ICPMS-Based Specific Quantification of Phosphotyrosine: A Gallium Tagging and Tyrosine-Phosphatase Mediated Strategy
ICPMS-Based Specific Quantification of Phosphotyrosine: A Gallium Tagging and Tyrosine-Phosphatase Mediated Strategy
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基于 ICPMS 的磷酸酪氨酸特异性定量：镓标记和酪氨酸磷酸酶介导的策略
DOI：
10.1021/acs.analchem.6b02979
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发表时间：
2016
影响因子：
7.4
通讯作者：
Wang Qiuquan
中科院分区：
文献类型：
--
作者：
Tang Nannan;Li Zhaoxin;Yang Limin;Wang Qiuquan
Low-abundance tyrosine phosphorylation is crucial to not only normal but also aberrant life processes. We designed and synthesized a photocleavable magnetic nanoparticle-based gallium tag for tagging and enrichment as well as UV-release of the phosphate-bearing molecules/ions in cells. HPLC/71Ga species-unspecific isotope dilution (71Ga-SUID) ICPMS was subsequently developed for specific and absolute quantification of phosphotyrosine (pY) under the assistance of a protein tyrosine phosphatase-1B (PTP-1B).pYquantification was thus achieved via determination of Ga in the Ga-phosphate complexes that come exclusively from the Ga-taggedpY. In this way, the method detection limit ofpYreached down to 30 amol with the RSD lower than 5.70% (n= 5 at pmol level). Feasibility of this proposed method was validated using VNQIGTLSEpYIK, VNQIGTLpSEpYIK, and extracellular regulated protein kinase 1 peptide (-pTEpY-) standards with the recovery of more than 96% (n= 5). It was applied to the absolute quantification ofpYin human breast cancer MCF-7 cells, indicating thatpYincreased by 1.60 nmol (61.1%) in 3.0 × 106MCF-7 cells after 100 nM insulin stimulation. We believe that, not limited topYquantification, this element-tagging and protease-specific reaction mediated ICPMS methodology will pave a simple path for ever more applications of ICPMS to the studies of quantitative protein post-translational modifications (PTMs) when suitable element-tags are designed and specific proteases are available toward targeted PTMs.
