Identification of N-terminal protein processing sites by chemical labeling mass spectrometry.
Identification of N-terminal protein processing sites by chemical labeling mass spectrometry.
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通过化学标记质谱法鉴定 N 末端蛋白质加工位点。
DOI：
10.1002/rcm.8435
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Reilly,JamesP
中科院分区：
文献类型：
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作者：
Misal,SantoshA;Li,Sujun;Tang,Haixu;Radivojac,Predrag;Reilly,JamesP
蛋白质经过翻译后修饰和蛋白水解加工，可以影响其生物学功能。加工通常涉及单个残留物的损失。信号肽从N末端的切割通常与易位相关。最近的报道表明，其他加工位点也存在。aureusN 315用三氯乙酸（TCA）沉淀并用S-甲基硫代乙酰亚胺酯（SMTA）脒化。用胰蛋白酶消化酰胺化蛋白质，并与高分辨率orbitrap质谱仪进行分析。Results 16例ofStaphylococcus aureus分泌蛋白，失去了N-末端信号肽在其出口过程中，确定使用这种脒化的方法。鉴定了含和不含甲硫氨酸的蛋白质的N末端。意想不到的蛋白质裂解由于分选酶和未知的蛋白酶也uncovered.ConclusionsA简单的N-末端amidination为基础的质谱方法进行了描述，有利于识别的N-末端的成熟蛋白质和意想不到的加工位点的发现。
RationaleProteins undergo post‐translational modifications and proteolytic processing that can affect their biological function. Processing often involves the loss of single residues. Cleavage of signal peptides from the N‐terminus is commonly associated with translocation. Recent reports have suggested that other processing sites also exist.MethodsThe secreted proteins fromS. aureusN315 were precipitated with trichloroacetic acid (TCA) and amidinated withS‐methyl thioacetimidate (SMTA). Amidinated proteins were digested with trypsin and analyzed with a high‐resolution orbitrap mass spectrometer.ResultsSixteen examples ofStaphylococcus aureussecretory proteins that lose an N‐terminal signal peptide during their export were identified using this amidination approach. The N‐termini of proteins with and without methionine were identified. Unanticipated protein cleavages due to sortase and an unknown protease were also uncovered.ConclusionsA simple N‐terminal amidination based mass spectrometry approach is described that facilitates identification of the N‐terminus of a mature protein and the discovery of unexpected processing sites.