Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
J Proteomics. 2009 Dec 1;73(2):240-51. doi: 10.1016/j.jprot.2009.09.007. Epub 2009 Sep 23.
In this article we present a simple method to enrich peptides containing in vivo N(alpha)-modified protein N-termini. We demonstrate that CNBr-activated Sepharose, a commercial amine reactive matrix, can selectively couple peptides via the alpha-NH(2) group under mild conditions. Following digestion by trypsin, a simple incubation step with the CNBr-activated Sepharose by which the free alpha-NH(2) containing peptides are coupled with matrix through a covalent bond, allows the separation of N(alpha)-modified peptides from massive free alpha-NH(2) containing peptides. The removal of contaminant peptides with artificial N(alpha)-modifications, like cyclization of N-terminal S-carbamoylmethylcysteine and glutamine, are also discussed. Application of this method to tryptic digests of HeLa cell proteins resulted by a single LC-MS/MS analysis in the identification of 588 in vivo N(alpha)-modified peptides, of which 507 contain IPI (International Protein Index) annotated protein N-termini and 81 contain IPI unannotated protein N-termini. Most of the identified modifications are acetylations with only a few formylations and propionylations present. Furthermore, Lys-N digestion was also applied and resulted in the identification of 394 in vivo N(alpha)-modified peptides, of which 371 contain IPI annotated protein N-termini and 23 contain IPI unannotated protein N-termini. Combination of the two datasets leads to the identification of 675 N(alpha)-modified IPI annotated protein N-termini and 88 N(alpha)-modified IPI unannotated protein N-termini. Our results suggest that N-terminal acetyltransferases (NATs) may function as N-terminal formyltransferases (NFTs) and N-terminal propionyltransferases (NPTs) in vivo.
在本文中，我们提出了一种简单的方法来富集含有体内 N(alpha)-修饰蛋白 N 末端的肽。我们证明，CNBr 活化的琼脂糖，一种商业胺反应基质，可以在温和条件下通过 α-NH(2) 基团选择性地偶联肽。用胰蛋白酶消化后，通过与 CNBr 活化的琼脂糖简单孵育，将含有游离 α-NH(2)的肽通过共价键与基质偶联，从而可以将 N(alpha)-修饰肽从大量含有游离 α-NH(2)的肽中分离出来。还讨论了去除含有人工 N(alpha)-修饰的杂质肽，如 N-末端 S-氨甲酰基甲基半胱氨酸和谷氨酰胺的环化。该方法应用于 HeLa 细胞蛋白的胰蛋白酶消化物，通过单次 LC-MS/MS 分析鉴定了 588 种体内 N(alpha)-修饰肽，其中 507 种含有 IPI（国际蛋白质索引）注释蛋白 N 末端，81 种含有 IPI 未注释蛋白 N 末端。大多数鉴定的修饰是乙酰化，只有少量的甲酰化和丙酰化。此外，还应用了 Lys-N 消化，并鉴定了 394 种体内 N(alpha)-修饰肽，其中 371 种含有 IPI 注释蛋白 N 末端，23 种含有 IPI 未注释蛋白 N 末端。两个数据集的组合鉴定了 675 个 IPI 注释蛋白 N 末端和 88 个 IPI 未注释蛋白 N 末端的 N(alpha)-修饰。我们的结果表明，N 端乙酰转移酶（NATs）可能在体内作为 N 端甲酰转移酶（NFTs）和 N 端丙酰转移酶（NPTs）发挥作用。
