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.
•Sialylated N-glycans are known to be difficult to analyze due to negative charge.•This study qualitatively and quantitatively investigated sialylated N-glycans.•ProA is favored for LC (-MS/MS) detection but not reliable for MS quantification.•HILIC and AEX-HILIC each had strengths in peak separability and sialylation profiling.•Increased SA level decreased the ionization efficiency on LC-MS compare to UPLC.
Bovine submaxillary mucin (BSM) is a natural polymer used in biomaterial applications for its viscoelasticity, lubricity, biocompatibility, and biodegradability.
-glycans are important for mucin ...stability and function, but their structures have not been fully characterized, unlike that of
-glycans. In this study, BSM
-glycans were investigated using liquid chromatography-tandem mass spectrometry. The microheterogeneous structures of 32
-glycans were identified, and the quantities (%) of each
-glycan relative to total
-glycans (100%) were obtained. The terminal
-acetylgalactosamines in 12
-glycans (sum of relative quantities; 27.9%) were modified with mono- (10 glycans) and disulfations (2 glycans). Total concentration of all sulfated
-glycans was 6.1 pmol in BSM (20 µg), corresponding to 25.3% of all negatively charged glycans (sum of present
-glycans and reported
-glycans). No
-glycans with sialylated or phosphorylated forms were identified, and sulfate modification ions were the only negative charges in BSM
-glycans. Mucin structures, including sulfated
-glycans located in the hydrophobic terminal regions, were indicated. This is the first study to identify the structures and quantities of 12 sulfated
-glycans in natural mucins. These sulfations play important structural roles in hydration, viscoelasticity control, protection from bacterial sialidases, and polymer stabilization to support the functionality of BSM via electrostatic interactions.
Bovine submaxillary mucin (BSM) is a gel-forming glycoprotein polymer, and Ser/Thr-linked glycans (
-glycans) are important in regulating BSM's viscoelasticity and polymerization. However, details of
...-glycosylation have not been reported. This study investigates the structural and quantitative characteristics of
-glycans and identifies
-glycosylation sites in BSM using liquid chromatography-tandem mass spectrometry. The
-glycans (consisting of di- to octa-saccharides) and their quantities (%) relative to total
-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%)
-glycans, which were characterized based on their constituents (sialylation (14
-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.
-glycosylation sites were identified using
-glycopeptides (bold underlined;
GE
R
VI,
H
GR
R
I,
G
P
V
AEQI,
RP
YGAL,
Q
LGPL,
M
R
VVV, and
RPEDN
AVA) 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
-glycans. This is the first study to characterize
-glycans and identify
-glycosylation sites in BSM.
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.
•BSM is used in various biomaterial applications and as a biochemical reagent.•BSM contains abundant O-glycans, but N-glycans have not been reported to date.•This study characterized 17 N-glycans and their relative quantities in BSM.•BSM contains core-fucosylated and sulfated N-glycans but not sialylated glycans.•BSM N-glycans are less-branched structures compared to human mucins.
•N-glycans containing O-acetylated SA affect therapeutic glycoprotein efficacy.•O-acetylated glycans of Myozyme for treating Pompe disease have not been reported.•This study investigated O-acetylated ...glycans on Myozyme using LC-ESI-HCD-MS/MS.•Four types of 17 O-acetylated glycans constitute 6.4% of Myozyme’s total glycans.•Glycopeptide analysis by nano-LC-ESI-HCD-MS/MS confirmed six O-acetylation sites.
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.
•Plant core-fucosylated glycans may elicit an immune response in humans.•Rice cell-derived rhGAAs were produced under eight different culture conditions.•N-glycans of eight rhGAAs were analyzed using ...UPLC and LC-ESI-HCD-MS/MS.•15 N-glycans were characterized as core-, de-core-, and non-core-fucosylated glycans.•The lowest core-fucosylated condition of rhGAA was 2-FF combined with PF-68.
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.
This study investigated the creep stability enhancement of Inconel 718 by induction heating during the laser-directed energy deposition. The induction heating during the deposition fully dissolved ...the Laves phase, mitigated Nb segregation, and formed directionally solidified columnar grains. Based on the results of nanoindentation creep testing, the deposit utilizing induction heating exhibited high creep stability.
In this study, various heat treatments were conducted on Inconel 718 (IN718) products deposited by wire arc additive manufacturing (WAAM) to investigate the formation kinetics of phases at ...microstructural locations. The formation kinetics of the secondary phases precipitated by heat-treating of WAAM products differ from those of wrought or cast products because of the heat that accumulates during layer-by-layer deposition. Therefore, the secondary phases in the heat-treated WAAM products differ from those in conventional products according to the microstructural locations, e.g., the dendritic region, interdendritic region, and grain boundaries. To investigate such phase differences, a 100-layer IN718 wall was deposited using cold metal transfer (CMT)-based WAAM and then heat-treated under various conditions. In each heat-treated specimen, the δ, γ’, and γ” phases were identified by microstructural analyses. Based on the results, time–temperature–transformation diagrams were constructed for each microstructural location, which confirmed that the secondary phases were precipitated faster in the interdendritic region and at grain boundaries than those in the dendritic region. In addition, the applicability of single-aging, which can be used to satisfy the desired mechanical properties in less time with less processing, was evaluated by the hardness and tensile tests in the WAAM products.
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•Various heat treatments designed by modifying the standard specification were conducted on IN718 WAAM deposit.•Modified heat treatments fully dissolved the Laves phase and selectively precipitated the grain boundary δ phase.•According to microstructural locations, formation kinetics of secondary phases during the heat treatments were investigated.•The mechanical properties of the single- and double-aged did not show significant difference despite the difference of 8 h.
This study investigates the effect of the ring beam-driven flow and the temperature gradient on the solidification pattern in coaxial dual-beam laser welding of aluminum alloy. The microstructure of ...the weld metal is significantly influenced by the heat transfer and fluid flow in the molten pool, and the thermal and fluid flow characteristics of the molten pool in coaxial dual-beam laser welding differ from those in the conventional single-beam laser welding process. Therefore, a numerical model simulating the coaxial dual-beam laser welding of aluminum alloy was developed to investigate the solidification pattern and reveal the influence of the temperature gradient and circulation flow in the molten pool induced by the ring beam on keyhole behavior, mechanical properties, and behavior of the molten pool surface. Accordingly, the solidification pattern induced by the temperature gradient and ring beam-driven flow was quantitatively characterized. Additionally, the microstructure morphology according to the power of the ring beam was elucidated by comparing the columnar-to-equiaxed-transition (CET) position from the EBSD results of the weld metal and the numerical model.
•A numerical model of coaxial dual-beam laser welding was developed and validated.•Ring beam-driven flow stabilized keyhole and mitigated flow of molten pool surface.•Solidification pattern based on thermal and fluid flow characteristics was analyzed.•Ring beam decreased temperature gradient and increased amount of equiaxed grains.
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