Whole Proteome Profiling of N-Myristoyltransferase Activity and Inhibition Using Sortase A

Abstract

A new method to quantify cellular myristoylation at physiological levels and in response to N -myristoyltransferase inhibition is presented and validated. Sortase A is an effective tool to label protein glycine N-termini across the whole proteome, and its specificity is determined in this context. Generally applicable improvements to the biotin/avidin affinity enrichment protocol are described that effectively eliminate avidin-derived tryptic peptide contaminants.

Wheat pathogenZymoseptoria tritici N-myristoyltransferase inhibitors: on-target antifungal activity and an unusual metabolic defense mechanism

“…By quantitative analysis, we found 25 proteins significantly enriched relative to samples without myristic acid competition ( Figure 2D). We excluded those lacking an N-terminal glycine which is known to be essential for NMT catalysis of N-myristoylation 35,36 , leaving 22 putative NMT substrate proteins; we speculate that non-myristoylated proteins may be enriched by non-covalent interactions, in line with observations in previous studies in human cells 31 . To complement the analysis, we applied a predictive web-based tool 'Myristoylator' 34 to analyze myristoylation by an orthogonal method; this predictor is trained primarily on an experimental yeast (S. cerevisiae) NMT substrate set, so we expected it to be a reasonable predictor of myristoylation for Z. tritici.…”

Discovery of a Potent and Selective Covalent Inhibitor and Activity-Based Probe for the Deubiquitylating Enzyme UCHL1, with Antifibrotic Activity

“…To determine selectivity across the proteome, unbiased quantitative chemical proteomic profiling ( Figure 2 a) was employed in HEK293 cells treated with 2 (2, 20, or 200 nM) or vehicle (DMSO) control, for 10, 60, or 180 min. CuAAC ligation of proteins to azide-arginine-biotin (AzRB, Figure S5 ) capture reagent 11 and enrichment on dimethylated NeutrAvidin-agarose beads were followed by on-resin digestion with LysC followed by trypsin 20 and labeling with tandem mass tags (TMT) to enable relative quantification between conditions following nanoLC-MS/MS analysis. UCHL1 was significantly enriched by 2 in a concentration-dependent manner, with marginal enrichment of UCHL3 at higher concentrations and no significant enrichment of any other DUB ( Figures 4 a and S11 ); UCHL1 enrichment was similar at all time points, in line with in-gel fluorescence analysis ( Figure S7 ).…”

Metabolic precision labeling enables selective probing of O-linkedN-acetylgalactosamine glycosylation

“…Biotin-containing, acid-cleavable alkynyl probe 12 served to enrich azide-containing glycoproteins from the de-N-glycosylated secretome of HepG2 cells. Samples were digested with Lysyl endopeptidase (LysC) after enrichment on Lys-dimethylated Neutravidin beads with enhanced LysC resistance (41). Following glycopeptide release, tandem MS was used to sequence glycopeptides.…”

“…RapiGest, urea and PBS supernatants were combined, and samples were diluted with PBS to 2 mL. Dimethylated Sera-Mag SpeedBeads Neutravidin Magnetic Beads (150 µL slurry = 75 µL settled resin) were washed with PBS twice in LoBind tubes (Eppendorf) and added to the lysate (25, 26). Samples were incubated for 16 h at 4 °C under rotation.…”