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Reagents for Isobaric Labeling Peptides in Quantitative Proteomics
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-09-27 00:00:00 , DOI: 10.1021/acs.analchem.8b00321 Yan Ren 1 , Yanbin He 1 , Zhilong Lin 1 , Jin Zi 1 , Huanming Yang 1, 2 , Shenyan Zhang 1 , Xiaomin Lou 3 , Quanhui Wang 4 , Shuwei Li 5 , Siqi Liu 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-09-27 00:00:00 , DOI: 10.1021/acs.analchem.8b00321 Yan Ren 1 , Yanbin He 1 , Zhilong Lin 1 , Jin Zi 1 , Huanming Yang 1, 2 , Shenyan Zhang 1 , Xiaomin Lou 3 , Quanhui Wang 4 , Shuwei Li 5 , Siqi Liu 1
Affiliation
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Currently, the commercial reagents for isobaric peptides labeling (TMT and iTRAQ) have some drawbacks, such as high cost in experiments, especially in quantitation for the modified peptides, and inconvenient handling for variable sizes of samples. Herein, we developed a set of 10-plex isobaric tags (IBT) with high stability and low cost. The labeled peptides were sensitively detected on Orbitrap Q Exactive MS with an MS2 resolution of 35 000 at 30% NCE, while the peptides were efficiently labeled over 97% by IBT at a ratio of 10:1 of reagent/peptide (w/w) in 200 mM TEAB buffer for 2 h. The IBT labeling was demonstrated with a wide dynamic range of 50-fold without obvious matrix effects on quantification. Importantly, there was little quantification bias found among the individual IBT tags, indicating that the peptides labeled by different tags were quantitatively comparable. The IBT 10-plex reagents were applied for dynamically monitoring the quantitative responses of phosphoproteome stimulated by EGF treatment in HeLa cells. In total, 5 361 unique phosphopeptides were identified, which reached a similar conclusion as others reported. The IBT reagents were therefore experimentally proven as a new type of reagents for isobaric peptides labeling and useful in a large quantity peptides of quantitative proteomics.
中文翻译：
定量蛋白质组学中等压标记肽的试剂
当前，用于同量异位肽标记的商业试剂（TMT和iTRAQ）具有一些缺点，例如实验成本高，尤其是修饰肽的定量，以及不方便处理可变大小的样品。在这里，我们开发了一组具有高稳定性和低成本的10重等压标签（IBT）。在Orbitrap Q Exactive MS上以30％的NCE在MS2分辨率为35000的条件下灵敏地检测到标记的肽，而IBT则以10：1的试剂/肽（w / w）比率有效地标记了超过97％的肽在200 mM TEAB缓冲液中放置2小时。证明了IBT标记具有50倍的宽动态范围，对定量没有明显的基质影响。重要的是，单个IBT标签之间几乎没有量化偏差，表明用不同标签标记的肽在数量上是可比的。IBT 10-plex试剂用于动态监测HeLa细胞中EGF处理刺激的磷酸化蛋白质组的定量反应。总共鉴定出5 361种独特的磷酸肽，得出的结论与其他报道相似。因此，IBT试剂在实验上被证明是用于等压肽标记的新型试剂，可用于大量的定量蛋白质组学肽。
更新日期：2018-09-27
中文翻译：
定量蛋白质组学中等压标记肽的试剂
当前，用于同量异位肽标记的商业试剂（TMT和iTRAQ）具有一些缺点，例如实验成本高，尤其是修饰肽的定量，以及不方便处理可变大小的样品。在这里，我们开发了一组具有高稳定性和低成本的10重等压标签（IBT）。在Orbitrap Q Exactive MS上以30％的NCE在MS2分辨率为35000的条件下灵敏地检测到标记的肽，而IBT则以10：1的试剂/肽（w / w）比率有效地标记了超过97％的肽在200 mM TEAB缓冲液中放置2小时。证明了IBT标记具有50倍的宽动态范围，对定量没有明显的基质影响。重要的是，单个IBT标签之间几乎没有量化偏差，表明用不同标签标记的肽在数量上是可比的。IBT 10-plex试剂用于动态监测HeLa细胞中EGF处理刺激的磷酸化蛋白质组的定量反应。总共鉴定出5 361种独特的磷酸肽，得出的结论与其他报道相似。因此，IBT试剂在实验上被证明是用于等压肽标记的新型试剂，可用于大量的定量蛋白质组学肽。
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