Versatile Strategy for Biochemical, Electrochemical and Immunoarray Detection of Protein Phosphorylations

Abstract

Protein kinases catalyze the phosphorylation of cellular proteins involved in the regulation of many cellular processes and have emerged as promising targets for the treatment of several diseases. Conventional assays to monitor protein kinase activity are limited because they typically rely on transfer of radioactive phosphate or phospho-specific antibodies that recognize specific substrates or sequence motifs. To overcome the limitations of conventional assays, we have developed a versatile approach based on transfer of ferrocene-phosphate that can be readily monitored using electrochemical detection or detection with antiferrocene antibodies in an immunoarray format. This assay is readily adapted to multiplex arrays and can be employed for monitoring kinase activity in complex mixtures and for kinase inhibitor profiling.

The generality of kinase-catalyzed biotinylation

“…5 Recently, kinase-catalyzed labeling using γ-phosphate modified ATP analogs has emerged as a tool for studying phosphorylation (Figure 1a). 6 A diversity of functional tags has been attached to γ-phosphate of ATP and transferred to proteins/peptide substrates by kinases, including biotin, 6–10 dansyl, 11 ferrocene, 12, 13 BODIPY, 14 azide, 15, 16 alkyne, 16 thiol, 17, 18 acetyl, and sulfonyl groups. 19 Among these analogs, ATP-biotin (Figure 1b) is an attractive probe for characterizing phosphorylation by affixing a biotin handle onto phosphoproteins, which can be used for subsequent visualization.…”

Gold nanoparticles-based electrochemical method for the detection of protein kinase with a peptide-like inhibitor as the bioreceptor

“…[24][25][26] Moreover, the use of ATP analogs as the cosubstrates (eg, ferrocene [Fc]-ATP, biotin-ATP, and adenosine 5′-[γ-thio] triphosphate [ATP-S]) can also facilitate the development of various novel electrochemical kinase biosensors. [27][28][29][30][31][32] However, most of these methods require catalyzed phosphorylation reaction to a specific peptide substrate. Thus, a relatively long reaction time is required to obtain a large amount of phosphorylated products for signal accumulation.…”

A Cell‐Permeable ATP Analogue for Kinase‐Catalyzed Biotinylation

“…One powerful approach exploits analogs of the universal co-substrate of kinases, adenosine-5′-triphosphate (ATP, Figure 1B). Multiple ATP analogs have been employed in kinase research, including base modified [6] , sugar modified [7] , and triphosphate modified analogs [8] . Our lab and others have utilized γ-phosphate modified ATP analogs to label kinase substrates for subsequent purification and analysis.…”

“…Our lab and others have utilized γ-phosphate modified ATP analogs to label kinase substrates for subsequent purification and analysis. [8e-j, 9] For example, ATP-biotin (1 , Figure 1B) is promiscuously accepted as a cosubstrate by protein kinases to phosphorylbiotinylate substrates. [9b, 9d, 10] After kinase-catalyzed biotinylation with ATP-biotin, the biotin group facilitates analysis of phosphoproteins using various commercial streptavidin-conjugated reagents.…”