title: Evolution of a mass spectrometry-grade protease with PTM-directed specificity.
abstract: Mapping posttranslational modifications (PTMs), which diversely modulate biological functions, represents a significant analytical challenge. The centerpiece technology for PTM site identification, mass spectrometry (MS), requires proteolytic cleavage in the vicinity of a PTM to yield peptides for sequencing. This requirement catalyzed our efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a PTM implicated in epigenetic and immunological function, made an ideal first target, because citrullination eliminates arginyl tryptic sites. Bead-displayed trypsin mutant genes were translated in droplets, the mutant proteases were challenged to cleave bead-bound fluorogenic probes of citrulline-dependent proteolysis, and the resultant beads (1.3 million) were screened. The most promising mutant efficiently catalyzed citrulline-dependent peptide bond cleavage (kcat/KM= 6.9 × 105M-1⋅s-1). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y1ion corresponding to citrulline (176.1030 m/z) and diagnostic peak pairs in the extracted ion chromatograms of LC-MS/MS analysis. Using these signatures, we identified citrullination sites in protein arginine deiminase 4 (12 sites) and in fibrinogen (25 sites, two previously unknown). The unique mass spectral features of PTM-dependent proteolytic digest products promise a generalized PTM site-mapping strategy based on a toolbox of such mutant proteases, which are now accessible by laboratory evolution.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.
authors.forename: Duc T
authors.initials: DT
authors.lastname: Tran
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.
authors.forename: Valerie J
authors.initials: VJ
authors.lastname: Cavett
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.
authors.forename: Vuong Q
authors.initials: VQ
authors.lastname: Dang
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.
authors.forename: Héctor L
authors.initials: HL
authors.lastname: Torres
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458 briandna@gmail.com.
authors.forename: Brian M
authors.identifier: 0000-0002-6531-6693
authors.initials: BM
authors.lastname: Paegel
mesh_terms: D001120:Arginine / Q000737:chemistry; D002956:Citrulline / Q000737:chemistry; D019143:Evolution, Molecular; D006801:Humans; D013058:Mass Spectrometry; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010447:Peptide Hydrolases / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D012235:Rhodamines / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry*; D014362:Trypsinogen / Q000737:chemistry
chemical_list: D009841:Oligonucleotides; D010455:Peptides; D011506:Proteins; D012235:Rhodamines; D002956:Citrulline; D014362:Trypsinogen; D001120:Arginine; D010447:Peptide Hydrolases; D014357:Trypsin; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases
keywords: directed evolution; in vitro compartmentalization; posttranslational modification; protease; proteomics