Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; Graduate Institute of Metabolism and Obesity Sciences, Taipei Medical University, Taipei, Taiwan.
Mol Cell Proteomics. 2021;20:100003. doi: 10.1074/mcp.TIR120.002148. Epub 2020 Nov 24.
We developed a simple and rapid method to enrich protein N-terminal peptides, in which the protease TrypN is first employed to generate protein N-terminal peptides without Lys or Arg and internal peptides with two positive charges at their N termini, and then, the N-terminal peptides with or without N-acetylation are separated from the internal peptides by strong cation exchange chromatography according to a retention model based on the charge/orientation of peptides. This approach was applied to 20 μg of human HEK293T cell lysate proteins to profile the N-terminal proteome. On average, 1550 acetylated and 200 unmodified protein N-terminal peptides were successfully identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N termini registered in the Swiss-Prot database. Because this method involves only two steps, protein digestion and chromatographic separation, without the need for tedious chemical reactions, it should be useful for comprehensive profiling of protein N termini, including proteoforms with neo-N termini.
我们开发了一种简单快速的方法来富集蛋白质 N 端肽，该方法首先使用蛋白酶 TrypN 产生不含赖氨酸或精氨酸的蛋白质 N 端肽和 N 端带有两个正电荷的内部肽，然后根据基于肽电荷/取向的保留模型，用强阳离子交换色谱将带有或不带有 N-乙酰化的 N 端肽与内部肽分离。该方法应用于 20 μg 人 HEK293T 细胞裂解物蛋白，以分析 N 端蛋白质组。在单次 LC/MS/MS 运行中，平均成功鉴定了 1550 个乙酰化和 200 个未修饰的蛋白质 N 端肽，内部肽的污染小于 3%，即使我们只接受 Swiss-Prot 数据库中注册的经典蛋白质 N 端。由于该方法仅涉及两步，即蛋白质消化和色谱分离，无需繁琐的化学反应，因此它应该对包括具有新 N 端的蛋白异构体在内的蛋白质 N 端的全面分析有用。
