Selective Sensing of Phosphorylated Peptides and Monitoring Kinase and Phosphatase Activity with a Supramolecular Tandem Assay

Abstract

Simple tuning of a host:guest pair allows selective sensing of different peptide modifications, exploiting orthogonal recognition mechanisms. Excellent selectivity for either lysine trimethylations or alcohol phosphorylations is possible by simply varying the fluorophore guest. The phosphorylation sensor can be modulated by the presence of small (μM) concentrations of metal ions, allowing array-based sensing. Phosphorylation at serine, threonine, and tyrosine can be selectively sensed via discriminant analysis. The phosphopeptide sensing is effective in the presence of small-molecule phosphates such as ATP, which in turn enables the sensor to be employed in continuous optical assays of both serine kinase and tyrosine phosphatase activity. The activity of multiple different kinases can be monitored, and the sensor is capable of detecting the phosphorylation of peptides containing multiple different modifications, including lysine methylations and acetylation. A single deep cavitand can be used as a "one size fits all" sensor that can selectively detect multiple different modifications to oligopeptides, as well as monitoring the function of their post-translational modification writer and eraser enzymes in complex systems.

Monitoring the crosstalk between methylation and phosphorylation on histone peptides with host-assisted capillary electrophoresis

“…These features are beneficial for obtaining high separation efficiency with simple and lowexpense operation. Several supramolecular host molecules, including calixarenes [13,15,16], cucurbiturils [17], and cavitands [18][19][20], have shown selectivity for PTMs like methylation and phosphorylation, which supports that synthetic hosts can be exploited to improve CE-based PTM analysis.…”

“…This protein is involved in the regulation of mitosis events [28] during which it can phosphorylate serine 10. Dimethylation on lysine 9 can have an antagonistic effect on the activity of Aurora B [20,21]. Aurora B has an important role in the regulation of kinetochore to microtubule attachment [29], so inhibition of the enzyme can interfere with this process.…”

Label‐Free Fluorescent Kinase and Phosphatase Enzyme Assays with Supramolecular Host‐Dye Pairs

“…For example, metal‐ion chelating sites were previously engineered into peptide kinase substrates to enable an interaction of the metal cation with the phosphate group after phosphorylation, and we have previously used positively charged anion receptor macrocycles to follow the hydrolysis of the negatively charged cofactor adenosine triphosphate . Only recently, Hooley and coworkers have utilized a negatively charged cavitand to distinguish phosphorylated and unphosphorylated versions of cationic peptides and, thereby, to monitor the activity of kinases and phosphatases . This prompted us to explore this rather counterintuitive strategy further by investigating whether we can also monitor the enzymatic conversion of uncharged or even negatively charged molecules with a cation receptor.…”

A new, deep quinoxaline-based cavitand receptor for the complexation of benzene

“…In our group, we have been exploiting two main types of receptors, in which the bridging groups at the upper rim are either phosphonate RPO 3 moieties or quinoxaline ring systems. Both families have been extensively used in sensing in solution (Lee et al, 2018;Liu et al 2018) and in the gas phase (Melegari et al, 2013;Tudisco et al, 2016). Indeed, the demand for fast and reliable detection of biological and chemical hazards is rising continuously and optimal sensors for environmental, security and biomedical applications must be sufficiently responsive to allow detection of the target analyte at low concentrations, and selective enough to respond primarily to a single chemical species in the presence of interferents.…”