Profiling N-myristoyltransferase activity and inhibition in breast cancer cells (MD-MB-231) using Sortase-A labelling

ABSTRACT: We report a novel application for Sortase A (SrtA), labeling N-terminal Gly proteins across the proteome at endogenous levels. We combined SrtA labeling of whole-cell lysates with a potent and selective NMT inhibitor, delivering a method capable of quantifying changes in cellular NMT activity in multiple substrates at the whole proteome level. This method shows good overlap in substrates with a previously reported YnMyr metabolic tagging strategy (PXD002687), and a similar capability to measure cellular NMT activity. The SrtA-based approach described here is complementary to YnMyr metabolic tagging, since the increase in signal for newly synthesized proteins exposing an N-terminal glycine mirrors the loss of signal seen with YnMyr. Importantly, this new method overcomes the limitations imposed by metabolic tagging. Since SrtA labeling is performed post-lysis this labeling strategy provides a readout of the cell status in the absence of any external perturbation and is applicable to virtually any type of biological sample, giving clear advantages over other in-cell labeling strategies. We anticipate that this method will enable the exploration of new avenues for NMT as a therapeutic target by providing a method for de novo identification of substrates most responsive to NMT inhibition, and a multifuctional target engagement biomarker assay suited to both animal models and patient biopsies. Besides shotgun proteomics, we coupled this robust NMT activity assay to a variety of other detection techniques, including gel-based analyses, and ELISA. This submission concerns myristoylated proteomes of human origin.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Remigiusz Serwa  

LAB HEAD: Edward W Tate

PROVIDER: PXD010634 | Pride | 2018-10-25

REPOSITORIES: Pride