GTPase Enrichment Kit and Probes

Thermo Scientific Pierce GTPase Enrichment Kits use ActivX* GTP Probes to covalently label the active site of GTPases and the GTPase subunits of G-protein coupled receptors enabling their selective enrichment using the desthiobiotin tag.

ActivX* GTP Probes feature a desthiobiotin (biotin analog) tag that facilitates selective enrichment and recovery of labeled GTPases allowing identification and profiling of target enzyme classes across samples or to assess the specificity and affinity of enzyme inhibitors.

Highlights:

• Specific – label only the conserved active-site lysines of nucleotide-binding proteins
• Compatible – use for in vitro labeling of GTPase enzymes derived from cells or tissues.
• Flexible – use with Western blot or mass spectrometry (MS) workflows

Applications:

• Broad enrichment of GTP-binding proteins from tissues, cells and subcellular proteomes
• Enrichment of enzymes based on function
• Profiling of dozens to hundreds of inhibitor targets

Product Details:

The Thermo Scientific Pierce GTPase Enrichment Kits with ActivX* GTP Probes enable selective labeling and enrichment of small GTPases and large G-protein subunits. Thermo Scientific ActivX Desthiobiotin-GTP Probe structure consists of a modified biotin attached to the nucleotide by a labile acyl-phosphate bond. After removal of GTP or GDP nucleotides from enzymes, the desthiobiotin-GTP probe can be used to covalently modify conserved lysine residues in the GTPase nucleotide-binding site. Desthiobiotin-GTP can selectively enrich, identify and profile target enzyme classes in samples. Pre-incubation of samples with small-molecule inhibitors that compete with active-site probes can be used to determine inhibitor binding affinity and target specificity.

Assessment of active-site labeling can be accomplished by either Western blot or mass spectrometry (MS). For the Western blot workflow, desthiobiotin-labeled proteins are enriched for SDS-PAGE analysis and subsequent detection with specific antibodies. For the MS workflow, desthiobiotin-labeled proteins are reduced, alkylated and enzymatically digested to peptides. Only the desthiobiotin-labeled, active-site peptides are enriched for analysis by LC-MS/MS. Both workflows can be used for determining inhibitor target binding, but only the MS workflow can identify global inhibitor targets and off-targets.

A.
B.
Mechanism and chemical structures of Thermo Scientific Active Site Probes for GTPases. A. Nucleotide analogues bind to the active sites of GTPases and the biotin affinity tag is irreversibly transferred to highly conserved lysine residues in the active site. B. Desthiobiotin is attached to the GTP nucleotide through a labile acyl phosphate linkage, allowing efficient desthiobiotin label transfer to amines near the active site of GTPases. Desthiobiotin binding to streptavidin is easily reversible under acidic elution conditions, allowing high recovery of labeled proteins and peptides.

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.

Labeling and Enrichment of small GTPases with desthiobiotin-GTP probe using the Western blot workflow. A. A549 cell lysates (500µg) were treated with (+) or without (-) 20mM magnesium chloride after labeling with 20µM of desthiobiotin-GTP probe. Desthiobiotin-labeled proteins were denatured and enriched using streptavidin agarose before separation by SDS-PAGE and Western blotting with specific GTPase antibodies. B. Recombinant RAC1 was pretreated with GTPγS before labeling with desthiobiton-GTP in the presence (+) or absence (-) of 20mM magnesium. Samples were separated by SDS-PAGE and analyzed Western blot (Labeled RAC1) to detect biotinylation of the active site. GelCode Blue stain (Total RAC1) was used to stain a duplicate gel to show equal loading.

References:

1. Patricelli, M.P., et al. (2007). Functional Interrogation of the kinome using nucleotide acyl phosphates. Biochemistry 46:350-358.
2. Cravatt, B.F., Wright, A.T., and Kozarich, J.W. (2008). Activity-based protein profiling: From enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77:383–414.
3. Okerberg, E.S., et al. (2005). High-resolution functional proteomics by active-site peptide profiling. Proc Natl Acad Sci USA 102(14):4996-5001.

Related Resources:

Mass Spec Sample Handbook
Mass Spectrometry Sample Prep Workflow
Activity-Based Protein Profiling Technology Licensed from ActivX Biosciences, Inc. (opens pdf)
ActivX KiNativ* in situ Kinase Profiling Services (opens new window)
Review of sample preparation for mass spectrometry

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