Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research
Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research
复制标题
DOI：
10.1002/pmic.200790019
复制
发表时间：
2007
期刊：
影响因子：
3.4
通讯作者：
S. Wiese;K. Reidegeld;H. Meyer;B. Warscheid
中科院分区：
文献类型：
--
作者：
S. Wiese;K. Reidegeld;H. Meyer;B. Warscheid
提出了一种基于MS的蛋白质相对定量新方法，该方法依赖于使用同量异位素标签对完整蛋白质中的伯氨基进行衍生化，以进行相对和绝对定量（iTRAQ）。由于iTRAQ试剂的同量异位素质量设计，差异标记的蛋白质在质量上没有差异;因此，其相应的蛋白水解肽在MS扫描中显示为单峰。由于定量信息由同位素编码的报告离子提供，只能在MS/MS光谱中观察到，因此我们使用TOF/TOF和/或QTOF仪器分析了iTRAQ标记蛋白质生成的肽的ESI和MALDI离子的裂解行为。我们观察到在低能碰撞条件下单质子化肽的报告离子的有效释放。相比之下，需要增加碰撞能量以从双电荷肽的赖氨酸侧链释放iTRAQ标记，从而观察适合于以高准确度相对定量蛋白质的报告离子。然后，我们开发了一种定量策略，包括通过iTRAQ标记完整蛋白，然后进行凝胶电泳和肽MS/MS分析。作为原理证明，对不同浓度比的五种不同蛋白质的混合物进行了定量，证明了本文所述方法对定量基于MS的蛋白质组学的普遍适用性。
A novel, MS‐based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope‐encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ‐labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low‐energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS‐based proteomics.