Integrating titania enrichment, iTRAQ labeling, and Orbitrap CID-HCD for global identification and quantitative analysis of phosphopeptides
Integrating titania enrichment, iTRAQ labeling, and Orbitrap CID-HCD for global identification and quantitative analysis of phosphopeptides
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DOI：
10.1002/pmic.200900788
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发表时间：
2010-06-01
期刊：
影响因子：
3.4
通讯作者：
He, Tao
中科院分区：
文献类型：
--
作者：
Wu, Jiang;Warren, Peter;He, Tao
MS仪器的最新进展和磷酸肽富集的进展，结合更强大的数据分析工具，促进了数千个位点特异性磷酸化事件的公正表征。结合细胞培养代谢标记中的氨基酸稳定同位素标记，这些技术使得定量评估稳定细胞系中各种生理状态下的磷酸化变化成为可能。然而，在原代细胞和组织中的定量磷酸化蛋白质组学仍然是一个主要的技术挑战，由于缺乏足够的技术进行准确的定量。在这里，我们描述了一个综合的战略，允许大规模的定量分析磷酸肽在复杂的生物混合物。在该技术中，使用二氧化钛亲和柱对蛋白水解肽的混合物进行磷酸肽富集，随后用iTRAQ试剂标记纯化的磷酸肽。通过强阳离子交换进一步分级分离后，在Orbitrap质谱仪上通过LC-MS/MS分析肽，该质谱仪依次收集CID和高能碰撞解离（HCD）光谱用于肽鉴定和定量。我们证明，直接磷酸肽富集蛋白质组学的二氧化钛亲和层析显着提高磷酸肽蛋白质组学分析的效率和重现性，是兼容的下游iTRAQ标记。优化HCD标准化碰撞能量的条件，以使用iTRAQ报告离子的相对丰度平衡总体肽鉴定和定量。使用这种方法，我们能够确定3557个不同的磷酸肽从HeLa细胞裂解物，其中2709也从HCD扫描定量。
Recent advances in MS instrumentation and progresses in phosphopeptide enrichment, in conjunction with more powerful data analysis tools, have facilitated unbiased characterization of thousands of site-specific phosphorylation events. Combined with stable isotope labeling by amino acids in cell culture metabolic labeling, these techniques have made it possible to quantitatively evaluate phosphorylation changes in various physiological states in stable cell lines. However, quantitative phosphoproteomics in primary cells and tissues remains a major technical challenge due to the lack of adequate techniques for accurate quantification. Here, we describe an integrated strategy allowing for large scale quantitative profiling of phosphopeptides in complex biological mixtures. In this technique, the mixture of proteolytic peptides was subjected to phosphopeptide enrichment using a titania affinity column, and the purified phosphopeptides were subsequently labeled with iTRAQ reagents. After further fractionation by strong-cation exchange, the peptides were analyzed by LC-MS/MS on an Orbitrap mass spectrometer, which collects CID and high-energy collisional dissociation (HCD) spectra sequentially for peptide identification and quantitation. We demonstrate that direct phosphopeptide enrichment of protein digests by titania affinity chromatography substantially improves the efficiency and reproducibility of phosphopeptide proteomic analysis and is compatible with downstream iTRAQ labeling. Conditions were optimized for HCD normalized collision energy to balance the overall peptide identification and quantitation using the relative abundances of iTRAQ reporter ions. Using this approach, we were able to identify 3557 distinct phosphopeptides from HeLa cell lysates, of which 2709 were also quantified from HCD scans.