VIB Center for Medical Biotechnology, Ghent, Belgium.
Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
Methods Mol Biol. 2022;2477:293-309. doi: 10.1007/978-1-0716-2257-5_17.
Especially in eukaryotes, the N-terminal acetylation status of a protein reveals translation initiation sites and substrate specificities and activities of N-terminal acetyltransferases (NATs). Here, we discuss a bottom-up proteomics protocol for the enrichment of N-terminal peptides via strong cation exchange chromatography. This protocol is based on depleting internal tryptic peptides from proteome digests through their retention on strong cation exchangers, leaving N-terminally acetylated/blocked peptides enriched among the nonretained peptides. As such, one can identify novel N-terminal proteoforms and quantify the degree of N-terminal protein acetylation.
特别是在真核生物中，蛋白质的 N 端乙酰化状态揭示了翻译起始位点以及 N 端乙酰转移酶（NATs）的底物特异性和活性。在这里，我们讨论了一种通过强阳离子交换层析来富集 N 端肽的自下而上的蛋白质组学方案。该方案基于通过其在强阳离子交换剂上的保留来耗尽蛋白质酶解物中的内部胰蛋白酶肽，从而使 N 端乙酰化/封闭的肽在非保留肽中富集。因此，可以鉴定新的 N 端蛋白异构体并定量 N 端蛋白质乙酰化的程度。
