Tryptic digestion of proteins followed by liquid chromatography with tandem mass spectrometry analysis is an extensively used approach in proteomics research and biopharmaceutical product characterization, owing to the high level of cleavage fidelity produced with this technique. However, nonspecific trypsin cleavages have been frequently reported and shown to be related to a number of digestion conditions and predigestion sample treatments. In this work, we reveal that, for a number of commercial trypsins, reconstitution and storage conditions can have a significant impact on the occurrence of trypsin nonspecific cleavages. We analyzed the tryptic digestion of a variety of biotherapeutics, using trypsins reconstituted under different conditions. The results indicate that, for many commercial trypsins, commonly recommended reconstitution/storage conditions (mildly acidic, e.g., 50 mM acetic acid, 1 mM HCl) can actually promote nonspecific trypsin activities, which are time dependent and can be as high as 20% in total relative abundance. In contrast, using water for reconstitution and storage can effectively limit nonspecific cleavages to 1%. Interestingly, the performances of different commercial trypsins were found to be quite distinct in their levels of nonspecific cleavages and responses to the two reconstitution conditions. Our findings demonstrate the importance of choosing the appropriate trypsin for tryptic digestion and the necessity of assessing the impact of trypsin reconstitution and storage on nonspecific cleavages. We advocate for manufacturers of commercial trypsins to reevaluate manufacturing processes and reconstitution/storage conditions to provide good cleavage specificity.

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轻度酸性条件下胰蛋白酶重构引起的非特异性裂解

蛋白质的胰蛋白酶消化，然后进行液相色谱和串联质谱分析，是蛋白质组学研究和生物制药产品表征中广泛使用的方法，这是由于该技术产生的高裂解保真度。然而，非特异性胰蛋白酶裂解已被频繁报道，并显示出与许多消化条件和消化前样品处理有关。在这项工作中，我们揭示出，对于许多商业化的胰蛋白酶，重构和储存条件可能对胰蛋白酶非特异性裂解的发生具有重大影响。我们使用在不同条件下重构的胰蛋白酶分析了多种生物疗法的胰蛋白酶消化。结果表明，对于许多商业胰蛋白酶，通常推荐的重构/存储条件（中等酸性，例如50 mM乙酸，1 mM HCl）实际上可以促进非特异性胰蛋白酶活性，这是时间依赖性的，总相对丰度可能高达20％。相反，使用水进行重组和存储可以有效地将非特异性裂解限制在1％。有趣的是，发现不同商业胰蛋白酶的性能在其非特异性裂解水平和对两种重构条件的反应方面非常不同。我们的发现表明选择合适的胰蛋白酶进行胰蛋白酶消化的重要性，以及评估胰蛋白酶重构和储存对非特异性裂解的影响的必要性。