Reagents for the selective capture and enrichment of kinases using active-site probes.
Thermo Scientific Pierce Kinase Enrichment Kits utilize ActivX* ATP or ADP Probes to covalently label the active site of ATPases, including chaperones and metabolic enzymes, to enable their selective enrichment using a desthiobiotin tag.
ActivX ATP and ADP Probes feature an amine-reactive nucleotide analog and a desthiobiotin (biotin analog) tag that facilitates selective labeling of lysines in the kinase active site and then subsequent enrichment and recovery of labeled protein. These features allow identification and profiling of target enzyme classes across samples or assessment of the specificity and affinity of enzyme inhibitors.
Specific – label only the conserved active-site lysines of nucleotide-binding proteins
Flexible – use for in vitro labeling of ATPase enzymes derived from cells or tissues
Compatible – use with Western blot or mass spectrometry (MS) workflows
Profile small-molecule binding affinities and active-site inhibition in a dose-dependent manner
Identify dozens to hundreds of inhibitor targets and off-targets from tissues, cells and subcellular proteomes
Enrichment of enzymes based on function
Thermo Scientific ActivX Desthiobiotin-ATP and -ADP are nucleotide derivatives that covalently modify the active site of enzymes at conserved lysine residues in the nucleotide binding site. The structure of these probes consists of a modified biotin (desthiobiotin) attached to the nucleotide through a labile acyl-phosphate bond. Desthiobiotin is a biotin analog that binds less tightly to biotin-binding proteins resulting in binding that is easily reversed by biotin displacement, low pH or heat denaturation.
Depending on the position of the lysine within the enzyme active site, either desthiobiotin-ATP or -ADP might be better for labeling specific ATPases. Both desthiobiotin-ATP and -ADP probes can be used to selectively enrich, identify and profile target enzyme classes or assess the specificity of enzyme inhibitors. Because many ATPases and other nucleotide-binding proteins bind nucleotides or inhibitors even when they are enzymatically inactive, the desthiobiotin probes allow profiling of both inactive and active enzymes in a complex sample. Preincubation of samples with small-molecule inhibitors that compete for active sites can be used to determine inhibitor binding affinity. Active-site nucleotide probes also can be used to identify inhibitor off-targets.
Mechanism and chemical structures of Thermo Scientific Active Site Probes for kinases and other ATPases.A. Nucleotide analogues bind to the active sites of ATPases and the biotin affinity tag is irreversibly transferred to highly conserved lysine residues in the active site. B. Structures of desthiobiotin nucleotide analogues. Desthiobiotin binding to streptavidin is easily reversible under acidic elution conditions, allowing high recovery of labeled proteins and peptides. Desthiobiotin is attached to the nucleotide through a labile acyl phosphate linkage, allowing efficient desthiobiotin label transfer to amines near the active site. ATP and ADP nucleotide analogues label a complementary set of ATPases, which is likely due to differences in the proximity of the acyl phosphate linkage to conserved lysines near the active site.
Assessment of active-site labeling can be accomplished by Western blot or mass spectrometry (MS). For the Western blot workflow, desthiobiotin-labeled proteins are enriched, analyzed by SDS-PAGE and detected with specific antibodies. For the MS workflow, desthiobiotin-labeled proteins are reduced, alkylated and enzymatically digested. Only the desthiobiotin-labeled, active site peptides are enriched for LC-MS/MS analysis. Both workflows can be used to determine inhibitor target binding, but the MS workflow also can identify global inhibitor targets and off-targets and provide higher throughput for quantitative assays.
Comparison of desthiobiotin ATP and ADP probe labeling of kinases using the Western blot workflow. A549 cell lysates (500µg) were treated with (+) or without (-) 100µM staurosporine before labeling with 5µM of each probe in triplicate (A-C). Desthiobiotin-labeled proteins were denatured and enriched using streptavidin agarose before separation by SDS-PAGE and Western blotting with specific kinase antibodies. Unlabeled lysate (-/-) was used as a control to show streptavidin pulldown specificity.
Mass spectrometry analysis of desthiobiotin-ATP labeled peptides. Active-site peptides (13% of total peptides) from more than 100 kinases were identified using desthiobiotin-ATP peptide pull-down's. K562 cell lysates from two independent biological replicates were used. A Thermo Scientific LTQ Orbitrap Mass Spectrometer was used for analysis.