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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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Fast, sensitive, and simple methods for quantitative analysis of disparities in glycan expression between different biological samples are essential for studies of protein glycosylation patterns (glycomics) and the search for disease glycan biomarkers. Relative quantitation of glycans based on stable isotope labeling combined with mass spectrometric detection represents an emerging and promising technique. However, this technique is undermined by the complexity of mass spectra of isotope-labeled glycans caused by the presence of multiple metal ion adduct signals, which result in a decrease of detection sensitivity and an increase of difficulties in data interpretation. Herein we report a simplified quantitative glycomics strategy, which features nonreductive isotopic labeling of reducing glycans with either nondeuterated (d0-) or deuterated (d5-) Girard's reagent P (GP) without salts introduced and simplified mass spectrometric profiles of d0- and d5-GP derivatives of neutral glycans as molecular ions without complex metal ion adducts, allowing rapid and sensitive quantitative comparison between different glycan samples. We have obtained optimized GP-labeling conditions and good quantitation linearity, reproducibility, and accuracy of data by the method. Its excellent applicability was validated by comparatively quantitative analysis of the neutral N-glycans released from bovine and porcine immunoglobulin G as well as of those from mouse and rat sera. Additionally, we have revealed the potential of this strategy for the high-sensitivity analysis of sialylated glycans as GP derivatives, which involves neutralization of the carboxyl group of sialic acid by chemical derivatization.

Qualitative and quantitative studies of glycosylation patterns of various biologically important proteins represent a key field for the understanding of their complex structure-function relationships. However, the analysis of glycoprotein glycans is usually undermined by tedious sample processing steps prior to detection, including deproteination, desalting and removal of some other non-glycan impurities, which results in considerable sample loss and increased difficulty of quantitative analysis. Herein we report a facile and versatile method for the quantitative isolation of reducing glycans from complex samples using sulfonyl hydrazine-functionalized polystyrene (SHPS) beads, namely the SHPS-based glycan capturing procedure. This method allows the chemoselective and efficient condensation of the aldehyde group of reducing glycans with the active sulfonyl hydrazine group of SHPS beads under anhydrous conditions, resulting in the formation of sulfonyl hydrazone conjugates. The non-glycan components in samples, such as proteins, salts and some other impurities, can be completely removed by washing the sulfonyl hydrazone conjugates. Regeneration of the reducing glycans can be performed via mild hydrolysis of the washed sulfonyl hydrazone conjugates. This procedure is compatible with almost all the current techniques for the derivatization or detection of reducing glycans. We have obtained essential data for this method, including optimized reaction conditions, linearity and reproducibility for glycan quantitation, as well as a final glycan recovery ratio. Moreover, mass spectrometric analysis of the glycans from some complex biological samples, including milk, human blood plasma and fetal bovine serum, was achieved, demonstrating good applicability of this novel procedure.

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.)