title: Comparative analysis of Cu (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) in O-GlcNAc proteomics.
abstract: O-linked β-N-acetylglucosamine (O-GlcNAc) is emerging as an essential protein post-translational modification in a range of organisms. It is involved in various cellular processes such as nutrient sensing, protein degradation, gene expression, and is associated with many human diseases. Despite its importance, identifying O-GlcNAcylated proteins is a major challenge in proteomics. Here, using peracetylated N-azidoacetylglucosamine (Ac4 GlcNAz) as a bioorthogonal chemical handle, we described a gel-based mass spectrometry method for the identification of proteins with O-GlcNAc modification in A549 cells. In addition, we made a labeling efficiency comparison between two modes of azide-alkyne bioorthogonal reactions in click chemistry: copper-catalyzed azide-alkyne cycloaddition (CuAAC) with Biotin-Diazo-Alkyne and stain-promoted azide-alkyne cycloaddition (SPAAC) with Biotin-DIBO-Alkyne. After conjugation with click chemistry in vitro and enrichment via streptavidin resin, proteins with O-GlcNAc modification were separated by SDS-PAGE and identified with mass spectrometry. Proteomics data analysis revealed that 229 putative O-GlcNAc modified proteins were identified with Biotin-Diazo-Alkyne conjugated sample and 188 proteins with Biotin-DIBO-Alkyne conjugated sample, among which 114 proteins were overlapping. Interestingly, 74 proteins identified from Biotin-Diazo-Alkyne conjugates and 46 verified proteins from Biotin-DIBO-Alkyne conjugates could be found in the O-GlcNAc modified proteins database dbOGAP (http://cbsb.lombardi.georgetown.edu/hulab/OGAP.html). These results suggested that CuAAC with Biotin-Diazo-Alkyne represented a more powerful method in proteomics with higher protein identification and better accuracy compared to SPAAC. The proteomics credibility was also confirmed by the molecular function and cell component gene ontology (GO). Together, the method we reported here combining metabolic labeling, click chemistry, affinity-based enrichment, SDS-PAGE separation, and mass spectrometry, would be adaptable for other post-translationally modified proteins in proteomics.
authors: Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, USA.
authors: Shanshan
authors: S
authors: Li
authors: Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, USA.
authors: He
authors: H
authors: Zhu
authors: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin, P. R. China.
authors: Jiajia
authors: J
authors: Wang
authors: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin, P. R. China.
authors: Xiaomin
authors: X
authors: Wang
authors: Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, USA.
authors: Xu
authors: X
authors: Li
authors: Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, USA.
authors: Cheng
authors: C
authors: Ma
authors: Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, USA.
authors: Liuqing
authors: L
authors: Wen
authors: Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, USA.
authors: Bingchen
authors: B
authors: Yu
authors: Department of Pathology, Shanxi Tumor Hospital, Taiyuan, P. R. China.
authors: Yuehua
authors: Y
authors: Wang
authors: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin, P. R. China.
authors: Jing
authors: J
authors: Li
authors: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin, P. R. China. pwang11@gsu.edu.|Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, USA. pwang11@gsu.edu.
authors: Peng George
authors: PG
authors: Wang
mesh_terms: D000072283:A549 Cells; D000117:Acetylglucosamine / Q000737:chemistry*; D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D002384:Catalysis; D057930:Click Chemistry; D003300:Copper / Q000737:chemistry; D061565:Cycloaddition Reaction; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*
chemical_list: D000480:Alkynes; D001386:Azides; D003300:Copper; D000117:Acetylglucosamine
keywords: Bioorthogonal chemistry; Biotin-DIBO-Alkyne; Biotin-Diazo-Alkyne; O-GlcNAc; Proteomics