Department of Chemistry and Department of Chemical and Physical Sciences, University of Toronto, Mississauga, 3359 Mississauga Road North, Mississauga, Ontario, CanadaL5L1C6.
Analyst. 2017 Jun 26;142(13):2451-2459. doi: 10.1039/c6an02537d.
Proximal phosphorylation on proteins appears to have functional significance and has been associated with several diseases, including Alzheimer's and cancer. While much remains to be learned about the role of proximal phosphorylation in biological systems, no simple and/or affordable technique is available for its detection. To this end, we have previously developed a ProxyPhos chemosensor, which detects proximally phosphorylated peptides and proteins over mono- and non-phosphorylated motifs in aqueous solutions. In this follow-up work, we performed extensive characterization of peptide and protein ProxyPhos assay conditions to achieve enhanced detection, and further explored the selectivity of ProxyPhos, and its potential off-targets. As a result of characterization studies, selective sensing of proximally phosphorylated over mono-phosphorylated peptides and proteins was achieved. Moreover, studies demonstrated that ProxyPhos was compatible with the detection of all commonly phosphorylated residues (i.e. tyrosine, serine and threonine residues). Under optimized conditions, ProxyPhos efficiently discriminated between peptides derived from the activated (proximally phosphorylated, disease-relevant) and inactive (mono-phosphorylated) forms of JAK2, SYK and MAPK1 kinases. In addition, ProxyPhos can be used to probe phosphatase activity on peptides and proteins via detecting changes in proximal phosphorylation, demonstrating immediate utility of this chemosensing system.
蛋白质的近端磷酸化似乎具有功能意义，并与几种疾病有关，包括阿尔茨海默病和癌症。虽然关于近端磷酸化在生物系统中的作用还有很多需要了解，但目前还没有简单和/或经济实惠的技术可用于检测。为此，我们之前开发了一种 ProxyPhos 化学传感器，可在水溶液中检测到近磷酸化肽和蛋白质，而不会检测到单磷酸化和非磷酸化基序。在这项后续工作中，我们对肽和蛋白质 ProxyPhos 检测条件进行了广泛的表征，以实现增强的检测，并进一步探索了 ProxyPhos 的选择性及其潜在的非靶点。作为表征研究的结果，实现了对近磷酸化肽和蛋白质的选择性检测，而不是单磷酸化肽和蛋白质。此外，研究表明，ProxyPhos 与所有常见磷酸化残基（即酪氨酸、丝氨酸和苏氨酸残基）的检测兼容。在优化条件下，ProxyPhos 能够有效地区分源自激活（近磷酸化，与疾病相关）和非激活（单磷酸化）形式的 JAK2、SYK 和 MAPK1 激酶的肽。此外，ProxyPhos 可用于通过检测近端磷酸化的变化来探测肽和蛋白质上的磷酸酶活性，证明了这种化学传感系统的即时实用性。
