BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow
BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow
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BAC-DROP：通过可溶性聚丙烯酰胺凝胶电泳快速消化分级蛋白质组及其在自下而上蛋白质组学工作流程中的应用
DOI：
10.1021/acs.jproteome.0c00749
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发表时间：
2020
影响因子：
4.4
通讯作者：
Takemori Nobuaki
中科院分区：
文献类型：
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作者：
Takemori Ayako;Ishizaki Jun;Nakashima Kenji;Shibata Takeshi;Kato Hidemasa;Kodera Yoshio;Suzuki Tetsuro;Hasegawa Hitoshi;Takemori Nobuaki
GeLC-MS工作流程将低成本、易于使用的十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（SDS-PAGE）与液相色谱-质谱（LC-MS）相结合，在当前自下而上的蛋白质组学中非常流行。然而，GeLC-MS要求PAGE分离的蛋白质在凝胶中进行过夜酶消化，导致LC-MS的样品制备超过20小时。在这项研究中，我们克服了GeLC-MS的局限性，开发了一种快速消化的工作流程，使用N，N ′-双（丙烯酰基）胱胺（BAC）交联凝胶，可以通过还原处理增溶蛋白质的PAGE分离。利用被称为BAC-DROP（BAC-凝胶溶解以消化PAGE解析的目标蛋白质）的既定工作流程，通过BAC交联PAGE基于分子量对粗蛋白质组样品进行分级。分级分离后，在5分钟内将凝胶片段还原溶解，并且在70 ° C下在不到1小时内完成从凝胶释放的蛋白质的溶液内胰蛋白酶消化，相当于与常规的凝胶内胰蛋白酶消化相比减少了90 - 95%的时间。将BAC-DROP工作流程引入MS测定中，用于炎症生物标志物CRP和病毒标志物HBsAg，可在短短5小时内完成血清样品制备，证明成功定量了0.5 μ L人血清样品中的标志物。
The GeLC–MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography–mass spectrometry (LC–MS), is very popular in current bottom-up proteomics. However, GeLC–MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 h of sample preparation for LC–MS. In this study, we overcame the limitations of GeLC–MS by developing a rapid digestion workflow for PAGE separation of proteins usingN,N′-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 min, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 h at 70 °C, equivalent to a 90–95% reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 h, demonstrating successful marker quantification from a 0.5 μL sample of human serum.