Accurate multiplexed proteomics at the MS2 level using the complement reporter ion cluster.
Accurate multiplexed proteomics at the MS2 level using the complement reporter ion cluster.
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DOI：
10.1021/ac301962s
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发表时间：
2012-11-06
影响因子：
7.4
通讯作者：
Gygi, Steven P.
中科院分区：
文献类型：
--
作者：
Wuehr, Martin;Haas, Wilhelm;McAlister, Graeme C.;Peshkin, Leonid;Rad, Ramin;Kirschner, Marc W.;Gygi, Steven P.
同量异位素标记策略，如相对和绝对定量同量异位素标签（iTRAQ）或串联质量标签（TMT），已承诺显着增加定量蛋白质组学的能力。然而，当应用于复杂的混合物时，准确度和精密度都受到与靶肽共分离和共片段化的干扰肽离子的破坏。额外的气相分离步骤，如质子转移离子-离子反应（PTR）或高阶MS 3扫描，几乎可以完全消除这个问题。不幸的是，这些方法以降低采集速度和灵敏度为代价。在这里，我们提出了一种方法，允许在MS 2水平上准确定量TMT标记的肽，而无需额外的离子纯化。定量是基于携带大部分TMT质量平衡的碎片离子簇。与使用低m/z报告离子相反，这些补体TMT（TMTC）离子在光谱中的定位是特异性的;共洗脱肽通常不会影响感兴趣的TMTC离子簇的测量。与PTR或MS 3策略不同，该方法可以在广泛的高分辨率质谱仪上实施，如四极Orbitrap仪器（QExactive）。目前的方法的局限性是，TMTC离子形成的效率受到肽序列和肽离子电荷状态的影响，我们讨论了潜在的途径来克服这个问题。最后，我们表明，补体报告离子的方法允许并行化的多重量化，因此有可能在给定的时间范围内，可以量化的不同的肽的数量倍增。
Isobaric labeling strategies, such as isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tags (TMT), have promised to dramatically increase the power of quantitative proteomics. However, when applied to complex mixtures, both the accuracy and precision are undermined by interfering peptide ions that coisolate and cofragment with the target peptide. Additional gas-phase isolation steps, such as proton-transfer ion–ion reactions (PTR) or higher-order MS3 scans, can almost completely eliminate this problem. Unfortunately, these methods come at the expense of decreased acquisition speed and sensitivity. Here we present a method that allows accurate quantification of TMT-labeled peptides at the MS2 level without additional ion purification. Quantification is based on the fragment ion cluster that carries most of the TMT mass balance. In contrast to the use of low m/z reporter ions, the localization of these complement TMT (TMTC) ions in the spectrum is precursor-specific; coeluting peptides do not generally affect the measurement of the TMTC ion cluster of interest. Unlike the PTR or MS3 strategies, this method can be implemented on a wide range of high-resolution mass spectrometers like the quadrupole Orbitrap instruments (QExactive). A current limitation of the method is that the efficiency of TMTC ion formation is affected by both peptide sequence and peptide ion charge state; we discuss potential routes to overcome this problem. Finally, we show that the complement reporter ion approach allows parallelization of multiplexed quantification and therefore holds the potential to multiply the number of distinct peptides that can be quantified in a given time frame.
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影响因子：
7.4
作者：
Pichler P;Köcher T;Holzmann J;Mazanek M;Taus T;Ammerer G;Mechtler K
通讯作者：
Mechtler K
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：
4.4
作者：
Ow, Saw Yen;Cardona, Tanai;Wright, Phillip C.
通讯作者：
Wright, Phillip C.