In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling
In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling
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DOI：
10.1074/mcp.m900291-mcp200
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发表时间：
2010-01-01
影响因子：
7
通讯作者：
Heck, Albert J. R.
中科院分区：
文献类型：
--
作者：
Boersema, Paul J.;Foong, Leong Yan;Heck, Albert J. R.
已经出现了几种基于质谱的测定方法，用于细胞酪氨酸磷酸化的定量分析。理想情况下，这些方法应该揭示酪氨酸磷酸化的确切位点，是定量的，并且成本不高。后者通常是一个问题，因为通常需要几毫克（稳定同位素标记的）起始蛋白质材料才能检测低丰度磷酸酪氨酸肽。在这里，我们采用并改进了以肽为中心的免疫亲和纯化方法，将其与具有成本效益的稳定同位素二甲基标记方法相结合，用于酪氨酸磷酸化的定量分析。我们仅使用两次 LC-MS/MS 运行就能通过质谱法从约 4 mg 起始材料中鉴定出 HeLa 细胞中超过 1100 种独特的非冗余磷酸肽，而无需任何进一步的亲和富集，因为所鉴定的肽中接近 80% 是酪氨酸磷酸化肽。即使对于使用大量 (mg) 的肽材料，也可以在免疫亲和纯化之前掺入稳定同位素二甲基标记，从而能够量化过钒酸盐处理或表皮生长因子刺激后酪氨酸磷酸化的差异。对表皮生长因子刺激的 HeLa 细胞（一种常用的酪氨酸磷酸化模型系统）进行分析，对 73 种受调节的独特磷酸酪氨酸肽进行了定量。发现定量数据与文献非常一致，证明这种有针对性的定量磷酸蛋白质组学方法可以提供可重复的结果。一般来说，酪氨酸磷酸化肽的免疫亲和纯化与大规模稳定同位素二甲基标记的结合提供了一种经济高效的方法，可以减轻样品制备和分析中的变化，因为样品可以尽早组合。使用这种方法，可以生成酪氨酸磷酸化信号事件的相当完整的定性和定量图像。分子与细胞蛋白质组学 9：84-99，2010。
Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated. Molecular & Cellular Proteomics 9: 84-99, 2010.