基于二辛基化标记的蛋白质组N末端肽富集方法

Enrichment strategy of protein N-termini based on dioctyl labeling

蛋白质末端的研究不仅对于蛋白质的结构和功能注释十分必要,还能够为蛋白酶作用机理的揭示提供重要信息.针对传统蛋白质组N末端肽富集需要采用大量去除材料导致N末端肽回收率低,以及步骤繁琐、样品损失等问题,本文发展了一种基于二辛基化标记的蛋白质组N末端肽反向富集方法,可以在单次反相色谱分离中实现非N末端肽的去除.以酵母蛋白质酶解产物为样品对方法进行考察,结果表明,该方法具备较高的二辛基化标记效率(93%),且非N末端肽经二辛基化标记后能够显著偏移至强保留区.将其应用于酵母蛋白质N末端组的分析,共鉴定到237条蛋白原始N末端肽和133 neo-N末端肽,较富集前分别提高了2.1和3.3倍.此外,将该方法结合多种酶切方式,使N末端肽和neo-N末端肽的鉴定数目进一步提高37%和60%,促进了蛋白质N末端组鉴定覆盖度的提高.

The analysis of protein N-termini provides valuable information for protein structure and function annotation, and helps the profiling of proteases substrates and cleavage sites. However, most of the current N-terminal enrichment approaches involve multiple chromatographic separation processes and require a large amount of scavenger materials, which make it time and labor consuming and may induce significant sample loss. Herein, we develop a negative N-termini enrichment strategy based on dioctyl labeling. With yeast lysate digests as the sample, the strategy showed a high efficiency in dioctyl labeling and retention shift. Such a strategy was applied for the enrichment of N-terminal peptides from yeast cell lysates and enabled the identification of 237 original N-termini and 133 neo-N-termini, which was improved by 100% and 480% compared with direct analysis. Furthermore, with the combination of multi-proteases digestion, the number of protein N-termini and neo-N-termin was improved by 50% and 90%, respectively, facilitating the in-depth N-terminome analysis.