SECRET AGENT O-GlcNAcylates Hundreds of Proteins Involved in Diverse Cellular Processes in Arabidopsis

• Published in: Molecular & cellular proteomics 2024-04, Vol.23 (4), p.100732-100732, Article 100732
• Main Authors: Shrestha, Ruben, Karunadasa, Sumudu, Grismer, TaraBryn S., Reyes, Andres V., Xu, Shou-Ling
• Format: Article
• Language: English
• Subjects: lectin-weak affinity chromatography; mass spectrometry; O-GlcNAcylation; PRM; SILIA
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1016/j.mcpro.2024.100732

O-GlcNAcylation is a critical post-translational modification of proteins observed in both plants and animals and plays a key role in growth and development. While considerable knowledge exists about over 3000 substrates in animals, our understanding of this modification in plants remains limited. Unlike animals, plants possess two putative homologs: SECRET AGENT (SEC) and SPINDLY, with SPINDLY also exhibiting O-fucosylation activity. To investigate the role of SEC as a major O-GlcNAc transferase in plants, we utilized lectin-weak affinity chromatography enrichment and stable isotope labeling in Arabidopsis labeling, quantifying at both MS1 and MS2 levels. Our findings reveal a significant reduction in O-GlcNAc levels in the sec mutant, indicating the critical role of SEC in mediating O-GlcNAcylation. Through a comprehensive approach, combining higher-energy collision dissociation and electron-transfer high-energy collision dissociation fragmentation with substantial fractionations, we expanded our GlcNAc profiling, identifying 436 O-GlcNAc targets, including 227 new targets. The targets span diverse cellular processes, suggesting broad regulatory functions of O-GlcNAcylation. The expanded targets also enabled exploration of crosstalk between O-GlcNAcylation and O-fucosylation. We also examined electron-transfer high-energy collision dissociation fragmentation for site assignment. This report advances our understanding of O-GlcNAcylation in plants, facilitating further research in this field. [Display omitted] •SILIA/PRM reveals the prominent role of SEC in O-GlcNAcylation in Arabidopsis.•Remarkable expansion of O-GlcNAc substrates with the addition of 227 new targets.•O-GlcNAc found in similar protein family regions without consensus.•One-third targets overlap with O-fucosylation targets.•The study of EThcD underscores the need for ongoing algorithm optimization. O-GlcNAcylation, crucial for plants but poorly understood, was explored using LWAC and SILIA–PRM quantitative proteomics. We unveiled the prominent roles of SEC in O-GlcNAcylation. Through extensive fractionations, we expanded the O-GlcNAcome by adding 227 new targets, revealing broad regulatory roles and overlapping role with O-fucosylation.