A method for N-terminal de novo sequencing of N^α-blocked proteins by mass spectrometry
A method for N-terminal de novo sequencing of N^α-blocked proteins by mass spectrometry
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一种利用质谱法对 N^α 封闭蛋白进行 N 端从头测序的方法
DOI：
10.1039/c0an00384k
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发表时间：
2011
期刊：
影响因子：
4.2
通讯作者：
and Susumu Tsunasawa
中科院分区：
文献类型：
--
作者：
Chihiro Nakajima;Hiroki Kuyama;Takashi Nakazawa;Osamu Nishimura;and Susumu Tsunasawa
提出了一种通过质谱 (MS) 对 Nα 封闭蛋白进行从头测序的方法。该方法包括酶消化 Nα 封闭的蛋白质、通过从消化池中去除非 N 末端肽来回收 N 末端肽，以及选择性衍生化分离的 N 末端肽的 C 末端 α-羧基。根据恶唑酮化学，N 端肽的 C 端 α-羧基选择性地用 3-氨基丙基-三(2,4,6-三甲氧基苯基)溴化鏻 (TMPP-丙胺) 衍生化。试剂 TMPP-丙胺旨在通过质量和电荷标记促进 MALDI-MS 的序列分析。通过几乎完全允许 y 型离子，成功分析了两种 Nα-乙酰化蛋白（兔磷酸化酶 b 和牛钙调蛋白）和人食欲素 A（其 N 末端具有焦谷氨酸）的所有身份和 N 末端序列。
A method for de novo sequencing of Nα-blocked proteins by mass spectrometry (MS) is presented. The approach consists of enzymatic digestion of Nα-blocked protein, recovery of N-terminal peptide by depletion of non-N-terminal peptides from the digest pool, and selective derivatization of a C-terminal α-carboxyl group of isolated N-terminal peptide. The C-terminal α-carboxyl group of the N-terminal peptide was selectively derivatized with 3-aminopropyl-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine), according to oxazolone chemistry. The reagent TMPP-propylamine was designed to facilitate sequence analysis with MALDI-MS by mass- and charge-tagging. All of the identities and N-terminal sequences of two Nα-acetylated proteins (rabbit phosphorylase b and bovine calmodulin) and human orexin A, which has pyroglutamic acid at the N-terminus, were successfully analyzed by allowing for the y-type ions almost exclusively.