Cellzome GmbH, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
J Proteome Res. 2013 Aug 2;12(8):3586-98. doi: 10.1021/pr400098r. Epub 2013 Jul 2.
Isobaric mass tagging (e.g., TMT and iTRAQ) is a precise and sensitive multiplexed peptide/protein quantification technique in mass spectrometry. However, accurate quantification of complex proteomic samples is impaired by cofragmentation of peptides, leading to systematic underestimation of quantitative ratios. Label-free quantification strategies do not suffer from such an accuracy bias but cannot be multiplexed and are less precise. Here, we compared protein quantification results obtained with these methods for a chemoproteomic competition binding experiment and evaluated the utility of measures of spectrum purity in survey spectra for estimating the impact of cofragmentation on measured TMT-ratios. While applying stringent interference filters enables substantially more accurate TMT quantification, this came at the expense of 30%-60% fewer proteins quantified. We devised an algorithm that corrects experimental TMT ratios on the basis of determined peptide interference levels. The quantification accuracy achieved with this correction was comparable to that obtained with stringent spectrum filters but limited the loss in coverage to <10%. The generic applicability of the fold change correction algorithm was further demonstrated by spiking of chemoproteomics samples into excess amounts of E. coli tryptic digests.
等压质量标记（例如，TMT 和 iTRAQ）是一种在质谱中精确且灵敏的多肽/蛋白质定量技术。然而，肽的共碎裂会损害复杂蛋白质组样品的准确定量，导致定量比的系统低估。无标记定量策略不会受到这种准确性偏差的影响，但不能进行多重定量，且精度较低。在这里，我们比较了这些方法在化学蛋白质组学竞争结合实验中获得的蛋白质定量结果，并评估了谱纯度测量值在调查谱中用于估计共碎裂对测定的 TMT 比值的影响的效用。虽然应用严格的干扰过滤器可以实现更准确的 TMT 定量，但这会导致定量的蛋白质减少 30%-60%。我们设计了一种算法，根据确定的肽干扰水平来校正实验 TMT 比值。这种校正所达到的定量准确性与严格的谱过滤器相当，但将覆盖率的损失限制在<10%。通过将化学蛋白质组学样品掺入过量的大肠杆菌胰蛋白酶消化物中，进一步证明了折叠变化校正算法的通用适用性。
