オーラルセッション
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2C-O7-1025（2P-29） PDF
ゲル支援LCMSにおける高速・多検体プロテオーム解析法EasyGelの開発
SDS-PAGE is an analytical technique that separates proteins based on their molecular masses in an electric field. GeLCMS (Gel-enhanced LC/MS/MS) has been widely used as a shotgun proteomic approach and involves slicing the SDS-PAGE gel into multiple pieces, digesting proteins into peptides in gel and analyzing peptides from individual gel pieces by LC/MS/MS. In theory, protein-level fractionation can more effectively reduce the complexity and wide dynamic range of proteome samples rather than peptide-level fractionation. Therefore, GeLCMS is, in principle, a powerful approach to achieve protein-level separation with high-resolving power and the deeper proteome coverage. However, it is necessary to remove SDS which hinders trypsin activity and mass spectrometric detection of the peptides, resulting in repeating multiple washing and dehydration steps. Here, we have developed a new high-throughput GeLCMS platform, called EasyGel, which uses MS-compatible detergents that do not inhibit trypsin activity, allowing us to simplify the in-gel digestion protocol without the requirement of washing and dehydration steps. Using EasyGel, we demonstrated that bovine serum albumin (BSA) can be successfully separated based on its molecular mass and digested in gel and that the identification numbers of BSA peptides and the recovery rate was comparable between EasyGel and the conventional GeLCMS method.