ICPMS-Based Specific Quantification of Phosphotyrosine: A Gallium-Tagging and Tyrosine-Phosphatase Mediated Strategy.

ABSTRACT

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). pY quantification was thus achieved via determination of Ga in the Ga-phosphate complexes that come exclusively from the Ga-tagged pY. In this way, the method detection limit of pY reached 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 of pY in human breast cancer MCF-7 cells, indicating that pY increased by 1.60 nmol (61.1%) in 3.0 × 106 MCF-7 cells after 100 nM insulin stimulation. We believe that, not limited to pY quantification, 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.