Peptide labeling with isobaric tags yields higher identification rates using iTRAQ 4-plex compared to TMT 6-plex and iTRAQ 8-plex on LTQ Orbitrap.
Peptide labeling with isobaric tags yields higher identification rates using iTRAQ 4-plex compared to TMT 6-plex and iTRAQ 8-plex on LTQ Orbitrap.
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DOI：
10.1021/ac100890k
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发表时间：
2010-08-01
影响因子：
7.4
通讯作者：
Mechtler K
中科院分区：
文献类型：
--
作者：
Pichler P;Köcher T;Holzmann J;Mazanek M;Taus T;Ammerer G;Mechtler K
使用同量异序标签进行肽标记已成为定量鸟枪法蛋白质组学中的流行技术。使用两个不同的样本，即。通过比较蛋白质混合物和 HeLa 提取物，我们发现三种市售同量异位标签在肽识别率方面存在差异：iTRAQ 4-plex 识别的蛋白质和肽数量最多，其次是 TMT 6-plex，iTRAQ 8-plex 识别的蛋白质和肽数量最少。在所有实验中，我们采用了先前描述的方法，其中在 LTQ Orbitrap 上为每个前体采集两次扫描：标准设置下的 CID 扫描用于识别，HCD 扫描用于定量。当使用 Mascot 或 Sequest 搜索数据时，观察到的识别率差异相似。我们认为这些发现是多种因素综合作用的结果，最显着的是 CID 光谱中突出的离子，这是由于前体离子中标签碎片丢失的结果。当前的搜索引擎无法解释这些碎片离子，并且观察到这些碎片离子对肽分数有负面影响。
Peptide labeling with isobaric tags has become a popular technique in quantitative shotgun proteomics. Using two different samples viz. a protein mixture and HeLa extracts, we show that three commercially available isobaric tags differ with regard to peptide identification rates: The number of identified proteins and peptides was largest with iTRAQ 4-plex, followed by TMT 6-plex, and smallest with iTRAQ 8-plex. In all experiments, we employed a previously described method where two scans were acquired for each precursor on an LTQ Orbitrap: A CID scan under standard settings for identification, and a HCD scan for quantification. The observed differences in identification rates were similar when data was searched with either Mascot or Sequest. We consider these findings to be the result of a combination of several factors, most notably prominent ions in CID spectra as a consequence of loss of fragments of the label tag from precursor ions. These fragment ions cannot be explained by current search engines and were observed to have a negative impact on peptide scores.
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