Evolution of a mass spectrometry-grade protease with PTM-directed specificity

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 113; no. 51; pp. 14686 - 14691
• Main Authors: Tran, Duc T., Cavett, Valerie J., Dang, Vuong Q., Torres, Héctor L., Paegel, Brian M.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 20.12.2016
• Subjects: Biochemistry; Biological Sciences; Chromatography; Genes; Mass spectrometry; Mutation; Peptides; Physical Sciences; Proteases; protease; proteomics; posttranslational modification; directed evolution; in vitro compartmentalization
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1609925113

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 PTMto 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 (k cat/K M = 6.9 × 10⁵ M−1·s−1). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y₁ ion 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.