Abstract 1796 Uncovering Archaeal Glycoproteomes via Large-Scale Bioinformatic Analysis of Public Proteomic Data

Protein glycosylation, a complex and ubiquitous post-translational modification (PTM), is crucial for numerous cellular processes in archaea, such as motility, biofilm formation, and cell morphology.However, analyzing this PTM is challenging due to the diversity and structural complexity of glycans and the heterogeneity across glycosylation sites.This study aims to identify glycosylated proteins across diverse archaeal species, thereby enhancing our understanding of the significant roles that protein glycosylation plays in archaeal biology.To achieve this, we conducted bioinformatic reanalyses -under the Archaeal Proteome Project framework -of publicly available large-scale mass spectrometry datasets from PRIDE.Integrating knowledge of previously characterized glycans from ProGlycProt enabled us to perform systematic data processing on these large datasets to identify comprehensive glycoproteomes.In addition, for species where such data was not available, open modifications searches allowed us to characterize glycan compositions.Our results revealed 95 glycosites and 61 glycoproteins across six archaeal species, representing a 2.5-fold increase in known glycosites and a five-fold increase in glycoproteins for these species.Additionally, we characterized the N-glycan composition in Sulfolobus islandicus and identified 27 glycoproteins with 57 glycosites within this organism.These findings significantly expand the known scope of archaeal glycoproteins and establish a robust proteomic data mining approach for studying protein glycosylation in prokaryotes.In conclusion, this study highlights the utility of reanalyzing mass spectrometry datasets for system-wide characterization of glycosylation patterns.The approach we developed is broadly applicable for exploring glycoproteomes across archaea and bacteria, paving the way for glycosylation studies in diverse species with biomedical and biotechnological relevance.