Time-Resolved Luminescence Detection of Spleen Tyrosine Kinase Activity through Terbium Sensitization

Abstract

Disruption of regulatory protein phosphorylation can lead to disease, and is particularly prevalent in cancers. Inhibitors that target deregulated kinases are therefore a major focus of chemotherapeutic development. Achieving sensitivity and specificity in high-throughput compatible kinase assays is key to successful inhibitor development. Here we describe the application of time-resolved luminescence detection to the direct sensing of Syk kinase activity and inhibition using a novel peptide substrate. Chelation and luminescence sensitization of Tb3+ allowed the direct detection of peptide phosphorylation without any antibodies or other labeling reagents. Characterizing the Tb3+ coordination properties of the phosphorylated vs. unphosphorylated form of the peptide revealed that an inner-sphere water was displaced upon phosphorylation, which likely was responsible for both enhancing the luminescence intensity and also extending the lifetime, which enabled gating of the luminescence signal to improve the dynamic range. Furthermore, a shift in the optimal absorbance maximum for excitation was observed, from 275 nm (for the unphosphorylated tyrosine peptide) to 266 nm (for the phosphorylated tyrosine peptide). Accordingly, time-resolved measurements with excitation at 266 nm via a monochromator enabled a 16-fold improvement in base signal to noise for distinguishing phosphopeptide from unphosphorylated peptide. This led to a high degree of sensitivity and quantitative reproducibility, demonstrating the amenability of this method to both research laboratory and high-throughput applications.

KINATEST-ID: A Pipeline To Develop Phosphorylation-Dependent Terbium Sensitizing Kinase Assays

“…ELISA), light scattering based methods using SERS and RLS, and fluorescence-based methods such as chelation enhanced fluorescence (CHEF), FRET and fluorescence quenching. 9-19 In particular, CHEF methods that sensitize lanthanide ions such as terbium (Tb 3+ ) in a phosphorylation-dependent manner 19-22 can enable high sensitivity and analytical reproducibility. Previously, we described the application of a kinase specific peptide substrate (SAStide) for the sensitive detection of spleen tyrosine kinase (Syk) activity in vitro through phosphorylation-dependent enhanced sensitization of Tb 3+ luminescence.…”

“…Previously, we described the application of a kinase specific peptide substrate (SAStide) for the sensitive detection of spleen tyrosine kinase (Syk) activity in vitro through phosphorylation-dependent enhanced sensitization of Tb 3+ luminescence. 22 The luminescence signal is generated when phosphorylation of the tyrosine residue results in exclusion of water and completion of the Tb 3+ coordination sphere. Phosphorylation also alters the excitation wavelength of the aromatic side chain, increases the binding affinity for the peptide, and increases the luminescence lifetime, 22 resulting in a large increase in signal to noise (16-fold in the case of SAStide).…”

A time-resolved luminescence biosensor assay for anaplastic lymphoma kinase (ALK) activity

“…The binding curve was hyperbolic and reached saturation at 20 µM of Tb 3+ , suggesting one-site binding behavior of the pALAStide (Figure 2d). The calculated K d for this pALAStide-Tb 3+ complex was 0.8 ± 0.2 µM, which is quite high Tb 3+ affinity in the context of other known Tb 3+ binding sequences 15,17,23 .…”

“…As a strategy to overcome such problems, peptide-based approaches can be integrated into various analytical workflows for developing HTS compatible kinase assays 12–15 . Several examples of detecting tyrosine phosphorylation directly by lanthanide luminescence (rather than the antibody-dependent technologies) have been demonstrated 17–20 , either by using sensitizer-labeled peptides or peptides with inherent lanthanide-sensitizing sequences.…”

“…We previously employed time-resolved lanthanide luminescence to design a high-throughput screening (HTS) compatible biosensor assay (Scheme 1) for Syk kinase activity 15 . However, lanthanide sensitization by the Syk substrate we developed was fortuitous based on the inherent substrate sequence, and not necessarily applicable to other substrates, which may not have the same advantageous chelating residues.…”