α-Amine Ligation Profiling Informing N-terminal modification Enzyme selection
ALPINE is a web-based tool that enables algorithmic selection of subtiligase mutants for protein and peptide N-terminal modification applications including site-specific bioconjugation and N-terminomics studies. Enzyme-catalyzed N-terminal modification is a powerful tool for site-specific protein bioconjugation, but its usefulness can be limited by strict or poorly characterized enzyme-substrate specificity. To overcome this limitation, we developed PILS (Proteomic Identification of Ligation Sites), a proteomic approach for comprehensive characterization of peptide ligase prime-side specificity. Using PILS, we characterized the ligation efficiency for >20,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase, identifying mutants with activity toward N-terminal sequences that were previously refractory to modification. ALPINE enables exploration of PILS datasets for 72 subtiligase mutants, as well as analysis of new PILS datasets for other peptide ligases or N-terminal modification reactions. Using ALPINE, one can algorithmically select the best subtiligase mutant for modification of a particular protein or peptide sequence, create a custom subtiligase cocktail for maximum coverage of a user-defined sequence space, or analyze new PILS datasets.
PILS utilizes database-searchable, proteome-derived peptide libraries as ligase substrates for specificity characterization. These peptides are subjected to N-terminal modification by the peptide ligase of interest and a substrate containing a biotin group for affinity enrichment, a TEV protease cleavage site for selective elution, and an aminobutyric acid mass tag for positive identification of bona fide substrates. Following selective isolation of ligated peptides and sequencing by LC-MS/MS, positional enrichment or de-enrichment of each amino acid at each prime-side position can be rapidly determined. We have applied PILS to over 70 subtiligase mutants, demonstrating its utility for rapid specificity profiling of peptide ligases.
