Influence of N-Linked glycosylation on proteolytic processing

The proteolytic process is crucial for a multitude of proteomic analysis by Mass Spectrometry (MS). One of the factors influencing this process is the presence of Post Translational Modifications (PTMs), such as N-linked as well as O-linked glycosylation, on specific amino acid residues. The N-linked glycosylation and proteolytic cleavage processes are explored in their individual contexts and interrelated dynamics. We aim to elucidate how N-linked glycosylation influences protease cleavage, especially within the framework of proteomics bottom-up approach. Here, we have systematically investigated how differently N-glycosylated proteins and synthetic peptides influence trypsin cleavage. The results provide evidence for this influence and hint for the direct effects of specific glycostructures near to protease recognition sites. This work identifies current challenges in understanding this complex relationship and suggests future research directions that could significantly impact widely used glycoproteomics workflows. By providing a detailed analysis of the interplay between N-linked glycosylation and protease cleavage, this work also contribute to a deeper understanding of their roles in protein processing and molecular biology, underscoring the importance of these processes in cellular functions and disease mechanisms.