Improved precision of iTRAQ and TMT quantification by an axial extraction field in an Orbitrap HCD cell.
Improved precision of iTRAQ and TMT quantification by an axial extraction field in an Orbitrap HCD cell.
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DOI：
10.1021/ac102265w
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发表时间：
2011-02-15
影响因子：
7.4
通讯作者：
Mechtler, Karl
中科院分区：
文献类型：
--
作者：
Pichler, Peter;Koecher, Thomas;Holzmann, Johann;Moehring, Thomas;Ammerer, Gustav;Mechtler, Karl
提高分析精度是定量差异蛋白质组学的一个主要目标，因为高精度可确保异常值数量较少，而异常值是定量方面的误报来源。此外，更高的精度会增加统计功效，即检测到显着差异的概率。通过使用同量异位标签进行相对和绝对定量 (iTRAQ) 或串联质量标签 (TMT) 试剂的化学标记，定量基于从串联质谱 (MS/MS) 谱中提取报告离子。我们比较了两个版本的 LTQ Orbitrap 高能碰撞解离 (HCD) 池在报告离子定量方面有和没有轴向电场的性能。具有轴向电场的 HCD 单元旨在将碎片离子推入 C 型陷阱，该版本安装在当前的 Orbitrap XL ETD 和 Orbitrap Velos 仪器中。我们的目标是评估所谓的离子传输改进是否对使用同量异位标签进行肽标记的基于 MS/MS 的定量精度产生可测量的影响。我们表明，轴向电场导致 HCD 谱的百分比增加，其中检测到完整的报告离子集，更重要的是，导致总体方差的减少，即提高了所获取数据的分析精度。值得注意的是，即使对于复杂生物样品的低前体离子强度，基于 HCD 的定量也能保持足够的精度。这些发现可能有助于研究人员使用同量异位标签设计定量蛋白质组学研究，并在 LTQ Orbitrap 上建立基于 HCD 的定量方法，作为定量蛋白质组学的高精度方法。
Improving analytical precision is a major goal in quantitative differential proteomics as high precision ensures low numbers of outliers, a source of false positives with regard to quantification. In addition, higher precision increases statistical power, i.e., the probability to detect significant differences. With chemical labeling using isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tag (TMT) reagents, quantification is based on the extraction of reporter ions from tandem mass spectrometry (MS/MS) spectra. We compared the performance of two versions of the LTQ Orbitrap higher energy collisional dissociation (HCD) cell with and without an axial electric field with regard to reporter ion quantification. The HCD cell with the axial electric field was designed to push fragment ions into the C-trap and this version is mounted in current Orbitrap XL ETD and Orbitrap Velos instruments. Our goal was to evaluate whether the purported improvement in ion transmission had a measurable impact on the precision of MS/MS based quantification using peptide labeling with isobaric tags. We show that the axial electric field led to an increased percentage of HCD spectra in which the complete set of reporter ions was detected and, even more important, to a reduction in overall variance, i.e., improved analytical precision of the acquired data. Notably, adequate precision of HCD-based quantification was maintained even for low precursor ion intensities of a complex biological sample. These findings may help researchers in their design of quantitative proteomics studies using isobaric tags and establish HCD-based quantification on the LTQ Orbitrap as a highly precise approach in quantitative proteomics.
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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：
14.8
作者：
Ong, Shao-En;Mann, Matthias
通讯作者：
Mann, Matthias
DOI：
10.1016/s1044-0305(02)00384-7
发表时间：
2002-06-01
影响因子：
3.2
作者：
Schwartz, JC;Senko, MW;Syka, JEP
通讯作者：
Syka, JEP
DOI：
10.1016/j.jasms.2009.03.019
发表时间：
2009-08-01
影响因子：
3.2
作者：
Zhang, Yi;Ficarro, Scott B.;Marto, Jarrod A.
通讯作者：
Marto, Jarrod A.
影响因子：
7
作者：
Ross, PL;Huang, YLN;Pappin, DJ
通讯作者：
Pappin, DJ