A strategy for identification and quantitation of phosphopeptides by liquid chromatography/tandem mass spectrometry
A strategy for identification and quantitation of phosphopeptides by liquid chromatography/tandem mass spectrometry
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DOI：
10.1006/abio.2000.4837
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发表时间：
2000-12-01
影响因子：
2.9
通讯作者：
Lee, SC
中科院分区：
文献类型：
--
作者：
Tsay, YG;Wang, YH;Lee, SC
液相色谱/串联质谱（LC/MS/MS）是一种最先进的肽/蛋白质结构分析方法。在这里，以激活转录因子-2 （ATF2）为例，我们报告了如何处理LC/MS/MS数据来生成选择的离子示踪，用于基于磷酸化肽与其非磷酸化类似物的平行洗脱行为来鉴定磷酸化肽。通过这种方法，我们验证了ATFS的氨基酸残基Thr-69、Thr-71和Ser-90是c-Jun激酶的体外靶点。选择离子示踪法定量测定肽的磷酸化状态。我们证明了Thr-69/Thr-71的磷酸化在紫外线照射下增加，特别是在亚融合培养中，而不是在融合培养中。约24%的含Thr-69/ thr -71的片段在亚融合培养中被单一磷酸化，而在融合培养中则发生少量磷酸化。相反，Ser-112磷酸化不受细胞密度的影响。这种策略可以应用于研究各种调节细胞过程中看到的各种修饰。(C) 2000年学术出版社。
Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is a state-of-the-art method of structural analysis of peptides/proteins. Here, using activating transcription factor-2 (ATF2) as an example, we report how LC/MS/MS data were processed to generate selected ion tracings for identification of phosphorylated peptides based on their parallel elution behavior with their nonphosphorylated analogs. Via this approach, we verified that amino acid residues Thr-69, Thr-71, and Ser-90 of ATFS were the in vitro targets for c-Jun kinase. Selected ion tracing method was also used to quantitatively determine phosphorylation states of peptides. We demonstrated that the phosphorylation of Thr-69/Thr-71 was increased in response to ultraviolet irradiation specifically in subconfluent but not in confluent cultures. About 24% of Thr-69/Thr-71-containing segment were singly phosphorylated in subconfluent cultures, while minimal phosphorylation occurred in confluent cultures. In contrast, Ser-112 phosphorylation remained unaffected by cell densities. This strategy could be applied to the studies of a variety of modifications seen in various regulated cellular processes. (C) 2000 Academic Press.