A novel 2D‐electrophoresis method for the simultaneous visualization of phosphorylated and O‐GlcNAcylated proteoforms of a protein

• Published in: Electrophoresis 2024-09, Vol.45 (17-18), p.1618-1629
• Main Authors: Bulangalire, Nathan, Claeyssen, Charlotte, Douffi, Sana, Agbulut, Onnik, Cieniewski‐Bernard, Caroline
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - analysis; Acetylglucosamine - chemistry; Acetylglucosamine - metabolism; Animals; Cartography; Desmin - analysis; Desmin - chemistry; Desmin - metabolism; Electrophoresis; Electrophoresis, Gel, Two-Dimensional - methods; Glycosylation; Humans; Life Sciences; O‐N‐acetyl‐β‐d‐glucosaminylation; Phosphorylation; Protein Processing, Post-Translational; Proteins; proteoform; two‐dimensional electrophoresis; Visualization; αB‐crystallin
• ISSN: 0173-0835; 1522-2683; 1522-2683
• DOI: 10.1002/elps.202400043

Post‐translational modifications (PTMs), such as phosphorylation and O‐N‐acetyl‐β‐d‐glucosaminylation (O‐GlcNAcylation), are involved in the fine spatiotemporal regulation of protein functions, and their dynamic interplay is at the heart of protein language. The coexistence of phosphorylation and O‐GlcNAcylation on a protein leads to the diversification of proteoforms. It is therefore essential to decipher the phosphorylation/O‐GlcNAcylation interplay on protein species that orchestrates cellular processes in a specific physiological or pathophysiological context. However, simultaneous visualization of phosphorylation and O‐GlcNAcylation patterns on a protein of interest remains a challenge. To map the proteoforms of a protein, we have developed an easy‐to‐use two‐dimensional electrophoresis method with a single sample processing permitting simultaneous visualization of the phosphorylated and the O‐GlcNAcylated forms of the protein of interest. This method, we termed 2D‐WGA‐Phos‐tag‐PAGE relies on proteoforms retardation by affinity gel electrophoresis. With this novel approach, we established the cartography of phospho‐ and glycoforms of αB‐crystallin and desmin in the whole extract and the cytoskeleton protein subfraction in skeletal muscle cells. Interestingly, we have shown that the pattern of phosphorylation and O‐GlcNAcylation depends of the subcellular subfraction. Moreover, we have also shown that proteotoxic stress condition increased the complexity of the pattern of PTMs on αB‐crystallin.