A Direct Nanoflow Liquid Chromatography-Tandem Mass Spectrometry System for Interaction Proteomics
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- 作者：Tohru Natsume ; Yoshio Yamauchi ; Hiroshi Nakayama ; Takashi Shinkawa ; Mitsuaki Yanagida ; Nobuhiro Takahashi ; and Toshiaki Isobe
- 刊名：Analytical Chemistry
- 出版年：2002
- 出版时间：September 15, 2002
- 年：2002
- 卷：74
- 期：18
- 页码：4725 - 4733
- 全文大小：235K
- 年卷期：v.74,no.18(September 15, 2002)
- ISSN：1520-6882
文摘
One of the strategies of functional proteomics, researchaiming to discover gene function at the protein level, isthe comprehensive analysis of protein-protein interactions related to the functional linkage among proteins andanalysis of functional cellular machinery to better understand the basis of cell functions. Here, we describe thedirect nanoflow LC (DNLC) system, which is equippedwith a fritless high-resolution electrospray interface column packed with 1-
m reversed-phase (RP) beads and anovel splitless nanoflow gradient elution system to operatethe column. Using RP-DNLC at an extremely slow flowrate, <50 nL/min, combined with data-dependent collision-induced dissociation tandem MS (MS/MS) and computer-assisted retrieval of spectra, we identified ~100protein components in a biological complex such as apremature mammalian ribosome pull-down from culturedcells when we used an epitope-tagged protein as bait.Because this analysis is most sensitive, requires ~0.2 gof total protein, and is a fully automated 1-h process, weanticipated that it should be an excellent tool for analyzinga limited amount of functional multi-protein complexesin cells.
m reversed-phase (RP) beads and anovel splitless nanoflow gradient elution system to operatethe column. Using RP-DNLC at an extremely slow flowrate, <50 nL/min, combined with data-dependent collision-induced dissociation tandem MS (MS/MS) and computer-assisted retrieval of spectra, we identified ~100protein components in a biological complex such as apremature mammalian ribosome pull-down from culturedcells when we used an epitope-tagged protein as bait.Because this analysis is most sensitive, requires ~0.2 gof total protein, and is a fully automated 1-h process, weanticipated that it should be an excellent tool for analyzinga limited amount of functional multi-protein complexesin cells.