Faculty Collaboration Database

Studying the O-GlcNAcome of human placentas using banked tissue samples. Glycobiology 2024 Apr 10;34(4)

Date

01/23/2024

Pubmed ID

38253038

Pubmed Central ID

PMC11005170

DOI

10.1093/glycob/cwae005

Scopus ID

2-s2.0-85190109720 (requires institutional sign-in at Scopus site)

Abstract

O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 μg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.

Author List

Luna S, Malard F, Pereckas M, Aoki M, Aoki K, Stichelen SO

Authors

Kazuhiro Aoki PhD Associate Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of Wisconsin
Stephanie Olivier-Van Stichelen PhD Assistant Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Acetylglucosamine
Female
Humans
Phosphorylation
Placenta
Pregnancy
Protein Processing, Post-Translational
Proteins