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A simple and efficient method for selectively imaging and monitoring in situ the expression of sialylated glycoproteins on living cells has been developed. Treating living cells by mild periodate oxidation to selectively generate aldehydes on sialylated glycoproteins, followed by direct labeling of aldehydes with a commercially available fluorescent tag, fluorescein-5-thiosemicarbazide (FTSC), allows in situ imaging and quantification of sialylated glycoproteins on living cells. Under optimum reaction conditions, the periodate oxidation-based FTSC ligation (PF) strategy could be completed within 40 min. The cells undergoing the PF assay revealed a 91% viability and a fairly high-level of metabolic activity. Compared with current labeling methods, the PF assay proved to be a simpler and faster means of imaging sialylated glycoproteins on living cells. The PF assay has been successfully applied to imaging the location and quantification of the abundance of sialylated glycoproteins on tumor and normal cells. Our results demonstrated the methodological significance in clinical diagnosis and functional elucidation studies.

Qualitative and quantitative analysis of naturally-occurring complex glycans is essential for glycomics, which focuses on the studies of structure-function correlations of saccharides. We previously reported a one-pot procedure for the non-reductive release from glycoproteins and in situ labeling with 1-phenyl-3-methyl-5-pyrazolone (PMP) of O-glycans (C. Wang et al., Proteomics 11 (2011) 4229). Here we describe an HPLC-based O-glycan analytical strategy that combines a range of techniques including the one-pot procedure, independent separation by hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC) and identification by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). The complex mixtures of both neutral and sialylated O-glycans as bis-PMP derivatives released from glycoproteins using the one-pot procedure could be well separated by HILIC based on their size or by RP-HPLC depending on the type of linkage and their resulting three-dimensional (3D) structure, and their structure could be characterized by the ESI-MS and MS/MS analysis of the eluted glycan fractions. The validity of the current strategy was confirmed by the analysis of O-glycans released by the one-pot procedure from some standard glycoproteins, including porcine stomach mucin (PSM), bovine submaxillary mucin (BSM) and bovine fetuin. The applicability of the current method to complex biological samples was also demonstrated by the analysis of mucin-type glycans from fetal bovine serum (FBS) and frog egg-jelly coat (FEC). This strategy, a powerful analytical tool that features the combination of different techniques, is useful for the qualitative and quantitative O-glycan analysis of more complex biological samples and has a potential for constructing an O-glycan analysis and structure database., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A novel strategy is proposed, using cost-saving chemical reactions to generate intact free reducing N -glycans and their fluorescent derivatives from glycoproteins for subsequent analysis. N -Glycans without core α-1,3-linked fucose are released in reducing form by selective hydrolysis of the N -type carbohydrate–peptide bond of glycoproteins under a set of optimized mild alkaline conditions and are comparable to those released by commonly used peptide- N -glycosidase (PNGase) F in terms of yield without any detectable side reaction (peeling or deacetylation). The obtained reducing glycans can be routinely derivatized with 2-aminobenzoic acid (2-AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), and potentially some other fluorescent reagents for comprehensive analysis. Alternatively, the core α-1,3-fucosylated N -glycans are released in mild alkaline medium and derivatized with PMP in situ, and their yields are comparable to those obtained using commonly used PNGase A without conspicuous peeling reaction or any detectable deacetylation. Using this new technique, the N -glycans of a series of purified glycoproteins and complex biological samples were successfully released and analyzed by electrospray ionization mass spectrometry (ESI–MS) and tandem mass spectrometry (MS/MS), demonstrating its general applicability to glycomic studies. [ABSTRACT FROM AUTHOR]