Highly sensitive two-dimensional profiling of N-linked glycans by hydrophilic interaction liquid chromatography and dual stacking capillary gel electrophoresis.

Background: N-Glycosylation is one of the most important post-translational modifications in proteins. As the N-glycan profiles in biological samples are diverse and change according to the pathological condition, various profiling methods have been developed, such as liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry. However, conventional analytical methods have limitations in sensitivity and/or resolution, hindering the discovery of minor but specific N-glycans that are important both in the basic glycobiology research and in the medical application as biomarkers.

A rapid and convenient enzyme digestion method for the analysis of N-glycans using exoglycosidase-impregnated polyacrylamide gels fabricated in an automatic pipette tip.

Efficient enzymatic digestion methods are critical for the characterization and identification of glycans. Glycan hydrolysis enzymes are widely utilized for the identification of glycoprotein or glycolipid glycans. The commonly utilized in solution glycan hydrolysis methods require several hours of incubation with enzymes for complete removal of their target monosaccharides.

In Situ Pinpoint Photopolymerization of Phos-Tag Polyacrylamide Gel in Poly(dimethylsiloxane)/Glass Microchip for Specific Entrapment, Derivatization, and Separation of Phosphorylated Compounds.

An improved method for the online preconcentration, derivatization, and separation of phosphorylated compounds was developed based on the affinity of a Phos-tag acrylamide gel formed at the intersection of a polydimethylsiloxane/glass multichannel microfluidic chip toward these compounds. The acrylamide solution comprised Phos-tag acrylamide, acrylamide, and -methylene-bis-acrylamide, while 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] was used as a photocatalytic initiator. The Phos-tag acrylamide gel was formed around the channel crossing point via irradiation with a 365 nm LED laser.

High-throughput N-glycan screening method for therapeutic antibodies using a microchip-based DNA analyzer: a promising methodology for monitoring monoclonal antibody N-glycosylation.

N-Glycosylation of therapeutic antibodies is a critical quality attribute (CQA), and the micro-heterogeneity affects the biological and physicochemical properties of antibodies. Therefore, the profiling of N-glycans on antibodies is essential for controlling the manufacturing process and ensuring the efficacy and safety of the therapeutic antibodies. To monitor N-glycosylation in recombinant proteins, a high-throughput (HTP) methodology for glycan analysis is required to handle bulk samples in various stages of the manufacturing process.

Nylon Monofilament Mold Three-dimensional Microfluidic Chips for Size-exclusion Microchip Electrophoresis: Application to Specific Online Preconcentration of Proteins.

We present a lithography-free procedure for fabricating intrinsically three-dimensional microchannels within PDMS elastomers using nylon monofilament molds. We embedded nylon monofilaments in an uncured PDMS composite to fabricate straight channels of desired length, for use as molds to form the microchannels. Next, we fabricated two layer devices consisting of dialysis membranes, which preconcentrate specific proteins in accordance with molecular weight, in between two layers of PDMS substrates with embedded microchannels.

Age-Related Changes in O-Acetylation of Sialic Acids Bound to N-Glycans of Male Rat Serum Glycoproteins and Influence of Dietary Intake on Their Changes.

O-Acetylation of sialic acids has been widely found in eukaryotic cells. Such modifications of sialic acids are tissue-specific and seem to be developmentally regulated. In this study, we performed comprehensive analysis of age-related changes in the serum N-glycans of male rats using capillary electrophoresis (CE) and investigated the changes in the O-acetylation of sialic acids bound to N-glycans with aging and different diets.

Analysis of 2-aminopyridine labeled glycans by dual-mode online solid phase extraction for hydrophilic interaction and reversed-phase liquid chromatography.

Quantitative analysis of glycans released from glycoproteins using high-performance liquid chromatography (HPLC) requires fluorescent tag labeling to enhance sensitivity and selectivity. However, the methods required to remove large amounts of excess labeling reagents from the reaction mixture are time-consuming. Furthermore, these methods, including solvent extraction and solid phase extraction (SPE), often impair quantitative analysis.

A practical method for preparing fluorescent-labeled glycans with a 9-fluorenylmethyl derivative to simplify a fluorimetric HPLC-based analysis.

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.

A fast and convenient solid phase preparation method for releasing N-glycans from glycoproteins using trypsin- and peptide-N-glycosidase F (PNGase F)-impregnated polyacrylamide gels fabricated in a pipette tip.

An efficient deglycosylation process is a key requirement for the identification and characterization of glycosylation during the production and purification of therapeutic antibodies. PNGase F is widely used for the deglycosylation of N-linked glycans. The commonly-used in-solution deglycosylation method is relatively time-consuming and requires several hours up to overnight for complete removal of all N-linked glycans.

On-line microchip electrophoresis-mediated preconcentration of cationic compounds utilizing cationic polyacrylamide gels fabricated by in situ photopolymerization.

A simple and efficient method was developed for the fabrication of a cationic sample preconcentrator on a channel of a commercial poly(methyl methacrylate) (PMMA) microchip. This approach is based on a simple photochemical copolymerization for the fabrication of a permselective preconcentrator. The intersection of the PMMA microchip was filled with a gel solution comprising acrylamide, N,N-methylene-bis-acrylamide, (3-acrylamidopropyl) trimethylammonium, and riboflavin that functioned as a photocatalytic initiator.

High-performance liquid chromatographic separation of 8-aminopyrene-1,3,6-trisulfonic acid labeled N-glycans using a functional tetrazole hydrophilic interaction liquid chromatography column.

8-Aminopyrene-1,3,6-trisulfonate (APTS) is one of the most frequently used reagent in capillary electrophoresis. Three sulfonate groups in APTS generate fast electrophoretic mobilities of derivatized glycans, therefore very suitable for CE-LIF applications. However, these groups also make separation with partition chromatography difficult.

Microchip electrophoresis utilizing an in situ photopolymerized Phos-tag binding polyacrylamide gel for specific entrapment and analysis of phosphorylated compounds.

A method was developed for the specific entrapment and separation of phosphorylated compounds using a Phos-tag polyacrylamide gel fabricated at the channel crossing point of a microfluidic electrophoresis chip. The channel intersection of the poly(methyl methacrylate)-made microchip was filled with a solution comprising acrylamide, N,N-methylene-bis-acrylamide, Phos-tag acrylamide, and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], which functioned as a photocatalytic initiator. In situ polymerization at the channel crossing point was performed by irradiation with a UV LED laser beam.

Plug-plug kinetic capillary electrophoresis for in-capillary exoglycosidase digestion as a profiling tool for the analysis of glycoprotein glycans.

An online exoglycosidase digestion was combined with a plug-plug kinetic mode of capillary electrophoresis (CE) for the analysis of glycoprotein-derived oligosaccharides. An exoglycosidase solution and a solution of glycoprotein glycans derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were introduced to a neutrally coated capillary previously filled with electrophoresis buffer solution containing 0.5w/v% hydroxypropylcellulose.

Current landscape of protein glycosylation analysis and recent progress toward a novel paradigm of glycoscience research.

This review covers the basics and some applications of methodologies for the analysis of glycoprotein glycans. Analytical techniques used for glycoprotein glycans, including liquid chromatography (LC), capillary electrophoresis (CE), mass spectrometry (MS), and high-throughput analytical methods based on microfluidics, were described to supply the essentials about biopharmaceutical and biomarker glycoproteins. We will also describe the MS analysis of glycoproteins and glycopeptides as well as the chemical and enzymatic releasing methods of glycans from glycoproteins and the chemical reactions used for the derivatization of glycans.

Capillary electrochromatography using monoamine- and triamine-bonded silica nanoparticles as pseudostationary phases.

Monoamine- and triamine-bonded silica nanoparticles were prepared using 3-aminopropyltrimethoxysilane and N(1)-(3-trimethoxysilylpropyl)diethylenetriamine, respectively, and used as pseudostationary phases for capillary electrochromatography. The amine-bonded silica nanoparticles were tightly adsorbed on the inner wall of a capillary and generated fast electro-osmotic flow (2.59 × 10(-4) cm(2) V(-1) s(-1)) toward the anode in an electric field.

Chiral separation of D/L-aldoses by micellar electrokinetic chromatography using a chiral derivatization reagent and a phenylboronic acid complex.

A novel method was developed for D/L-isomeric separation of aldopentoses and aldohexoses as their (S)-(+)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole derivatives using phenylboronate buffer containing sodium dodecyl sulfate as a background electrolyte. The combination of derivatization with a chiral labeling reagent and micellar electrokinetic chromatography with phenylboronate made possible the efficient separation of D/L isomers as well as epimeric isomers of aldopentoses and aldohexoses. Laser-induced fluorescence detection permitted the micromolar-level determination of monosaccharide derivatives.

A rapid and highly sensitive microchip electrophoresis of mono- and mucin-type oligosaccharides labeled with 7-amino-4-methylcoumarin.

A selective separation method using a poly(methylmethacrylate) microchip was developed for 7-amino-4-methylcoumarin-labeled saccharides in a crude reaction mixture. In an alkaline borate buffer, saccharide derivatives formed strong anionic borate complexes. These complexes moved from the cathode to the anode in an electric field and were detected near the anode.

Application of online preconcentration affinity capillary electrophoresis method to glycans labeled with 8-aminonaphthalene-1,3,6-trisulfonic acid using blue light emitting diode-induced fluorescence detection.

An online preconcentration technique, large volume sample stacking with an electroosmotic flow pump, was combined with partial filling affinity capillary electrophoresis (PFACE) to create a highly sensitive analysis of the interaction of glycoprotein-derived oligosaccharides with plant lectins. Oligosaccharides were derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) for use in a blue light emitting diode-induced fluorescence detection capillary electrophoresis system. ANTS-labeled oligosaccharides were delivered to an entire neutrally coated capillary, and lectin solution was then hydrodynamically introduced from the outlet of the capillary as a short plug.

[In situ photopolymerization of polyacrylamide-based preconcentrator on a microfluidic chip for capillary electrophoresis].

Microchip electrophoresis is widely used for microfluidics and has been studied extensively over the past decade. Translation of capillary electrophoresis methods from traditional capillary systems to a microchip platform provides rapid separation and easy quantitation of sample components. However, most microfluidic systems suffer from critical scaling problems.

Microchip electrophoresis of oligosaccharides using lectin-immobilized preconcentrator gels fabricated by in situ photopolymerization.

A lectin-impregnated gel was fabricated at the channel crossing point in a microfluidic chip made from polymethyl methacrylate (PMMA). The acrylamide containing lectin was photopolymerized to form a round gel (radius 60 μm) by irradiation with an argon laser, which was also used for fluorometric detection. This gel was applied to specific concentration, elution, and electrophoretic separation of fluorescent-labeled oligosaccharides.

Partial filling affinity capillary electrophoresis using large-volume sample stacking with an electroosmotic flow pump for sensitive profiling of glycoprotein-derived oligosaccharides.

An online preconcentration technique, large-volume sample stacking with an electroosmotic flow pump (LVSEP) was combined with partial filling affinity capillary electrophoresis (PFACE) to realize highly sensitive analysis of the interaction of glycoprotein-derived oligosaccharides with some plant lectins. Oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were delivered to an entire neutrally-coated capillary and then lectin solution was hydrodynamically introduced from the outlet of the capillary as a short plug. A negative voltage was then applied after immersion of both ends of the capillary in 100 mM Tris-acetate buffer, pH 7.

Application of partial-filling capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in a therapeutic antibody.

Oligosaccharides in therapeutic recombinant antibodies play important roles in regulation of various biological functions. To monitor the glycosylation profiles of antibody pharmaceuticals in the manufacturing process, a highly sensitive and specific method is required. We extended partial-filling techniques using lectins and exoglycosidases in capillary electrophoresis for the characterization of 8-aminopylene-1,3,6-trisulfonic acid labeled N-linked oligosaccharides derived from the therapeutic antibody rituximab.

Simultaneous concentration enrichment and electrophoretic separation of weak acids on a microchip, using in situ photopolymerized carboxylate-type polyacrylamide gels as the permselective preconcentrator.

A method for the simultaneous concentration and separation of weak acids using an acidic polyacrylamide gel, fabricated in the microfluidic channel of a commercial poly(methyl methacrylate)-made microchip, is reported. This approach is based on simple photochemical copolymerization for the fabrication of a permselective preconcentrator. The intersection of the poly(methyl methacrylate)-made microchip was filled with a gel solution comprising acrylamide, N,N'-methylene-bis-acrylamide, and 2-acrylamidoglycolic acid, with riboflavin as a photocatalytic initiator.