Identification and quantification of sialylated and core-fucosylated N-glycans in human transferrin by UPLC and LC-MS/MS.

Sialylated and core-fucosylated N-glycans in human transferrin (HTF) are used as glycan biomarkers due to their increased or decreased characteristics in certain diseases. However, their absolute quantities remain unclear. In this study, N-glycans of HTF were identified by UPLC and LC-MS/MS using fluorescence tags [2-aminobenzamide (AB) and procainamide (ProA)] and columns [HILIC and anion exchange chromatography-HILIC (AXH)]. The structures of 14 (including five core-fucosylated) N-glycans in total comprising two non-, six mono-, four di-, and two tri-sialylated N-glycans were identified. The quantities (%) of each N-glycan relative to the total N-glycans (100%) were obtained. HILIC and AXH were better for peak identification and separability except for desialylation, respectively. Specifically, sialylated (in ProA-HILIC and ProA-AXH by UPLC or LC-MS/MS) and core-fucosylated (in AB-HILIC and ProA-AXH by UPLC) N-glycans were efficiently identified. Seven neuraminidase-treated (including three core-fucosylated) N-glycans were efficiently identified in ProA-AXH, even their poor separation. Additionally, ProA-AXH was more efficient for the estimation of the absolute quantities of N-glycans from the results of fluorescence intensity (by UPLC) and relative quantity (by LC-MS/MS). These results first demonstrate that ProA is useful for identifying and quantifying sialylated, core-fucosylated, and neuraminidase-treated desialylated N-glycans in HTF using AXH by UPLC and LC/MS.

Structural and Quantitative Characterization of Mucin-Type O-Glycans and the Identification of O-Glycosylation Sites in Bovine Submaxillary Mucin.

Bovine submaxillary mucin (BSM) is a gel-forming glycoprotein polymer, and Ser/Thr-linked glycans (O-glycans) are important in regulating BSM's viscoelasticity and polymerization. However, details of O-glycosylation have not been reported. This study investigates the structural and quantitative characteristics of O-glycans and identifies O-glycosylation sites in BSM using liquid chromatography-tandem mass spectrometry. The O-glycans (consisting of di- to octa-saccharides) and their quantities (%) relative to total O-glycans (100%; 1.1 pmol per 1 µg of BSM) were identified with 14 major (>1.0%), 12 minor (0.1%-1.0%), and eight trace (<0.1%) O-glycans, which were characterized based on their constituents (sialylation (14 O-glycans; 81.9%, sum of relative quantities of each glycan), non-sialylation (20; 18.1%), fucosylation (20; 17.5%), and terminal-galactosylation (6; 3.6%)) and six core structure types [Gal-GalNAc, Gal-(GlcNAc)GalNAc, GlcNAc-GalNAc, GlcNAc-(GlcNAc)GalNAc, and GalNAc-GalNAc]. O-glycosylation sites were identified using O-glycopeptides (bold underlined; 56SGETRTSVI, 259SHSSSGRSRTI, 272GSPSSVSSAEQI, 307RPSYGAL, 625QTLGPL, 728TMTTRTSVVV, and 1080RPEDNTAVA) obtained from proteolytic BSM; these sites are in the four domains of BSM. The gel-forming mucins share common domain structures and glycosylation patterns; these results could provide useful information on mucin-type O-glycans. This is the first study to characterize O-glycans and identify O-glycosylation sites in BSM.

Profiles of plant core-fucosylated N-glycans of acid alpha-glucosidases produced in transgenic rice cell suspension cultures treated with eight different conditions.

Recombinant human acid alpha-glucosidase (rhGAA) from Chinese hamster ovary cells is the only approved treatment for patients with Pompe disease. In this study, rhGAAs were produced in transgenic rice cell suspension cultures under eight different conditions; untreated, 5 µM of 2-fluoro-l-fucose (2-FF), 50 µM of 2-FF, 100 µM of 2-FF, 100 µM of 2-FF + 0.5% Pluronic F-68 (PF-68), 100 µM of 2-FF + 0.05% Tween 20 (Tw 20), 0.5% PF-68, and 0.05% Tw 20. The N-glycans of eight rhGAAs were analyzed using ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry. The relative quantity (%) of each glycan was obtained from the corresponding UPLC peak area per the sum (100%) of individual UPLC peak area. Fifteen N-glycans, comprising seven core-fucosylated glycans (71.5%, sum of each relative quantities) that have immunogenicity-inducing potential, three de-core-fucosylated glycans (15.4%), and five non-core-fucosylated glycans (13.1%), were characterized with high mass accuracy and glycan-generated fragment ions. The increases or decreases of relative quantities of each glycan from seven rhGAAs were compared with those of untreated control. The percentages of the sum of the relative quantities of core-fucosylated glycans divided by the sums of those of de-core- (core-fucose removed) and non-core-fucosylated glycans were calculated, and the lowest percentage was obtained in 100 µM of 2-FF combined with 0.5% PF-68. These results indicate that the relative quantity of each glycan of rhGAA produced in rice cell suspension cultures is significantly affected by their culture condition. This study performed the comparison of the N-glycan profiles of rice cell-derived rhGAA to identify the core-fucosylated glycans using UPLC and tandem mass spectrometry.

N-glycans of bovine submaxillary mucin contain core-fucosylated and sulfated glycans but not sialylated glycans.

Bovine submaxillary mucin (BSM) is a heavily-glycosylated macromolecular (approximately 4 MDa) protein and is used in various biomaterial applications in light of its high viscosity and biocompatibility, in addition to use as a biochemical substrate or inhibitor as a result of its abundant O-glycans. Although it has been reported that N-glycosylation provides stability of human mucins, most BSM research has been focused on its O-glycans, while N-glycans have not been reported to date. In this study, a common N-glycan core component was detected by monosaccharide analysis of BSM, and the structures of the N-glycans and their relative quantities were determined by liquid chromatography-tandem mass spectrometry. Seventeen N-glycans comprising ten complex-type [Fucose0~2Hexose3~4N-acetylhexosamine1~6Sulfate0~1; 61.1% (the sum of the relative quantities of each N-glycan out of the total N-glycans)], two high-mannose-type (Hexose5~6N-acetylhexosamine2; 12.0%), and five paucimannose type (Fucose0~1Hexose3~4N-acetylhexosamine2~3; 26.9%) were identified, but no hybrid-type or sialylated N-glycans were found. Additionally, these are less-branched structures compared to human mucins. Of these, ten glycans (77.2%), including two sulfated glycans (8.0%), were core fucosylated, which confer unique biological functions to glycoproteins. The N-glycosylation sites were identified from the analysis of glycopeptides from BSM. This study is the first confirmation of N-glycan attachment to BSM.

Seventeen O-acetylated N-glycans and six O-acetylation sites of Myozyme identified using liquid chromatography-tandem mass spectrometry.

O-acetylated sialic acid (SA) attached to the N-glycans of therapeutic glycoproteins reportedly inhibit sialidase activity, increase protein half-life, decrease protein antigenicity, and stabilize protein conformation. Recombinant human acid α-glucosidase (Myozyme) is the only drug approved by the United States Food and Drug Administration for the treatment of Pompe disease. In this study, unreported N-glycans containing O-acetylated SA in Myozyme and the relative quantities of total glycans were investigated using liquid chromatography (LC)-electrospray ionization (ESI)-high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS). The 17 N-glycans (6.4% of total glycans) containing mono-, di-, mono/di-, and di/di-O-acetylated N-acetylneuraminic acid (Neu5Ac) were identified with mass accuracy, glycan-generated fragment ions, and the retention time on an LC column. The analysis of peptides containing mono- and/or di-O-acetylated Neu5Ac ions sorted from all peptides using nano-LC-ESI-HCD-MS/MS confirmed six O-acetylation sites (Asn 140, Asn 233, Asn 390, Asn 470, Asn 652, and Asn 882), at least five of which (Asn 140, Asn 233, Asn 390, Asn 470, and Asn 652) could contribute to the drug efficacy or cellular uptake of Myozyme. This is the first study to identify N-glycans containing O-acetylated Neu5Ac and O-acetylation sites in Myozyme.

Qualitative and quantitative characterization of sialylated N-glycans using three fluorophores, two columns, and two instrumentations.

Sialylation can influence the stability, half-life, and immunogenicity of glycoproteins, but sialylated N-glycans are known to be difficult to analyze. Human alpha1-acid glycoprotein (AGP) is reported to have glycans that consist of sialylated N-glycans. The N-glycan profiling of AGP is qualitatively and quantitatively investigated here by UPLC and LC-ESI-MS/MS. Three fluorescent tags (AB, AA, and ProA) and two separation columns (HILIC and AEX-HILIC) were adopted to confirm and compare each analytical characteristic. The results of AA were comparable to those of the well-established AB. The qualification of ProA was notable due to its superior fluorescence intensity and ionization efficiency, and ProA showed smaller quantitative or larger-sized fragments in LC-ESI-MS/MS compared to AB and AA. However, the MS quantification of ProA was distorted because the increased sialylation level decreased the LC-ESI-MS/MS ionization efficiency. HILIC had better peak separability, AEX-HILIC had an advantage in UPLC sialylation profiling, and each isomeric glycan could be identified by both columns in LC-ESI-MS/MS. In conclusion, ProA is favored for UPLC and LC-ESI-MS/MS detection but not reliable for MS quantification. This study firstly demonstrates the qualification and quantification of sialylated N-glycans by comparing the commonly used analytical conditions with different fluorescent tags, columns, and instruments.