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A practical method for preparing fluorescent-labeled glycans with a 9-fluorenylmethyl derivative to simplify a fluorimetric HPLC-based analysis
摘要： Analysis of glycans in glycoproteins is often performed by liquid chromatography (LC) separation coupled with fluorescence detection and/or mass spectrometric detection. Enzymatically or chemically released glycans from glycoproteins are usually labeled by reductive amination with a fluorophore reagent. Although labeling techniques based on reductive amination have been well-established as sample preparation methods for fluorometric HPLC-based glycan analysis, they often include time-consuming and tedious purification steps. Here, we reported an alternative fluorescent labeling method based on the synthesis of hydrazone and its reduction using 9-fluorenylmethyl carbazate (Fmoc-hydrazine) as a fluorophore reagent. Using isomaltopentaose and N-glycans from human IgG, we optimized the Fmoc-labeling conditions and purification procedure of Fmoc-labeled N-glycans and applied the optimized method for the analysis of N-glycans released from four glycoproteins (bovine RNase B, human fibrinogen, (cid:2)1-acid glycoprotein, and bovine fetuin). The complete workflow for preparation of fluorescent-labeled N-glycans takes a total of 3.5 h and is simple to implement. The method presented here lowers the overall cost of a fluorescently labeled N-glycan and will be practically useful for the screening of disease-related glycans or routine analysis at an early stage of development of biopharmaceuticals.
关键词： Fluorescence detection,HPLC,N-glycans,Fmoc-hydrazine
更新于2025-09-23 15:19:57
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Laser cleavable probes for <i>in situ</i> multiplexed glycan detection by single cell mass spectrometry
摘要： Glycans binding on the cell surface through glycosylation play a key role in controlling various cellular processes, and glycan analysis at a single-cell level is necessary to study cellular heterogeneity and diagnose diseases in the early stage. Herein, we synthesized a series of laser cleavable probes, which could sensitively detect glycans on single cells and tissues by laser desorption ionization mass spectrometry (LDI-MS). This multiplexed and quantitative glycan detection was applied to evaluate the alterations of four types of glycans on breast cancer cells and drug-resistant cancer cells at a single-cell level, indicating that drug resistance may be related to the upregulation of glycan with a b-D-galactoside (Galb) group and Neu5Aca2-6Gal(NAc)-R. Moreover, the glycan spatial distribution in cancerous and paracancerous human tissues was also demonstrated by MS imaging, showing that glycans are overexpressed in cancerous tissues. Therefore, this single-cell MS approach exhibits promise for application in studying glycan functions which are essential for clinical biomarker discovery and diagnosis of related diseases.
关键词： mass spectrometry,breast cancer,drug resistance,laser cleavable probes,single-cell analysis,glycans
更新于2025-09-16 10:30:52
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DNA-Stabilized Silver Nanoclusters for Label-Free Fluorescence Imaging of Cell Surface Glycans and Fluorescence Guided Photothermal Therapy
摘要： A multifunctional nanoplatform that enables the integration of biological detection, imaging diagnosis, and synergistic therapy into a single nanostructure holds great promise for nanoscience and nanomedicine. Herein, a novel theranostic platform was presented for label-free imaging of cell surface glycans based on DNA/silver nanoclusters (AgNCs) via hybridization chain reaction (HCR) and fluorescence guided photothermal therapy (PTT). In this strategy, a dibenzocyclooctyne (DBCO)-functionalized DNA and two hairpin structures of DNA/AgNCs probes were involved. Following metabolic glycan labeling, the binding of DBCO-functionalized DNA to cell surface initiated HCR, and then cell surface glycans were specifically labeled by DNA/AgNCs fluorescent probes. Furthermore, this signal amplification strategy was adopted in quantitative analysis, and the detection limit could be achieved as low as 20 cells in 200 μL binding buffer. Moreover, the remarkable photothermal properties of DNA/AgNCs via HCR, led to efficient killing of cancer cell and inhibited the tumor growth under imaging guide. In this strategy, DNA/AgNCs were utilized to detect the cellular glycans, which aided in overcoming the high cost and instability of fluorescent dyes. Simultaneously, the HCR process avoided the introduction of excessive azido-sugars under the precondition of ensuring apparent fluorescence. These results indicated that the developed nanoplatform has great potential for specific cell surface glycans imaging and fluorescence guided PTT.
关键词： DNA-stabilized silver nanoclusters,fluorescence guided photothermal therapy,hybridization chain reaction,cell surface glycans,label-free fluorescence imaging
更新于2025-09-09 09:28:46