A novel derivatization method with 5-bromonicotinic acid N-hydroxysuccinimide for determination of the amino acid sequences of peptides
A novel derivatization method with 5-bromonicotinic acid N-hydroxysuccinimide for determination of the amino acid sequences of peptides
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DOI：
10.1002/(sici)1097-0231(19980529)12:10
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发表时间：
1998-01-01
影响因子：
2
通讯作者：
Tsunasawa, S
中科院分区：
文献类型：
--
作者：
Miyagi, M;Nakao, M;Tsunasawa, S
我们开发了一种新方法，可有效识别肽串联质谱 (MS/MS) 分析中产生的 N 端产物离子，并结合使用 5-溴烟酸 N-羟基琥珀酰亚胺酯 (BrNA-NHS) 对 N 端氨基进行特异性衍生化。电喷雾电离与低能碰撞诱导解离 (CID) MS/MS 可以清楚地将用 5-溴烟基基团标记的 N 端产物离子与其他离子区分开来，这是基于具有双峰模式的 CID 峰的出现，该双峰模式的特征是由 Br-79 和 Br-81 的相等自然丰度产生的 2 个质量单位分开。通过追踪一系列含溴产物离子，可以轻松对肽进行氨基酸测序。使用Gln-Arg-Leu-Gln-Ser-Asn-Gln-Leu-Lys作为测试肽，我们发现在pH 6.5和37℃下30分钟内其α-氨基被BrNA-NHS完全酰化（肽：BrNA-NHS = 1:40；mol/mol）。 C-末端赖氨酸残基的ε-氨基在这些条件下不太可能被酰化，仅被部分修饰（约20%）。这表明有可能保持ε-氨基免于酰化。该方法成功应用于赖氨酰内肽酶和金黄色葡萄球菌VS蛋白酶消化产生的猪肾氨酰化酶I肽的氨基酸序列测定。 (C) 1998 约翰威利父子有限公司
We have developed a novel method that effectively identifies the N-terminal product ions produced in the tandem mass spectrometry (MS/MS) analysis of peptides done in conjunction with the specific derivatization of the N-terminal amino group using 5-bromonicotinic acid N-hydroxysuccinimide ester (BrNA-NHS). Electrospray ionization with low-energy collision-induced dissociation (CID) MS/MS clearly differentiated the N-terminal product ions labeled with the 5-bromonicotinyl group from other ions, on the basis of the appearance of CID peaks with a doublet pattern characteristically separated by 2 mass units produced by the equal natural abundances of Br-79 and Br-81. Th, tracing of a series of these bromine-containing product ions allows the easy amino acid sequencing of peptides. Using Gln-Arg-Leu-Gln-Ser-Asn-Gln-Leu-Lys as the test peptide, we found that within 30 minutes at pH 6.5 and 37 degrees C its alpha-amino group was completely acylated with BrNA-NHS (peptide: BrNA-NHS = 1:40; mol/mol). The epsilon-amino group of the C-terminal lysine residue was less likely to be acylated under these conditions, being only partly modified (about 20%). This suggests the possibility of keeping the epsilon-amino group free from acylation. The method was successfully applied to the determination of the amino acid sequences of peptides from porcine kidney aminoacylase I produced by digestion with lysyl endopeptidase and with Staphylococus aureus VS protease. (C) 1998 John Wiley & Sons, Ltd.