Detection and identification ofO-GlcNAc-modified proteins using 6-azido-6-deoxy-N-acetyl-galactosamine

Abstract

Ac36AzGalNAc, as a metabolic probe, can robustly label O-GlcNAc-modified proteins in cells with high selectivity.

Analytical and Biochemical Perspectives of Protein O-GlcNAcylation

“…Besides Ac 4 GlcNAz and Ac 4 GalNAz, many other unnatural monosaccharide analogues have been synthesized (Figure ), including 1,3,4,6-tetra- O -acetyl- N -4-pentynylglucosamine (Ac 4 GlcNAlk), 1,3,4-tri- O -acetyl-2-deoxy-2- N -acetyl-6-deoxy-azidoglucopyranose (Ac 3 6AzGlcNAc), 1,3,6-tri- O -acetyl-2-azidoacetamido-2,4-dideoxy- d -glucopyranose (Ac 3 4dGlcNAz), 1,3,4-tri- O -acetyl-2-deoxy-2- N -acetyl-6-deoxy-alkynylglucopyranose (Ac 3 6AlkGlcNAc), 1,3,4,6-tetra- O -acetyl-2-azido-2-deoxyglucose (Ac 4 2AzGlc), , 1,3,4-tri- O -acetyl-2-azido-2-deoxyglucose (Ac 3 2AzGlc), 1,3,4,6-tetra- O -acetyl-6-azido-6-deoxy-glucose (Ac 4 6AzGlc), 1,3,4-tri- O -acetyl-2-acetamido-6-azido-2,6-dideoxy- d -galactopyranoside (Ac 3 6AzGalNAc), a glucosamine nitrobenzoxadiazole (GlcN-NBD) precursor (i.e., Ac 3 GlcN-β-Ala-NBD-α-1-P­(Ac-SATE) 2 ), a diazirine-modified GlcNAc analogue Ac 3 GlcNDAz-1-P­(Ac-SATE) 2 , and a methylcyclopropene GlcNAc analogue (Ac 4 GlcNCyoc). , After feeding cells with these metabolic probes, the unnatural analogues will partially replace the natural UDP-GlcNAc for O-GlcNAcylation. The resulting chemical handles on proteins can be derivatized via bioorthogonal reactions (e.g., with alkynyl- or azido-conjugated biotin) with subsequent imaging (using fluorescent or radionuclide-labeled avidins) for the detection of O-GlcNAcylation on proteins.…”

“…Although the detection is mostly performed in vitro (e.g., on cell lysates) with in-gel imaging, derivatization can occur in situ by using cellular compatible bioorthogonal reactions followed by tagging with fluorescent dyes for imaging with fluorescence microscopy. To avoid the cytotoxicity of Cu + in CuAAC, several metabolic probes have been coupled with Cu + -free SPAAC (e.g., by using DIBO-biotin) and fluorescent reagents (e.g., neutravidin-conjugated Alexa Flour dyes) for imaging intracellular O-GlcNAcylation. ,, To reduce nonspecific reactivity of strained alkynes used in SPAAC and increase intracellular click reaction kinetics, inverse-electron-demand Diels–Alder reaction (IEDDA or DA INV ), the latest generation of bioorthogonal reaction, has been purposed to detect metabolically labeled proteins by Ac 4 GlcNCyoc with cellular imaging . Of special note, metabolic labeling has also been coupled with the genetically encoded FRET-based sensor, a method specifically designed to visualize the O-GlcNAc status on specific proteins in living cells .…”

The Metabolic Chemical Reporter Ac46AzGal Could Incorporate Intracellular Protein Modification in the Form of UDP-6AzGlc Mediated by OGT and Enzymes in the Leloir Pathway

“…Finally, biotin or the fluorescent tag is labeled on the glycan-modified protein through bio-orthogonal click chemical reaction to realize visualization and enrichment of glycoproteins. Currently, an increasing number of peracetylated O-GlcNAc analogues for O-GlcNAc metabolic markers have been developed and characterized, such as modification at the 2-position of HexNAc ( Vocadlo et al, 2003 ; Boyce et al, 2011 ; Tan et al, 2018 ) or at the 4-/6-sites ( Chuh et al, 2014 ; Li et al, 2016 ; Chuh et al, 2017 ; Darabedian et al, 2018 ; Guo et al, 2019 ), and all the reported functionalized GlcNAc analogues demonstrated satisfactory selectivity and efficiency, which exhibited a wide range of substrate tolerance of OGT ( Li et al, 2018 ). Meanwhile, any nonanalogues of GlcNAc, 6-azido-6-deoxy-glucose (6AzGlc) ( Darabedian et al, 2018 ), and 2-azido-2-deoxy-glucose (2AzGlc) ( Zaro et al, 2017 ) are also reported as the substrates of OGT to modify O-GlcNAc proteins.…”

Mass Spectrometry for O-GlcNAcylation

“…for subsequent enrichment, as shown in Figure 1 . Generally, cells were cultured with metabolic chemical reporters (MCRs) such as Ac 4 GlcNAz ( Sprung et al, 2005 ), Ac 4 GlcNAlk ( Zaro et al, 2011 ), Ac 3 6AzGlcNAc ( Chuh et al, 2014 ), Ac 3 4dGlcNAz ( Li et al, 2016 ), Ac 3 6AlkGlcNAc ( Chuh et al, 2017 ), Ac 4 6AzGlc ( Darabedian et al, 2018 ), Ac 3 6AzGalNAc ( Guo et al, 2019 ) and 1,3-Pr 2 GalNAz ( Hao et al, 2019 ), etc., to synthesize O-GlcNAcylated proteins with active reactive groups. Then, the biotin probes with corresponding reactive groups ( Figure 1B ) were introduced to tag the labeled O-GlcNAcylations through staudinger linkage, copper-catalyzed azido-alkyne cycloaddition (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC).…”