Yamaguchi Minoru, Obama Takashi, Kuyama Hiroki, Nakayama Daisuke, Ando Eiji, Okamura Taka-Aki, Ueyama Norikazu, Nakazawa Takashi, Norioka Shigemi, Nishimura Osamu, Tsunasawa Susumu
Life Science Laboratory, Shimadzu Corporation, Kyoto 604-8511, Japan.
Rapid Commun Mass Spectrom. 2007;21(20):3329-36. doi: 10.1002/rcm.3215.
A new method to determine N-terminal amino acid sequences of multiple proteins at low pmol level by a parallel processing has been developed. The method contains the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling with sulfosucccimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate(sulfo-NHS-SS-biotin) to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease; (4) specific isolation of N-terminal fragments of proteins by affinity capture using the biotin-avidin system; (5) de novo sequence analysis of peptides by MALDI-TOF-/MALDI-TOF-PSD mass spectrometry with effective utilization of the CAF (chemically assisted fragmentation) method.1 This method is also effective for N-terminal sequencing of each protein in a mixture of several proteins, and for sequencing components of a multiprotein complex. It is expected to become an essential proteomics tool for identifying proteins, especially when used in combination with a C-terminal sequencing method.
已开发出一种通过并行处理在低皮摩尔水平测定多种蛋白质N端氨基酸序列的新方法。该方法包括以下五个步骤：（1）对目标蛋白质进行还原、S-烷基化和胍基化；（2）将磺基琥珀酰亚胺-2-（生物素酰胺基）乙基-1,3-二硫代丙酸酯（磺基-NHS-SS-生物素）与蛋白质的N（α）-氨基偶联；（3）用适当的蛋白酶消化修饰后的蛋白质；（4）使用生物素-抗生物素蛋白系统通过亲和捕获特异性分离蛋白质的N端片段；（5）通过MALDI-TOF-/MALDI-TOF-PSD质谱法对肽段进行从头测序，并有效利用CAF（化学辅助裂解）方法。1该方法对于几种蛋白质混合物中每种蛋白质的N端测序以及多蛋白复合物的组分测序也有效。预计它将成为鉴定蛋白质的重要蛋白质组学工具，特别是与C端测序方法结合使用时。
