Improved protease digestion conditions for membrane protein detection.
Improved protease digestion conditions for membrane protein detection.
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改善了膜蛋白检测的蛋白酶消化条件。
DOI：
10.1002/elps.201400579
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发表时间：
2015-08
期刊：
影响因子：
2.9
通讯作者：
Lee KH
中科院分区：
文献类型：
--
作者：
Min L;Choe LH;Lee KH
这项工作提出了改进的蛋白酶消化条件，膜蛋白检测。研究了具有7个跨膜结构域（TMDs）的模型膜蛋白细菌视紫红质（BR）的酶解过程。最初的凝胶内消化鉴定出17%的BR序列覆盖率，包括第七TMD的一部分。为了提高序列覆盖率，用不同浓度的RapiGest、甲醇（MeOH）和SDS（胰酶或凝乳胰酶）检测BR消化酶。选择0.01% SDS和10% MeOH + 0.01% RapiGest两种改良条件。两种条件下胰蛋白酶消化的BR序列覆盖率均超过40%，而标准消化条件下为0%。在相同条件下，从胰蛋白酶和胰凝乳酶消化中检测到的肽被组合在一起以最大化序列覆盖率。同样的条件应用于不同的膜蛋白，其中含有一种TMD，硒蛋白S和大肠杆菌蛋白。对于硒蛋白S，与典型条件相比，改进条件下检测到更高的序列覆盖率，包括来自TMD的肽。将这两种改良条件应用于大肠杆菌的膜蛋白片段，鉴定出309个（SDS）和329个（MeOH/RapiGest）独特蛋白，其中140/309和148/329为膜蛋白。
This work presents improved protease digestion conditions for membrane protein detection. The enzymatic digest of bacteriorhodopsin (BR), a model membrane protein with seven transmembrane domains (TMDs) was investigated. An initial in-gel digestion identified 17% BR sequence coverage, including part of the seventh TMD. To improve sequence coverage, BR digest was tested with different concentrations of RapiGest, methanol (MeOH) and SDS using either trypsin or chymotrypsin. Two improved conditions, 0.01% SDS or the combination of 10% MeOH and 0.01% RapiGest, were chosen. Trypsin digestions in both conditions achieved more than 40% BR sequence coverage compared to 0% using standard digestion conditions. Peptides detected from trypsin and chymotrypsin digestions in the same condition were combined to maximize sequence coverage. The same conditions were applied to a different membrane protein with one TMD, Selenoprotein S, and proteins from Escherichia coli. For Selenoprotein S, a higher sequence coverage, including a peptide from the TMD, was detected from the improved condition compared to the typical condition. The application of both improved conditions to a membrane protein fraction of Escherichia coli resulted in the identification of 309 (SDS) and 329 (MeOH/RapiGest) unique proteins of which 140/309 and 148/329 were membrane proteins.