Increasing the coverage of the N-terminome with LysN amino terminal enrichment (LATE).

The field of N-terminomics has been advancing with the development of novel methods that provide a comprehensive and unbiased view of the N-terminome. Negative selection N-terminomics enables the identification of free and naturally modified protein N-termini. Here, we present a streamlined protocol that combines two negative selection N-terminomics methods, LATE and HYTANE, to increase N-terminome coverage by 1.5-fold compared to using a single methodology. Our protocol includes sample preparation and data analysis of both methods and can be applied to studying the N-terminome of diverse samples. The suggested approach enables researchers to achieve a more detailed and accurate understanding of the N-terminome.