Abstract
Transglutaminases (TGases, E.C. 2.3.2.13) are a large family of enzymes that catalyze an acyl group transfer between protein-bound glutamine and primary amines, including the ε-amino group of lysine to generate crosslinked proteins. This unique capacity for site-specific modification of proteins has led to widespread applications of TGases in many fields (e.g., food, biomaterials, medicine), yet TGase substrate specificity remains poorly understood. Thus, methods to improve the detection, quantification and characterization of TGase-mediated protein modifications were developed as described herein. First, a two-step tagging method to append fluorous tags (e.g., perfluoroalkyl chains) onto TGase substrate glutamines was developed, which allowed fluorous-tagged peptides to be enriched following fluorous solid phase extraction (F-SPE). Next, a novel method for site-specific protein photocaging (e.g., removable by light) was developed via TGase-mediated installation of amine-bearing photolabile groups (e.g., o-nitrobenzyl and o-nitrophenylethyl) into substrate glutamines. The utility of this photocaging method toward modulation of protein activity was demonstrated using the Escherichia coli protein UmuD. Additionally, the identification and characterization of TGase-crosslinked peptides was performed via our previously described workflow XChem-Finder (Anal. Chem. 2014, 86, 4940-4948; Anal. Chem. 2013, 85, 5900-5908), 18O-labeling and mass spectrometry (MS) analysis; even crosslinked peptides generated from non-specific cleavages were identified, demonstrating the robust nature of our workflow. Lastly, a method for absolute quantification of protein crosslinking was developed via dual-tagging of crosslinked peptides (e.g., installation of two different tags) and affinity capture; the method is pan-specific (e.g., without a priori knowledge of crosslinking chemistry) and thus is applicable for unknown crosslinking chemistry as well.
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