通过化学标记质谱法鉴定蛋白质N端加工位点
Identification of N-terminal protein processing sites by chemical labeling mass spectrometry.
作者信息
Misal Santosh A, Li Sujun, Tang Haixu, Radivojac Predrag, Reilly James P
机构信息
Department of Chemistry, Indiana University, Bloomington, Indiana, USA.
School of Informatics, Computing, and Engineering, Indiana University, Bloomington, Indiana, USA.
出版信息
Rapid Commun Mass Spectrom. 2019 Jun 15;33(11):1015-1023. doi: 10.1002/rcm.8435.
RATIONALE
Proteins 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.
METHODS
The secreted proteins from S. aureus N315 were precipitated with trichloroacetic acid (TCA) and amidinated with S-methyl thioacetimidate (SMTA). Amidinated proteins were digested with trypsin and analyzed with a high-resolution orbitrap mass spectrometer.
RESULTS
Sixteen examples of Staphylococcus aureus secretory 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.
CONCLUSIONS
A 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.
原理
蛋白质会经历翻译后修饰和蛋白水解加工，这会影响其生物学功能。加工过程通常涉及单个残基的丢失。从N端切割信号肽通常与转运相关。最近的报告表明还存在其他加工位点。
方法
用三氯乙酸（TCA）沉淀金黄色葡萄球菌N315分泌的蛋白质，并用S-甲基硫代乙脒（SMTA）进行 amidination。用胰蛋白酶消化 amidinated 蛋白质，并用高分辨率轨道阱质谱仪进行分析。
结果
使用这种 amidination 方法鉴定出16个在输出过程中失去N端信号肽的金黄色葡萄球菌分泌蛋白实例。鉴定了有和没有甲硫氨酸的蛋白质的N端。还发现了由分选酶和一种未知蛋白酶导致的意外蛋白质切割。
结论
描述了一种基于简单N端 amidination 的质谱方法，该方法有助于鉴定成熟蛋白质的N端并发现意外的加工位点。
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