Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses.
Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses.
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DOI：
10.1021/ac301572t
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发表时间：
2012-09-04
影响因子：
7.4
通讯作者：
Gygi, Steven P.
中科院分区：
文献类型：
--
作者：
McAlister, Graeme C.;Huttlin, Edward L.;Haas, Wilhelm;Ting, Lily;Jedrychowski, Mark P.;Rogers, John C.;Kuhn, Karsten;Pike, Ian;Grothe, Robert A.;Blethrow, Justin D.;Gygi, Steven P.
定量质谱法提供近乎全面的蛋白质组覆盖；然而，这些方法在样本吞吐量方面仍然受到影响。通过等压化学标记（例如，TMT和iTRAQ）进行多重定量为基于质谱的蛋白质组定量实验提供了一条途径，使其从简单的二元比较转向更大的并行化。在这里，我们展示了一种直接的方法，可以立即将TMT等压试剂的吞吐量从6-plex扩大到8-plex。这种方法是基于我们解决了用15N代替13C所导致的同位素位移的能力。为了适应ETD的首选裂解途径，TMT-127和-129试剂最近进行了修改，将13C换成了15N。由于这种取代，新的TMT报告离子轻了6.32 mDa。尽管这些报告离子同位素物之间的质量差异非常小，但现代高分辨率和质量精度分析仪可以分辨这些离子。基于我们解析和精确测量这些等压报告离子的相对强度的能力，我们证明我们能够通过结合含有13C和15N的报告离子同时对8个样品进行量化。考虑到TMT报告离子的结构，我们相信这项工作可以作为将TMT试剂的多路复用能力扩展到至少10-plex甚至可能高达18-plex的蓝图。
Quantitative mass spectrometry methods offer near-comprehensive proteome coverage; however, these methods still suffer with regards to sample throughput. Multiplex quantitation via isobaric chemical tags (e.g., TMT and iTRAQ) provides an avenue for mass spectrometry based proteome quantitation experiments to move away from simple binary comparisons and towards greater parallelization. Herein, we demonstrate a straightforward method for immediately expanding the throughput of the TMT isobaric reagents from 6-plex to 8-plex. This method is based upon our ability to resolve the isotopic shift that results from substituting a 15N for a 13C. In an accommodation to the preferred fragmentation pathways of ETD, the TMT-127 and -129 reagents were recently modified such that a 13C was exchanged for a 15N. As a result of this substitution, the new TMT reporter ions are 6.32 mDa lighter. Even though the mass difference between these reporter ion isotopologues is incredibly small, modern high-resolution and mass accuracy analyzers can resolve these ions. Based on our ability to resolve and accurately measure the relative intensity of these isobaric reporter ions, we demonstrate that we are able to quantify across 8 samples simultaneously by combining the 13C and 15N containing reporter ions. Considering the structure of the TMT reporter ion, we believe this work serves as a blueprint for expanding the multiplexing capacity of the TMT reagents to at least 10-plex and possibly up to 18-plex.
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影响因子：
9.9
作者：
通讯作者：
--
影响因子：
64.5
作者：
Huttlin EL;Jedrychowski MP;Elias JE;Goswami T;Rad R;Beausoleil SA;Villén J;Haas W;Sowa ME;Gygi SP
通讯作者：
Gygi SP
影响因子：
7.3
作者：
Dephoure N;Gygi SP
通讯作者：
Gygi SP
影响因子：
3.4
作者：
Boersema, Paul J.;Aye, Thin Thin;Mohammed, Shabaz
通讯作者：
Mohammed, Shabaz
DOI：
10.1016/j.jasms.2009.01.005
发表时间：
2009-08-01
影响因子：
3.2
作者：
Makarov, Alexander;Denisov, Eduard;Lange, Oliver
通讯作者：
Lange, Oliver