Enrichment strategy of protein N-termini based on dioctyl labeling

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.