A Systematic Investigation of Proteoforms with N‐Terminal Glycine and Their Dynamics Reveals Its Impacts on Protein Stability

The N‐termini of proteins can regulate their degradation, and the same protein with different N‐termini may have distinct dynamics. Recently, it was found that N‐terminal glycine can serve as a degron recognized by two E3 ligases, but N‐terminal glycine was also reported to stabilize proteins. Here we developed a chemoenzymatic method for selective enrichment of proteoforms with N‐terminal glycine and integrated dual protease cleavage to further improve the enrichment specificity. Over 2000 unique peptides with protein N‐terminal glycine were analyzed from >1000 proteins in human cells, and most of them are previously unknown, indicating the effectiveness of the current method to capture low‐abundance proteoforms with N‐terminal glycine. The degradation rates of proteoforms with N‐terminal glycine were quantified along with those of proteins from the whole proteome. Bioinformatic analyses reveal that proteoforms with N‐terminal glycine with the fastest and slowest degradation rates have different functions and localizations. Membrane proteins with N‐terminal glycine and proteins with N‐terminal glycine from the N‐terminal methionine excision degrade more rapidly. Furthermore, the secondary structures, adjacent amino acid residues, and protease specificities for N‐terminal glycine are also vital for protein degradation. The results advance our understanding of the effects of N‐terminal glycine on protein properties and functions.