Lipid raft proteomics: Analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry
Lipid raft proteomics: Analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry
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DOI：
10.1002/pmic.200400832
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发表时间：
2004-10-01
期刊：
影响因子：
3.4
通讯作者：
Li, L
中科院分区：
文献类型：
--
作者：
Li, N;Shaw, ARE;Li, L
脂筏是富含糖脂和胆固醇的膜微区，参与膜信号传导和运输。脂筏蛋白的高度疏水性造成了溶解和回收的重大问题，这阻碍了质谱（MS）分析，并可能低估了脂筏的组成。在以前的研究单核细胞脂筏中的蛋白质消化后，用聚丙烯酰胺凝胶电泳，我们确定了52个蛋白质。在这里，我们报告了十二烷基硫酸钠（SDS）辅助的方法，其中蛋白质在溶液中消化，并通过高效液相色谱-基质辅助激光解吸/电离-串联质谱（HPLC-MALDI-MS/MS）使用一种新的LC-MALDI接口，从而避免了需要在凝胶上分离蛋白质检查的发展。使用这种方法，我们确定了71个蛋白质的脂筏，其中45个没有检测到使用凝胶内消化。在新的蛋白质中有α-和β-微管蛋白，微管蛋白特异性伴侣蛋白A，一种参与微管蛋白二聚体组装的折叠蛋白，和KIF 13，一种微管马达蛋白，表明参与微管组装和运输的蛋白质更容易使用溶液方法检测。为了研究为什么微管蛋白不能通过凝胶内消化鉴定，我们比较了在SDS-凝胶上分离之前和之后α-微管蛋白和筏标记物flotillin-2在浮力密度梯度中的分布。两种蛋白质都存在于筏级分中，但微管蛋白在SDS-凝胶上分离后选择性地丢失。因此，细胞骨架蛋白与脂筏的组装可以使用溶液中消化来解决，这将是使用基于凝胶的方法所错过的。
Lipid rafts are glycolipid- and cholesterol-enriched membrane microdomains implicated in membrane signaling and trafficking. The highly hydrophobic nature of lipid raft proteins pose significant problems of solubilization and recovery that hinder analysis by mass spectrometry (MS) and may under-report the composition of lipid rafts. In a previous investigation of the monocyte lipid raft in which proteins were digested with trypsin following polyacrylamide gel electrophoresis we identified 52 proteins. Here we report the development of a sodium dodecyl sulfate (SDS)-aided approach in which proteins are digested in solution and examined by high-performance liquid chromatography-matrix-assisted laser desorption/ionization-tandem mass spectrometry (HPLC-MALDI-MS/MS) using a novel LC-MALDI interface thereby circumventing the need to separate proteins on gels. Using this approach we identified 71 proteins in the lipid raft, 45 of which were not detected using in-gel digestion. Among the new proteins are alpha- and beta-tubulin, tubulinspecific chaperone A, a folding protein involved in tubulin dimer assembly, and KIF13, a microtubule motor protein indicating that proteins involved in microtubule assembly and trafficking are more readily detected using an in-solution approach. To investigate why tubulin was not identified by in-gel digestion, we compared the distribution of alpha-tubulin and the raft marker flotillin-2 in buoyant density gradients before and after separation on SDS-gels. Both proteins were present in the raft fractions, but tubulin was selectively lost following separation on SDS-gels. Assemblies of cytoskeletal proteins with lipid rafts may therefore be resolved using in-solution digestion that would be missed using gel-based approaches.