High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry
High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry
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DOI：
10.1021/ac048776w
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发表时间：
2005-01-15
影响因子：
7.4
通讯作者：
Norioka, S
中科院分区：
文献类型：
--
作者：
Yamaguchi, M;Nakazawa, T;Norioka, S
建立了一种利用基质辅助激光解吸电离源后衰变（PSD）技术对蛋白质N端进行高通量测序的方法。在PVDF膜上印迹的蛋白质被依次还原、S-烷基化和胍基化后，其N-氨基与生物素基半胱氨酸偶联。然后从膜中提取蛋白质并用胰蛋白酶消化。衍生的N-末端片段，然后专门从胰蛋白酶消化与亲和素树脂分离，其从头测序成功地进行PSD利用磺酸基团引入到N末端。
A high-throughput method for sequencing of N termini of proteins by using postsource decay (PSD) of matrix-assisted laser desorption/ionization mass spectrometry has been developed. After a protein blotted on the PVDF membrane was successively reduced, S-alkylated, and guanidinated, its N-amino group was coupled to biotinylcysteic acid. The protein was then extracted from the membrane and digested with trypsin. The derivatized N-terminal fragment was then specifically isolated from the tryptic digest with avidin resins, and its de novo sequencing was successfully performed by PSD utilizing a sulfonic acid group introduced to the N terminus.