Abstract：
Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as the main transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5040 plant proteins within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. This analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor-like kinases. Transient expression of double-tagged candidate proteins confirmed processing events in vivo. This large proteomic dataset implicates an elaborate proteolytic machinery shaping the proteome of N. benthamiana.
摘要：
蛋白酶的加工不可逆地调节植物蛋白的命运，并阻碍植物中重组蛋白的生产，然而，只有很少的处理事件在农业渗透的烟草benthamiana中被描述，已成为植物科学和分子制药中主要的瞬时蛋白质表达平台。这里,我们使用凝胶内消化和质谱来监测蛋白质凝胶中5040种植物蛋白的迁移和形貌。通过在凝胶切片上绘制肽，我们生成了胃蛋白酶图，揭示了在蛋白质凝胶中检测到每种蛋白质的哪一部分。这些数据发现，60％的检测到的蛋白质具有低于预测分子量的蛋白质形式，涉及广泛的蛋白水解加工。该分析证实了大多数但不是所有蛋白酶的自抑制前结构域的蛋白水解去除和降解，并揭示了果胶甲基酯酶和脂肪酶家族中的差异加工。该分析还揭示了糖苷酶的复杂加工，并揭示了胞外域脱落可能对于多种受体样激酶是常见的。双标记候选蛋白的瞬时表达证实了体内加工事件。这个大的蛋白质组数据集暗示了形成N.benthamiana的蛋白质组的复杂的蛋白水解机制。
