Metabolic labeling and click chemistry detection of glycoprotein markers of mesenchymal stem cell differentiation.

Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.

• Level A · Stronger Clinical Evidence
• Level B · Emerging clinical evidence with positive signals
• Level C · Early human research exploring benefits
• Level D · Scientific groundwork from lab and animal studies
• Emerging · Emerging topic under active research

Abstract (original English)

Human mesenchymal stem cells (hMSCs) are multipotent stem cells that can differentiate into a variety of cell types in vitro including osteoblasts, adipocytes, and chondrocytes. Here we apply a metabolic labeling approach to characterize changes in cellular glycoprotein expression during hMSC differentiation and to identify glycoprotein markers unique to differentiated cell types. The two-step labeling method involves the metabolic incorporation of unnatural azido-modified sugars into protein glycans and subsequent ligation with fluorescent azide-reactive detection probes utilizing the copper (I)-catalyzed cycloaddition reaction between azides and alkynes, or "click" chemistry. Metabolic labeling of cell surface O-linked or sialic acid-containing glycoproteins, or intracellular O-GlcNAc-modified proteins was accomplished by feeding cells the tetraacetylated azide-modified sugar precursors, GalNAz, ManNAz, or GlcNAz, respectively, for 48-72 h prior to harvesting the cells. The cells were then lysed, and protein extracts were reacted with a fluorescent alkyne detection probe. Labeled glycoproteins were analyzed by 1D and 2D gel electrophoresis and detected by fluorescence imaging. Our results demonstrate highly sensitive labeling of O-linked, sialic acid-containing, and O-GlcNAc modified proteins in all cell types without affecting cell growth or morphology. Selective labeling of

Evidence level

Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.

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