Evolution of a mass spectrometry-grade protease with PTM-directed specificity
Evolution of a mass spectrometry-grade protease with PTM-directed specificity
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DOI：
10.1073/pnas.1609925113
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发表时间：
2016-12-20
影响因子：
11.1
通讯作者：
Paegel, Brian M.
中科院分区：
文献类型：
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作者：
Tran, Duc T.;Cavett, Valerie J.;Paegel, Brian M.
映射翻译后修饰（PTM），这是一个重要的分析挑战。用于PTM位点鉴定的核心技术质谱法（MS）需要在PTM附近进行蛋白水解切割以产生用于测序的肽。这一要求催化了我们努力进化MS级突变PTM指导的蛋白酶。瓜氨酸是一种与表观遗传和免疫功能有关的PTM，是理想的第一个靶点，因为瓜氨酸消除了乙酰基胰蛋白酶位点。将珠粒展示的胰蛋白酶突变基因翻译成液滴，挑战突变蛋白酶以切割瓜氨酸依赖性蛋白水解的珠粒结合的荧光探针，并筛选所得珠粒（130万）。最有希望的突变体有效催化瓜氨酸依赖性肽键裂解（k（cat）/K-M = 6.9 x 10（5）M-1）。s（-1））。所得C-末端瓜氨酸化肽在MALDI-TOF MS中产生特征性同位素模式，并且在LC-MS/MS分析的提取离子色谱图中产生对应于瓜氨酸（176.1030 m/z）的裂解产物y（1）离子和诊断峰对。利用这些特征，我们确定了蛋白精氨酸脱亚胺酶4（12个位点）和纤维蛋白原（25个位点，两个以前未知）中的瓜氨酸酶位点。PTM依赖性蛋白水解消化产物的独特质谱特征承诺基于这样的突变蛋白酶的工具箱的广义PTM位点定位策略，其现在可通过实验室进化获得。
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 (k(cat)/K-M = 6.9 x 10(5) M-1 . s(-1)). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y(1) 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.