Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Mol Cell Proteomics. 2012 Jun;11(6):M111.014423. doi: 10.1074/mcp.M111.014423. Epub 2011 Dec 30.
Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ™ or TMT™ allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, nonisobaric labeling with mTRAQ™ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of nonregulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification.
将含有稳定同位素的试剂标记到肽上的一级胺是定量质谱中常用的技术。等重标记技术（如 iTRAQ™ 或 TMT™）允许根据在通过前体离子碎裂产生的光谱的低 m/z 区域中的报告离子的比率来相对定量肽。相比之下，mTRAQ™ 的非等重标记会产生具有不同质量的前体，可以在 MS1 光谱中直接定量。在这项研究中，我们比较了基于 iTRAQ 和 mTRAQ 的来自 EGF 刺激的 HeLa 细胞的肽和磷酸肽的定量。这两种标签具有相同的化学结构，因此基于前体离子和碎片离子的定量可以直接比较。我们的结果表明，iTRAQ 标记对前体强度具有加性效应，而 mTRAQ 标记会导致由于用不同的 mTRAQ 标签触发相同的肽而导致更多冗余的 MS2 扫描事件。我们发现，iTRAQ 标记定量的磷酸肽（12,129 对 4,448）和蛋白质（2,699 对 1,597）几乎分别是 mTRAQ 标记的三倍和两倍。尽管两种技术都可以定量 EGFR 信号网络中的大多数关键蛋白，但 iTRAQ 标记可以定量两倍的激酶。iTRAQ 通过报告离子定量的准确性受到在相同前体分离窗口中共同碎裂的肽的影响，使观察到的比率向 1 靠拢。然而，由于 iTRAQ 比值分布更紧密，通过 iTRAQ 和 mTRAQ 检测到的统计学上有显著调节的磷酸肽和蛋白质的百分比相似。我们观察到对数 iTRAQ 与 mTRAQ 比值在两个数量级上的线性相关性，表明可以通过平均压缩因子来校正 iTRAQ 比值。使用具有定义比率的肽在非调节肽的背景下进行的 Spike-in 实验表明，iTRAQ 定量的准确性较低，但不如 mTRAQ 定量的变化大。
