Metabolic Labeling of Glycans with Azido Sugars for Visualization and Glycoproteomics

Laughlin, Scott T.; Agard, Nicholas J.; Baskin, Jeremy M.; Carrico, Isaac S.; Chang, Pamela V.; Ganguli, Anjali S.; Hangauer, Matthew J.; Lo, Anderson; Prescher, Jennifer A.; Bertozzi, Carolyn R.

doi:10.1016/s0076-6879(06)15015-6

Cited by 129 publications

(99 citation statements)

References 42 publications

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“…Furthermore, using alkyne or phosphine-based fluorescent reagents, we anticipate that this approach can be extended to a solution or gel-based assay to determine the concentration of sulfenic acids in purified proteins in vitro. [43][44][45] To evaluate the specificity of the DAz-1 probe in the context of a different protein, we used human serum albumin (HSA). HSA is the most abundant protein in plasma and is proposed to have an antioxidant role in biological systems.…”

Section: Resultsmentioning

confidence: 99%

A chemical approach for detecting sulfenic acid-modified proteins in living cells

et al. 2008

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Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO 2 H) and sulfonic acids (Cys-SO 3 H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acidmodified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting protein sulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteine residues.

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Section: Resultsmentioning

confidence: 99%

A chemical approach for detecting sulfenic acid-modified proteins in living cells

et al. 2008

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“…The cells were then harvested and lysed. Azide-modified glycoprotein samples were labeled with a biotin-alkyne and precipitated (30,31). The precipitated sample was resolubilized and incubated on streptavidin-bound Dynabeads (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Sialic Acids Attached to O-Glycans Modulate Voltage-gated Potassium Channel Gating

Schwetz

Norring

Ednie

et al. 2011

Journal of Biological Chemistry

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“…This involved metabolic labeling of primary cultured HUVECs exposed or not to gaseous hypoxia by peracetylated Ac 4 ManNAz, resulting in the incorporation of modified N-azidoacetylsialic acid into the cell glycans (10,16). The metabolically modified sialoglycoproteins react chemically on the surface of living cells with biotinylated triarylphosphine via Staudinger ligation through their reactive functional azido groups.…”

Section: Analysis Of Sialoglycoproteome In Huvecs-mentioning

confidence: 99%

Targeted Identification of Sialoglycoproteins in Hypoxic Endothelial Cells and Validation in Zebrafish Reveal Roles for Proteins in Angiogenesis

Delcourt

Quevedo

Nonne

et al. 2015

Journal of Biological Chemistry

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Background: Target identification on tumor microvascularization is an opportunity for cancer therapy. Results: Membrane and secreted glycoproteins were identified on endothelial cells under hypoxia, and their function was assessed in zebrafish. Conclusion: Three novel hypoxia-regulated glycoproteins involved in angiogenesis have been identified and validated. Significance: We describe an approach that can be used to rapidly identify novel angiogenesis-related genes and their protein products.

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Metabolic Labeling of Glycans with Azido Sugars for Visualization and Glycoproteomics

Cited by 129 publications

References 42 publications

A chemical approach for detecting sulfenic acid-modified proteins in living cells

A chemical approach for detecting sulfenic acid-modified proteins in living cells

Sialic Acids Attached to O-Glycans Modulate Voltage-gated Potassium Channel Gating

Targeted Identification of Sialoglycoproteins in Hypoxic Endothelial Cells and Validation in Zebrafish Reveal Roles for Proteins in Angiogenesis

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