostate cancer cells. The protocol involves only a single digestion and 2.5 h of LC-MS/MS analysis and identified 178 membrane proteins. In comparison, the same membrane fraction was resolved by SDS-PAGE, and 20 gel slices were excised and individually digested and analyzed by LC-MS/MS. The more elaborate effort demanded more than 50 h of LC-MS/MS analysis and identified 268 proteins. The new Tube-Gel digestion protocol is an alternative method for high throughput analysis of membrane proteins.