化学
串联质谱法
色谱法
蛋白质组学
质谱法
电喷雾
洗脱
蛋白质-蛋白质相互作用
串联
自上而下的蛋白质组学
选择性反应监测
生物物理学
生物化学
基因
生物
复合材料
材料科学
作者
Tohru Natsume,Yoshio Yamauchi,Hiroshi Nakayama,Takashi Shinkawa,Mitsuaki Yanagida,Nobuhiro Takahashi,Toshiaki Isobe
摘要
One of the strategies of functional proteomics, research aiming to discover gene function at the protein level, is the comprehensive analysis of protein-protein interactions related to the functional linkage among proteins and analysis of functional cellular machinery to better understand the basis of cell functions. Here, we describe the direct nanoflow LC (DNLC) system, which is equipped with a fritless high-resolution electrospray interface column packed with 1-microm reversed-phase (RP) beads and a novel splitless nanoflow gradient elution system to operate the column. Using RP-DNLC at an extremely slow flow rate, <50 nL/min, combined with data-dependent collision-induced dissociation tandem MS (MS/MS) and computer-assisted retrieval of spectra, we identified approximately 100 protein components in a biological complex such as a premature mammalian ribosome pull-down from cultured cells when we used an epitope-tagged protein as bait. Because this analysis is most sensitive, requires approximately 0.2 microg of total protein, and is a fully automated 1-h process, we anticipated that it should be an excellent tool for analyzing a limited amount of functional multi-protein complexes in cells.
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