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GTPase enrichment using a new active-site probe
Introducing a desthiobiotin nucleotide probe for studying GTPases.
The Thermo Scientific Pierce GTPase Enrichment Kit with ActivX* GTP Probe enables selective labeling and enrichment of small GTPases and large G-protein subunits. The Thermo Scientific ActivX Desthiobiotin-GTP Probe† structure consists of a modified biotin attached to the nucleotide by a labile acyl-phosphate bond (Figure 1A). 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 (Figure 1B) and selectively enrich, identify and profile target enzyme classes in samples.1-3 To study inhibitor binding affinity and target specificity, samples are preincubated with small-molecule inhibitors that compete with the probe.
Figure 1. Mechanism and chemical structure of the desthiobiotin-GTP probe for GTPases. Panel A: 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 using acidic elution conditions, allowing high recovery of labeled proteins and peptides. Panel B: 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.
Active-site labeling is assessed by either Western blot or mass spectrometry (MS) (Figure 2). For the Western blot workflow, desthiobiotin-labeled proteins are enriched for SDS-PAGE analysis and subsequent detection. For the MS workflow, desthiobiotinlabeled 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.
Figure 2. Assessment of active-site labeling is accomplished by Western blot or mass spectrometry. 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.
RESULTS and DISCUSSION:
Small GTPase labeling and inhibitor profiling
GTPases are important in signal transduction at the intracellular domain of transmembrane receptors, vesicle transport, nuclear protein export and protein biosynthesis. Heterotrimeric GTPases are typically associated with G-protein-coupled receptors (GPCRs) and have different signaling isoforms that determine receptor signaling. The small GTPase superfamily consists of several hundred proteins, which are further subdivided into Ras, Rho/Rac, Rab, Arf and Ran families. These monomeric GTPases function as molecular switches in intracellular signaling events depending on whether they are bound to GTP or GDP.
To demonstrate the utility and specificity of the active-site probe for GTPase labeling, we labeled A549 cell lysates with desthiobiotin- GTP and enriched before Western blot analysis (Figure 3). Using the Western blot workflow (Figure 2) revealed that cell lysates treated with magnesium chloride enabled specific labeling of the small GTPases Ras, Cdc42 and Rho A. Additionally, we used GTPγS, a non-hydrolyzable form of GTP, to inhibit recombinant Rac1 before labeling with desthiobiotin-GTP. These results show that desthiobiotin-GTP probes specifically label GTPase active sites.
Figure 3. Desthiobiotin-GTP probe specifically labels small GTPases. Panel A: A549 cell lysates (500µg) were treated with (+) or without (-) 20mM of MgCl2 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. Panel B: Recombinant Rac1 was treated with GTPγS before labeling with desthiobiotin-GTP probe. Labeling was performed in the presence (+) or absence (-) of 20mM MgCl2. Samples were separated by SDS-PAGE and analyzed by Western blot (Labeled) to detect biotinylation of the active site. Thermo Scientific GelCode Blue Stain Reagent (Total) was used to stain a duplicate gel to show equal loading.
GTPase enrichment for mass spectrometry identification
Although the desthiobiotin-GTP probe does not preferentially label active GTPases, this compound can be used to specifically label GTPases for global proteomic enrichment studies. Protein targets can be identified by analyzing desthiobiotin-labeled, active-site peptides. Using the MS workflow, we identified over 500 active-site peptides corresponding to 66 different GTPases from both large G-protein subunits and small GTPase families (Table 1). Generating proteomic target lists by this approach enables comparison of GTPase expression for multiple cell and tissue types.
Table 1. GTPases identified by mass spectrometry.
||Total GTPases per family
The Pierce Desthiobiotin GTPase Kits and Probes are highly versatile tools enabling the selective labeling and global enrichment of GTPases for profiling by Western blot or mass spectrometry.
- Cravatt, B.F., et al. (2008). Activity-based protein profiling: From enzyme chemistry to proteomic chemistry. Ann Rev Biochem 77:383-414.
- Patricelli, M.P. (2002). Activity-based probes for functional proteomics. Brief Funct Genomic Proteomic 1(2):151-8.
- Okerberg, E.S., et al. (2005). High-resolution functional proteomics by active-site peptide profiling. Proc Natl Acad Sci U.S.A. 102(14):4996-5001.
†ActivX Desthiobiotin-GTP Probe is exclusively licensed from ActivX Biosciences Inc. to Thermo Fisher Scientific for research use only.