Abstract 5167: Profiling GTPases in native proteomes with a GTP acyl phosphate probe.

Abstract GTPases constitute an important class of regulatory proteins that participate as signal transducers in the capacity of molecular switches. The GTPase superfamily consists of 250 members in the human genome. These fall into several families, including the small GTPases (Ras Rho, Arf, Rab and Ran), heterotrimeric GTPases, elongation and initiation factors, and dynamins. In order to profile the distribution of GTPases in various cell lines and tissues, we have developed a chemical probe comprised of GTP linked to desthiobiotin through an acyl-phosphate bond. By treating complex proteomes with the probe, we are able to isolate probe-labeled peptides with streptavidin, and subsequently, identify the probe-labeled peptides by mass spectrometry. GTPases were identified from almost all known GTPase families indicating that this approach can be used to globally profile GTPases in various proteomes. The most common labeling site occurs at the conserved lysine residue in the phosphate binding loop (GXXXXGK) of the GTP-binding site. Significantly, the probe-labeled peptide for H-,K- and N-RAS includes amino acid positions 12 and 13, which are frequently mutated in cancers. Thus, the GTP probe can be used to directly quantify mutant RAS isoforms at the protein level. Citation Format: Tyzoon K. Nomanbhoy, Ann Shih, Arwin Aban. Profiling GTPases in native proteomes with a GTP acyl phosphate probe. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5167. doi:10.1158/1538-7445.AM2013-5167