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Plug-plug kinetic capillary electrophoresis for in-capillary exoglycosidase digestion as a profiling tool for the analysis of glycoprotein glycans
Accepted Manuscript Title: Plug-plug Kinetic Capillary Electrophoresis for In-capillary Exoglycosidase Digestion as a Profiling Tool for the Analysis of Glycoprotein Glycans Author: Maki Yamagami Yurie Matsui Takao Hayakawa Sachio Yamamoto Mitsuhiro Kinoshita Shigeo Suzuki PII: DOI: Reference:
S0021-9673(17)30385-0 http://dx.doi.org/doi:10.1016/j.chroma.2017.03.019 CHROMA 358364
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
5-12-2016 3-3-2017 10-3-2017
Please cite this article as: M. Yamagami, Y. Matsui, T. Hayakawa, S. Yamamoto, M. Kinoshita, S. Suzuki, Plug-plug Kinetic Capillary Electrophoresis for In-capillary Exoglycosidase Digestion as a Profiling Tool for the Analysis of Glycoprotein Glycans, Journal of Chromatography A (2017), http://dx.doi.org/10.1016/j.chroma.2017.03.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Plug-plug Kinetic Capillary Electrophoresis for In-capillary Exoglycosidase Digestion as a Profiling Tool
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for the Analysis of Glycoprotein Glycans
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Maki Yamagamia, Yurie Matsuia, Takao Hayakawaa, Sachio Yamamotoa, Mitsuhiro Kinoshitaa,b, Shigeo Suzukia,b,*
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Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka, Japan
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Antiaging Center, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka, Japan
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Running title: Exoglycosidase digestion ppkCE analysis of glycoprotein glycans
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Tel.: +81-6-4307-4004
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Fax: +81-6-6721-2353
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* Corresponding author: Shigeo Suzuki
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e-mail: [email protected]
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E-mail list of all authors: [email protected] (Maki Yamagami), [email protected]
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(Yurie Matsui), [email protected] (Takao Hayakawa), [email protected] (Sachio
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Yamamoto), [email protected] (Mitsuhiro Kinoshita)
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Keywords: Plug-plug kinetic capillary electrophoresis; exoglycosidase digestion; profiling of glycans,
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glycoprotein-derived oligosaccharides
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Highlights
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An online exoglycosidase digestion combined with a plug-plug kinetic mode of capillary electrophoresis
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(CE) for the analysis of APTS-oligosaccharides from glycoproteins
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Zero potential amplification for β-N-acetylhexosaminidase, and low mixing voltage application for
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α-fucosidase and α-mannosidase digestion
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A fully optimized CE conditions established for all of exoglycosidases commonly used for glycoprotein
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31 Abstract
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An online exoglycosidase digestion was combined with a plug-plug kinetic mode of capillary
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electrophoresis (CE) for the analysis of glycoprotein-derived oligosaccharides. An exoglycosidase
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solution and a solution of glycoprotein glycans derivatized with 8-aminopyrene-1,3,6-trisulfonic acid
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(APTS) were introduced to a neutrally coated capillary previously filled with electrophoresis buffer
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solution containing 0.5w/v% hydroxypropylcellulose. After immersion of both ends of the capillary in the
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buffer solutions, a negative voltage was applied for analysis. An APTS group of an oligosaccharide
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derivative has triply negative charges, which forced saccharide derivatives to anode with fast mobility and
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pass through the enzyme plug, which are detected at the anodic end. If the terminal monosaccharides of
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APTS-labeled oligosaccharides are released by the action of an exoglycosidase, the migration times of the
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oligosaccharides shift to those of digested oligosaccharides. We examined β-galactosidase,
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α-mannosidase, β-N-acetylhexosaminidase, α-neuraminidase, and α-fucosidase, and found only
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β-galactosidase and α-neuraminidase showed good reactivity toward APTS-labeled oligosaccharides; the
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reaction was completed by injecting a 3.6 cm long plug of 200 and 50 mU/mL concentration of
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exoglycosidases. In contrast, other exoglycosidases could not react with APTS labeled oligosaccharides at
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a concentration up to 5 U/mL. The β-N-acetylhexosaminidase reaction was successively followed by the
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electrophoretic mobility of APTS oligosaccharides and stopped for 10 min when saccharide derivatives
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were achieved in the enzyme plug. The reaction of α-fucosidase and α-mannosidase was completed by
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decreasing the electrophoretic voltage to –2 kV when the APTS oligosaccharides were passing through an
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exoglycosidase plug. We established the CE conditions for all of the glycosidic linkage analysis of
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glycoprotein glycans.
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53 1. Introduction
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Glycosylation of proteins is a typical post-translational modification. Oligosaccharides in glycoproteins
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are synthesized by the combined actions of glycosyltransferases and glycosidases, which creates several
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populations of glycans that may control the diverse functions of proteins [1]. Some of these play a pivotal
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role in many biological processes, including protein folding [2,3], physicochemical stability [4,5],
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immune response [6-11], and inflammation [12]. Some oligosaccharides function as biomarkers
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indicating malignancy and physiological state of specific diseases. Occupancy of a glycosylation site and
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the particular carbohydrate sequences and linkages at each site can vary considerably and contribute to
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heterogeneity [13,14]. The structures of saccharide chains have been determined by comprehensive
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studies using high resolution separation methods combined with mass spectrometry and chemical and
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biological reactions. However, the poor sensitivity of MS towards carbohydrates reduces the reliability of
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oligosaccharide analysis, which requires more convenient method for highly sensitive and quantitative
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determination of glycan profiles.
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CE has been emerged as a powerful and versatile separation tool for the study of minute quantities of
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samples with short analysis time. The most important feature of CE is that the high resolution can be
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obtained under physiological conditions, such as in a high ionic strength solution at neutral pH. This
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feature enables a biological assay called electrophoretically mediated microanalysis [15], in which the
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capillary is used as a microbioreactor and for the separation of substrates and products. Since then many
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reports concerned on in-capillary bioassay CE have been published and they are summarized in some
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reviews [16, 17]. Some of them were concerned with the field of carbohydrate analysis [18]. There are
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two ways to mix the reaction components in a capillary. One is the continuous mode, in which the
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capillary is initially completely filled with one of the reactants, and the second reactant is introduced as a
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plug or continuously from the inlet vial. Application of this mode includes lectin affinity CE of milk
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oligosaccharides [19] and glycoproteins [20], quantitative analysis of disaccharides derived from
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chondroitin sulfates by in-capillary enzymatic digestion [21], lectin affinity CE of glycoprotein glycans
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on a microfluidic chip [22]. Another mode of bioassay CE is called plug-plug kinetic CE (ppkCE), in which substrates and an
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enzyme are introduced into the capillary as different plugs and the reaction products generated in the
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enzyme plug are separated at the remaining part of the capillary [23]. Lectins specifically recognizes a
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wide variety of glycans, but the mixing of lectins causes aggregation because the most of lectins are
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glycoproteins [24]. Exoglycosidases are commonly used for sequence analysis of complex
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oligosaccharides. Serial injection of a series of exoglycosidase could directly indicates the sequence of
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oligosaccharides. Therefore ppkCE could be preferable for exoglycosidase and lectin CE. Accordingly,
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we previously applied the method to various purpose; the ppkCE analysis of glycoprotein glycans with a
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series of lectins enables simultaneous determination of binding constants of a series of closely related
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oligosaccharides [25], a sequential injection of four lectins to remove all peaks of complex type
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oligosaccharides from a mixture of high-mannose and complex type oligosaccharides derived from
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thyroglobulin, and a large volume sample stacking ppkCE for 800 times enhancement of the sensitivity in
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lectin ppkCE [26]. Archer-Hartmann et al. applied lectin and exoglycosidase ppkCE to profile
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glycoproteins in MCF7 cells [27], and therapeutic IgGs [28]. ppkCE was also applied for the detection of
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two types of epitopes, N-glycolylneuraminic acid and α-galactose (α-Gal) residues in IgG
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pharmaceuticals [29]. However, previously published data are limited to specific exoglycosidases or
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incomplete digestion. This will be due to slow kinetics of some exoglycosidases in ppkCE analysis.
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Here, we have focused on the reactivity in ppkCE of some exoglycosidases commonly used for
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structural analysis of glycoprotein glycans,
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β-N-acetylhexosaminidase, α-neuraminidase, and α-fucosidase, to establish the method for sequence
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which includes β-galactosidase,
α-mannosidase,
analysis of glycoprotein glycans.
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101 2. Materials and Methods
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2.1. Chemicals
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8-Aminopyrene-1,3,6-trisulfonic acid (APTS), α-fucosidase (bovine kidney), bovine pancreas
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ribonuclease B, human α1-acid glycoprotein, human transferrin, and human IgG were obtained from
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Sigma-Aldrich Japan (Tokyo, Japan). Peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F
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(PNGase F, EC 3.5.1.52) was from Roche Applied Science (Tokyo, Japan). Neuraminidase (Arthrobacter
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ureafaciens) was from Nacalai Tesque (Tokyo, Japan). β-Galactosidase, α-mannosidase, and
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β-N-acetylhexosaminidase from Jack bean meal were from Prozyme, Inc. (CA, USA). Sodium
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cyanoborohydride and iodoacetamide were obtained from Wako Pure Chemical Industries Limited
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(Osaka, Japan). The NAP5 column (Sephadex 25-packed short column) was from GE Health Care Japan
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(Hino, Japan). Other reagents and solvents were of the highest commercial grade.
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2.2. Preparation of APTS-labeled oligosaccharides [29]
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N-Linked oligosaccharides were prepared from 0.1 mg of lyophilized glycoprotein. A sample was
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dissolved in 50 µL of 50 mM phosphate buffer (pH 7.9) containing 0.1 w/v% sodium dodecyl sulfate and
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2% 2-mercaptoethanol. The solution was heated at 100 °C for 5 min. After cooling, the solution was
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mixed with 5 µL of 7.5 w/v% NP-40 and 2 mU (2 µL) of PNGase F and the reaction mixture incubated
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for 2 h at 37 °C. Deglycosylated proteins were precipitated by the addition of 180 µL of ice-cold ethanol
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and removed by centrifugation at 10,000 rpm for 5 min. The released oligosaccharides in the supernatant
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were transferred to another tube and dried in a centrifugal vacuum evaporator. To the resultant residue,
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each 2 µL of 0.2 M APTS in 15 v/v% acetic acid and 2 µL of 1 M NaBH3CN in tetrahydrofuran was
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added, and the mixture was heated for 1 h at 80 °C for neutral oligosaccharides or 1.5 h at 55 °C for
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sialylated oligosaccharides. The solution was then diluted with 85 µL of water and APTS-
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oligosaccharides were separated from excess APTS using a NAP5 column by collecting 0.6–1.3 mL of
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eluate. The eluate was evaporated to dryness by a centrifugal evaporator. The residue was dissolved in
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500 µL of water and used for analysis.
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In all CE separations, polydimethylsiloxane (PDMS)-coated capillaries (InertCap I®; GL Sciences Inc.,
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Tokyo, Japan) of 50 µm i.d. with an effective length of 40 cm (50 cm in total) was used with 50 mM
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Tris-acetate buffer containing 0.5 w/v% hydroxypropylcellulose as electrophoresis buffers, whose pHs
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were adjusted to optimum pH of each exoglycosidase : pH 7.0 for β-galactosidase and neuraminidase, pH
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6.0 for α-fucosidase and β-N-acetylhexosaminidase, and pH 4.5 for α-mannosidase. A P/ACE MDQ
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(Beckman Coulter, Brea, CA, USA) equipped with an argon laser-induced fluorimetric detection system
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with a band path filter for fluorescein was used for the CE analysis. The capillary was thermostated at
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25 °C. An exoglycosidase solution was injected to the capillary by applying a pressure of 3.45 kPa (0.5
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psi) for 60 s followed by an intermediate buffer plug for 2 s at 3.45 kPa and APTS-oligosaccharides
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solution for 5 s at 3.45 kPa. The APTS- oligosaccharides were separated at –15 kV. Three mixing modes
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in exoglycosidase phases were applied to provide maximum recovery for each exoglycosidase:
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β-Galactosidase and neuraminidase digestion were conducted by applying a separation voltage of –15 kV.
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β-N-Acetylhexosaminidase digestion was conducted by stopping the voltage after migrated for 1 min at
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–15 kV; after 10 min the separation was resumed. Capillary digestion using α-fucosidase and
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α-mannosidase was completed by applying –2 kV for 10 min, then applying –15 kV for separation of
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reaction products. After each run, an enzyme solution in the capillary was removed by introducing buffer
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solution from the outlet by pressure application (207 kPa, 5 min) and conditioned by rinsing with 0.1 M
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NaOH solution. The volume of enzyme solution injected in a capillary was calculated using CE Expert
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supplied by Beckman Coulter according to the following equation.
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V= Where V is the volume of delivered solution across the capillary (liter), ∆P denotes the pressure drop
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across the capillary (in Pascals), d signifies the internal diameter of the capillary (meters), t is the duration
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of pressure application (seconds), η represents the buffer viscosity (Pascal seconds), and L is the total
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capillary length (meters). Enzymatic activity was estimated from the concentration and the
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passing-through time of APTS-labeled oligosaccharides and the unit of an exoglycosidase in enzyme
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plug.
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156 3. Results and Discussion
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3.1. Principle of exoglycosidase digestion ppkCE
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The overall scheme of exoglycosidase digestion ppkCE is shown in Fig. 1. In this method, solutions of an
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exoglycosidase and a mixture of APTS-labeled oligosaccharides are introduced to the PDMS-coated
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capillary filled with an electrophoresis buffer in this order. Both ends of the capillary are then immersed
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in electrophoresis buffer and the separation voltage is applied. Because electroosmotic flow (EOF) in the
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PDMS-coated capillary is negligible, the enzyme plug could stay near the introduced position in the
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electric field. Whereas APTS-labeled oligosaccharides, which carry three negatively charged sulfonate
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groups, migrate faster toward the anode and contact with exoglycosidase. In this step, any
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oligosaccharides recognized by the glycosidase are converted to product oligosaccharides, which indicate
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the mobilities of the products according to size and charge ratio differences. By comparing separation
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profiles under the presence and absence of the glycosidase plug, we can find the presence or absence of
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terminal monosaccharide residues on each saccharide peak.
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PDMS-coated capillary and hydroxypropylcellulose in electrophoresis buffer yielded a good
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separation by suppressing EOF and preventing adsorption of glycosidases as well as APTS-labeled
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oligosaccharides onto the inner surface of the capillary. EOF generation was significantly suppressed by
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an EOF velocity of 6.3 × 10-5 cm2V-1s-1 from anode to cathode in 100 mM Tris/acetate, pH 7.0 containing
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0.5 w/v% hydroxypropylcellulose as electrophoresis buffer. Electrophoretic mobility of APTS
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oligosaccharides in the same buffer ranges between –2.5 × 10-3 and –1.5 × 10-4 cm2V-1s-1. Therefore, all of
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the APTS oligosaccharides moved and passed through the exoglycosidase plug in the ppkCE mode.
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Typically, 3.45 kPa for 5 s was applied for hydrodynamic injections of APTS-labeled saccharide mixture
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and 3.45 kPa for 1 min of exoglycosidase solution. The injection volumes calculated by CE Expert are
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5.94 nL and 71.3 nL, respectively, and their plug length of the enzyme is 3 mm and 36 mm, respectively,
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which induces ranges of contact time of APTS-labeled oligosaccharides between 1.0 min and 2.5 min at
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–15 kV.
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3.2. Optimization of the ppkCE digestion using α-neuraminidase and transferrin derived glycans as a
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model
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The efficiency of exoglycosidase digestion ppkCE was examined using α-neuraminidase and
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APTS-labeled oligosaccharides derived from human transferrin as a model system. Transferrin mainly
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contains biantennary complex-type oligosaccharides with two terminal α2,6-linked NeuAc residues [30].
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α-Neuraminidase action on the transferrin-derived oligosaccharides induces the removal of NeuAc and
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produces neutral biantennary oligosaccharides via monosialylated oligosaccharides. Transferrin has a
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molecular weight of around 80 kDa and contains two glycosylation sites [31]. Injected amount of
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oligosaccharides were estimated to be 28 fmol in total. Amount of neuraminidase (25 mU/mL injected for
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1 min) was calculated to be 1.8 µU/71 nL. The disialylated oligosaccharide may pass through the
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neuraminidase plug for 1.1 min, which means 2.0 pmol of sialic acid linkages are hydrolyzed.
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Fig. 2A shows selected ppkCE analysis of neuraminidase digestion of 28 fmol of a transferrin-derived
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oligosaccharide mixture. Under the absence of neuraminidase (trace R), the transferrin glycan mixture
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indicated disialylated biantennary oligosaccharides at 12.2 min with small peaks of monosialylated
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oligosaccharides at 13.9 min. A small peak at 12.3 min was identified as a core fucosylated disialylated
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saccharide. Injection of 25 mU/mL of neuraminidase for 5 s induced complete dissipation of the
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disialylated peak (12.2 min) and its conversion to a monosialylated saccharide (13.9 min) and an
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asialylated saccharide at 16.2 min (trace a). With the increase of injection time of neuraminidase, the
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asialylated saccharide peak (16.2 min) was increased, but the monosialylated peak remained. The
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completed conversion to asialylated oligosaccharides requires 30 s injection of 50 mU/mL of
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neuraminidase (trace f).
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Fig. 2B shows the effect of the concentration of neuraminidase injected for 1 min on the removal of
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sialic acids from transferrin-derived oligosaccharides. Disialylated oligosaccharide (28 fmol) can rapidly
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disappear by injecting 5 mU/mL of neuraminidase (trace j) and mainly converting to monosialylated
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oligosaccharides (13.9 min). In contrast, monosialylated oligosaccharides were somewhat slowly
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converted to neutral oligosaccharides (16.2 min) with increasing neuraminidase concentration, while the
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complete removal of sialic acids of 28 fmol of biantennary oligosaccharides requires more than 25
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mU/mL of neuraminidase (trace m).
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Fig. 2C shows the reaction varying the amount of saccharide within the 56–896 fmol range.
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Surprisingly, 25 mU/mL (148 nU) could hydrolyze more than 896 fmol of disialylated oligosaccharides
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(trace r) and converted to monosialylated oligosaccharides. In contrast, the monosialylated biantennary
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oligosaccharides
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oligosaccharides with low efficiency, and monosialylated oligosaccharides remained at amount of 56 fmol
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of sample (trace n).
resisted
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As a result, the neuraminidase reaction on disialylated oligosaccharides proceeded with two times
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higher reactivity estimated from the unit definition. High enzymatic activity of neuraminidase may be due
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to the effective mixing of substrate oligosaccharides and neuraminidase in the electric field. However,
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monosialylated oligosaccharides indicated low reactivity toward neuraminidase digestion and only 10%
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of the reaction efficiency. We examined the effect of capillary temperature on hydrolysis efficiency, but
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the recovery of glycosidase digestion did not change between 25 °C to 35 °C (see Figure S1). This will be
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due to higher temperatures inside the capillary from Joule heating. Previously Evenhuis et al. reported
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that the temperature of capillary insides with 75 µm i.d. is up to ca. 20°C higher than external temperature
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[32]. The low reactivity to NeuAc in monosialylated biantennary oligosaccharides may be due to
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substrate specificity.
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We also examined hydrolysis efficiency of other exoglycosidases commonly used for the analysis of
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glycoprotein-derived oligosaccharides and found that α- and β-galactosidase indicated only a few percent
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of the efficiency expected from enzyme unit definition, and the digestion of ca. 50 fmol of IgG
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oligosaccharides required 200 mU/mL of galactosidase [29]. The other glycosidases i.e., α-mannosidase,
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α-fucosidase, and β-N-acetylhexosaminidase, indicated very low reaction efficiency; the hydrolytic
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reaction could not be completed by using 5 U/mL of glycosidases. Increasing the concentration and/or
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injection time of glycosidases impaired the high resolution of oligosaccharides. To enhance the online
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incubation time, we examined two approaches; “zero potential amplification” [15] and the “low mixing
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voltage method” [33] for increasing the contact time of oligosaccharides with the exoglycosidases.
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3.3. Application of zero potential amplification for β-N-acetylhexosaminidase digestion of IgG glycans
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β-N-Acetylhexosaminidase from Jack bean meal is commonly used for sequence analysis of glycoprotein
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glycans, which can release both of the terminal β-linked GlcNAc and GalNAc, but the optimal pH
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differed for two saccharides and reported to be pH 5.0 to 6.0 for releasing GlcNAc and pH 3.5 to 4.0 for
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GalNAc [34]. Most N-linked oligosaccharides found in glycoproteins commonly possess β-linked
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GlcNAc. Therefore, we chose pH 6.0 buffer for the ppkCE analysis.
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The efficiency of acetylhexosaminidase ppkCE was examined using IgG-derived oligosaccharides.
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IgG oligosaccharides mainly consist of a series of biantennary core-fucosylated complex-type
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oligosaccharides terminated with zero, one, and two β-linked Gal residues, which means two GlcNAc
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residues are exposed in their non-reducing end of the agalactosylated saccharide and one GlcNAc residue
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on two monogalactosylated oligosaccharides. Therefore, β-N-acetylhexosaminidase digestion induces
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conversion of three peaks corresponding to two monogalactosylated to monoantennary oligosaccharides,
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and an agalactosylated to trimannosyl (Man3GlcNAc2Fuc1) oligosaccharides.
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As described in the previous section, enzymatic activity of β-N-acetylhexosaminidase is not high
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enough for complete digestion of saccharides when using 5 U/mL of this enzyme. Therefore, we applied a
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zero potential amplification method for ppkCE digestion of GlcNAc residues. Fig. 3 shows the results.
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We used somewhat increased concentrations of β-N-acetylhexosaminidase (5 U/mL). In-capillary
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digestion was conducted by applying –15 kV for 1 min to overlay APTS oligosaccharides near the end of
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the enzyme plug, then stopping applied voltage for 2 min (b) to 10 min (d), after which we again applied
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separation voltage. As shown in trace a, no application of zero potential indicated insufficient hydrolysis
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and 50% of agalactosylated, while 40% and 20% of two monogalactosylated peaks remain without
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digestion. Further, two isomeric oligosaccharides containing one GlcNAc residue derived from the
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agalactosylated oligosaccharide appeared at 17.3 and 17.5 min with a small peak of Man3GlcNAc2Fuc1 at
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15.9 min. Applying zero potential as the incubation time decreased undigested and partially digested
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oligosaccharide peaks. Undigested peaks almost disappeared following 10 min incubation (trace d).
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Further extension of stopping time causes broadening of oligosaccharide peaks. From these results, zero
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potential application for 10 min seems optimal for β-N-acetylhexosaminidase digestion.
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3.4. Application of low mixing voltage method
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3.4.1. α-Fucosidase digestion of asialylated oligosaccharides from α1-acid glycoproteins
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α-Fucosidase from bovine kidney is commonly used for releasing both α1,6-linked fucose (Fuc) bound to
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β-GlcNAc residues in reducing termini and α1,3-linked Fuc bound to lactosamine branches of complex
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type oligosaccharides. Optimal pH of this enzyme is 6.0. Therefore, an electrophoresis buffer of pH 6.0
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was used for in-capillary digestion with α-fucosidase and asialylated oligosaccharides derived from
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human α1–acid glycoprotein. We increased concentration of α-fucosidase to 5 U/mL in the ordinary
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plug-plug kinetic mode, but only ~1 fmol of Fuc residues were released from oligosaccharides (see Figure
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S2). Here, low mixing voltage method was applied to further enhance enzymatic digestion.
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Fig. 4 shows the effect of the mixing voltage until the APTS-oligosaccharides completely passed
276
through the α-fucosidase plug; it takes about 9.6 min for –5kV and 48 min for –1kV. Bottom
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electropherogram (trace R) indicates the separation of asialylated oligosaccharides from α1-acid
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glycoproteins without injecting fucosidase. Biantennary, triantennary, fucosylated triantennary,
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tetraantennary, and fucosylated tetraantennary oligosaccharides were completely separated. α-Fucosidase
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digestion of the saccharides decreased fucosylated oligosaccharides appearing at 23.3 min and 26 min and
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increased peak areas at 22.7 min and 25.1 min, respectively. As shown in Fig. 4, two peaks of fucosylated
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oligosaccharides at 23.3 min and 26 min were drastically reduced by decreasing voltage and disappeared
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by applying voltage lower than –2 kV. Further decrease of voltage (–1 kV) induced broadening of peaks
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as shown in trace d; –2 kV seems most suitable for potentially applying the low mixing voltage method.
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3.4.2. α-Mannosidase digestion of high-mannose type glycans from ribonuclease B
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Jack bean α-mannosidase can release all α-mannose residues from oligosaccharides in their non-reducing
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termini, but the specificity is somewhat complicated. This enzyme can hydrolyze α1,2- and α1,6-linked
289
mannose (Man) more quickly than α1,3-linked Man residues. The enzyme cannot release α1,6-linked
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Man from Manα1-6Manβ1-4GlcNAcβ1-4GlcNAc, but can release Man from α1,6-linked Man from
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Manα1-3Manβ1-4GlcNAcβ1-4GlcNAc [35]. This means the digestion of high-mannose oligosaccharides
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by this enzyme gave two products; Manα1-6Manβ1-4GlcNAcβ1-4GlcNAc and
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Manβ1-4GlcNAcβ1-4GlcNAc. As shown in Fig. 5, overnight digestion of APTS-labeled, high-mannose type oligosaccharides with
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α-mannosidase mainly produced a completely digested product (Manβ1-4GlcNAcβ1-4GlcNAc) and
296
minute
297
Manα1-6Manβ1-4GlcNAcβ1-4GlcNAc from the enzyme specificity. However, application of zero
298
potential for 1.5 min indicated Man2 to Man4 oligosaccharides (trace b), while the application of low
299
mixing voltage at –2 kV for 10 min for digestion indicated mainly single peaks corresponding to
300
Manβ1-4GlcNAcβ1-4GlcNAc (trace c). The difference in the content of Man2GlcNAc2 by overnight
301
digestion and in-capillary digestion is not certain, but the complete digestion in this method may be of use
302
in the analysis of oligosaccharide structure.
of
Man2GlcNAc2
(trace
a),
which
was
estimated
to
be
M
an
us
cr
amounts
ip t
294
303 4. Conclusion
305
We have optimized the conditions for online exoglycosidase digestion ppkCE for analyzing the
306
saccharide structures derived from glycoproteins. Neuraminidase digestion were completed with the plug
307
length of 36 mm (60 s injection) and applied to the separation voltage. Other exoglycosidases required
308
further optimization of other parameters to increase reaction time and produce an accurate overlap of
309
oligosaccharides with the enzyme plug. Full digestion could be obtained by zero potential amplification
310
for β-N-acetylhexosaminidase digestion and low mixing voltage application for α-fucosidase and
311
α-mannosidase digestion. Our method can be applied to other glycoprotein glycans. We believe the
312
information obtained by this method, as well as with lectin affinity ppkCE [24 -28], are useful for
313
profiling glycans in a glycoprotein. Low consumption (~10 nL) of sample solution in CE enables a series
314
of ppkCE analyses for online incubation with a series of exoglycosidases and lectins, which appears
315
straightforward in glycan analysis for minute amounts of glycoproteins.
Ac ce pt e
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316 317 318
Acknowledgements This work was supported by JSPS KAKENHI Grant Number 15K18852 and 16K08209. This work
319
also
320
Technology)-supported Program for the Strategic Research Foundation at Private Universities, 2014-2018
321
(S1411037)
MEXT
(Ministry
of
Education,
Culture,
Sports,
Science
and
ip t
by
cr
supported
Ac ce pt e
d
M
an
us
partly
14 Page 14 of 22
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8-aminopyrene-1,3,6-trisulfonic acid, J. Chromatogr. A 1218 (2011) 4772-4778.
electrophoresis
of
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oligosaccharides
derivatized
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[26] S. Gattu, C.L. Crihfield, L.A. Holland. Microscale measurements of Michaelis-Menten constants of
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affinity capillary electrophoresis using large-volume sample stacking with an electroosmotic flow
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pump for sensitive profiling of glycoprotein-derived oligosaccharides, J. Chromatogr. A 1246 (2012)
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processing and lectin capture of glycans with phospholipid assisted capillary electrophoresis
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separations, Anal. Chem. 83 (2011) 2740-2747.
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[29] S.A. Archer-Hartmann, C.L. Crihfield, L.A. Holland, Online enzymatic sequencing of glycans
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from Trastuzumab by phospholipid-assisted capillary electrophoresis, Electrophoresis 32 (2011)
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3491-3498.
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[30] Y. Yagi, S. Yamamoto, K. Kakehi, T. Hayakawa, Y. Ohyama, S. Suzuki, Application of partial-filling
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capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in
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a therapeutic antibody, Electrophoresis 32 (2011) 2979-2985.
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[31] Y. Satomi, Y. Shimonishi, T. Hase, T. Takao, Site-specific carbohydrate profiling of human
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transferrin by nano-flow liquid chromatography/electrospray ionization mass spectrometry, Rapid
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commun. Mass spectrom. 18 (2004) 2983-2988.
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[32] G. Spik, B. Bayard, B. Fournet, G. Strecker, S. Bouquelet, J. Montreuil, Studies on glycoconjugates.
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LXIV. Complete structure of two carbohyrate units of human serotransferrin, J. FEBS Lett. 50 (1975)
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dissipation in capillary electrophoresis. Anal. Chem. 78 (2006) 2684-2693. [34] A.E. Jones, E. Grushka, Nature of temperature gradients in capillary zone electrophoresis, J. Chromatogr. 466 (1989) 219-225.
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[33] C.J. Evenhuis, R.M. Guijt, M. Macka, P.J. Marriott, P.R. Haddad, Temperature profiles and heat
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cr
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[36] S.-C. Li, Y.-T. Li, Studies on the glycosidases of Jack bean meal III. Crystallization and properties of β-N-acetylhexosaminidase, J. Biol. Chem. 245 (1970) 5135-5160.
us
413
[37] H. Oku, S. Hase, T. Ikenaka, Separation of oligomannose-type sugar chains having one to five
423
mannose residues by high-performance liquid chromatography as their pyridylamino derivatives, Anal.
424
Biochem. 186 (1990) 331-334.
427 428
M
426
[38] Z.Szabo, A. Guttman, T. Rejtar, B.L. Karger, Improved sample preparation method for glycan analysis of glycoproteins by CE-LIF and CE-MS, Electrophoresis 31 (2010) 1389-1395. [39] L.A. Gennaro, O. Salas-Solano, S. Ma, Capillary electrophoresis-mass spectrometry as a characterization tool for therapeutic proteins, Anal. Biochem. 355 (2006) 249-258.
d
425
an
422
[40] F. Dang, K. Kakehi, J. Cheng, O. Tabata, M. Kurokawa, K. Nakajima, M. Ishikawa, Y. Baba, Hybrid
430
dynamic coating with n-dodecyl β-D-maltoside and methyl cellulose for high-performance
431
carbohydrate analysis on poly(methyl methacrylate) chips, Anal. Chem. 78 (2006) 1452-1458.
432 433 434
Ac ce pt e
429
[41] Z. Zhuang, J.A. Starkey, Y. Mechref, M.V. Novotny, S.C. Jacobson, Electrophoretic analysis of N-glycans on microfluidic devices, Anal. Chem. 79 (2007) 7170-7175.
18 Page 18 of 22
434 435
Figure captions
436
Fig. 1. An image of ppkCE for in-capillary digestion with exoglycosidases. Hydrodynamic injection for 1
437
min at 3.45 kPa introduces 71.26 nL of solution which occupies 36.3 mm of the capillary.
ip t
438 Fig. 2. α-Neuraminidase digestion ppkCE of transferrin-derived oligosaccharides. (A) Oligosaccharides
440
(28 fmol) were treated with 25 mU/mL of neuraminidase injected for 5 s (a), 10 s (b), and 20 s (c), or 50
441
mU/mL injected for 5 s (d), 15 s (e), and 30 s (f). (B) Neuraminidase was injected for 1 min at
442
concentrations of 0.5 mU/mL (g), 1 mU/mL (h), 2.5 mU/mL (i), 5 mU/mL (j), 7.5 mU/mL (k), 12.5
443
mU/mL (l), or 25 mU/mL (m). (C) Neuraminidase (25 mU/mL) was injected for 1 min for the digestion
444
of 56 fmol (n), 112 fmol (o), 224 fmol (p), 448fmol (q), and 896 fmol (r) of oligosaccharides. Peaks were
445
identified according to the reference [38].
us
cr
439
an
446
Fig. 3. β-N-Acetylhexosaminidase digestion ppkCE of IgG glycans by the zero potential method. 5 U/mL
448
solution of β-N-acetylhexosaminidase was used. Application of voltage was stopped at 1 min after
449
injection of APTS oligosaccharides for 0 min (a), 2 min (b), 5 min (c), and 10 min (d). ppkCE data were
450
shifted +1.5 min to align with the reference data indicated on the bottom trace. Electropherograms a to d
451
were shifted + 1 min to align them to reference data. Peaks were identified according to the reference
452
[39].
454
Fig. 4. α-Fucosidase digestion ppkCE analysis of asialo-oligosaccharides from human α1-acid
455
glycoprotein by applying a low mixing voltage at –5 kV for 9.6 min (a), –3 kV for 16 min (b), –2 kV for
456
24 min (c), and –1 kV for 48 min (d). α-Fucosidase was dissolved in 100 mM Tris acetate (pH 6.0) at a
457
concentration of 5 U/mL. Oligosaccharides containing terminal Fuc residue (▲) were reduced and
458
disappeared by decreasing mixing voltage. Electropherograms a to d were shifted + 3.2 min to align them
459
to reference data. Peaks were identified according to the reference [40].
460
Ac ce pt e
453
d
M
447
461
Fig. 5. α-Mannosidase digestion ppkCE analysis of oligosaccharides from ribonuclease B. From bottom,
462
R, separation of ribonuclease B-derived oligosaccharides as reference; a, overnight digestion with 150
463
mU of α-mannosidase; b, zero potential was applied for 10 min with 5 U/mL α-mannosidase; c,
464
application of mixing voltage at –2 kV for 10 min with 5 U/mL of α-mannosidase. Electropherograms b
465
and c were shifted +1 min and +1.3 min to align them to reference data. Peaks were identified according
466
to the reference [41].
19 Page 19 of 22
467 468
Fig. 1.
ip t
469
Fig. 2.
A
us
471
cr
470
60000
B
50000
40000
f
l
e
k
d
j
20000
c
10000
a
0
R
11 12 13 14 15 16 17
474 475 476 477
20000
h
10000
g
0
30000
r q
i
Ac ce pt e
b
M
20000
10000
472 473
40000
30000
d
Ffluorescence intensity
m 30000
C
an
40000
p o n
R
0
11 12 13 14 15 16 17
R 11 12 13 14 15 16 17
Migration time (min)
20 Page 20 of 22
477 478
Fig. 3.
2500000
ip t
2000000
c 1500000
cr
b 1000000
us
a 500000
R
0
16
18
20
22
Migration time (min)
M
479 480 481 482
an
Fluorescence intensity
d
Fig. 4.
d
2000000
1600000
d
1400000
c
Ac ce pt e
Fluorescence intensity
1800000
1200000
b
1000000
800000
a
600000 400000 200000
R
0
16 483 484
18
20
22
24
26
28
30
Migration time (min)
21 Page 21 of 22
484 Fig. 5
ip t
b
cr
400000
c
us
600000
a
200000
an
Fluorescence intensity
485
R
0
486
10
12
14
16
18
M
8
20
Migration time (min)
Ac ce pt e
488
d
487
22 Page 22 of 22
S0021-9673(17)30385-0 http://dx.doi.org/doi:10.1016/j.chroma.2017.03.019 CHROMA 358364
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
5-12-2016 3-3-2017 10-3-2017
Please cite this article as: M. Yamagami, Y. Matsui, T. Hayakawa, S. Yamamoto, M. Kinoshita, S. Suzuki, Plug-plug Kinetic Capillary Electrophoresis for In-capillary Exoglycosidase Digestion as a Profiling Tool for the Analysis of Glycoprotein Glycans, Journal of Chromatography A (2017), http://dx.doi.org/10.1016/j.chroma.2017.03.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Plug-plug Kinetic Capillary Electrophoresis for In-capillary Exoglycosidase Digestion as a Profiling Tool
2
for the Analysis of Glycoprotein Glycans
3 4 5
Maki Yamagamia, Yurie Matsuia, Takao Hayakawaa, Sachio Yamamotoa, Mitsuhiro Kinoshitaa,b, Shigeo Suzukia,b,*
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a
Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka, Japan
8
b
Antiaging Center, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka, Japan
cr
9 10
Running title: Exoglycosidase digestion ppkCE analysis of glycoprotein glycans
12
Tel.: +81-6-4307-4004
13
Fax: +81-6-6721-2353
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us
11
14
* Corresponding author: Shigeo Suzuki
16
e-mail: [email protected]
17
E-mail list of all authors: [email protected] (Maki Yamagami), [email protected]
18
(Yurie Matsui), [email protected] (Takao Hayakawa), [email protected] (Sachio
19
Yamamoto), [email protected] (Mitsuhiro Kinoshita)
21
d
Ac ce pt e
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15
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Keywords: Plug-plug kinetic capillary electrophoresis; exoglycosidase digestion; profiling of glycans,
23
glycoprotein-derived oligosaccharides
24 25
Highlights
26
An online exoglycosidase digestion combined with a plug-plug kinetic mode of capillary electrophoresis
27
(CE) for the analysis of APTS-oligosaccharides from glycoproteins
28
Zero potential amplification for β-N-acetylhexosaminidase, and low mixing voltage application for
29
α-fucosidase and α-mannosidase digestion
30
A fully optimized CE conditions established for all of exoglycosidases commonly used for glycoprotein
31
1 Page 1 of 22
31 Abstract
33
An online exoglycosidase digestion was combined with a plug-plug kinetic mode of capillary
34
electrophoresis (CE) for the analysis of glycoprotein-derived oligosaccharides. An exoglycosidase
35
solution and a solution of glycoprotein glycans derivatized with 8-aminopyrene-1,3,6-trisulfonic acid
36
(APTS) were introduced to a neutrally coated capillary previously filled with electrophoresis buffer
37
solution containing 0.5w/v% hydroxypropylcellulose. After immersion of both ends of the capillary in the
38
buffer solutions, a negative voltage was applied for analysis. An APTS group of an oligosaccharide
39
derivative has triply negative charges, which forced saccharide derivatives to anode with fast mobility and
40
pass through the enzyme plug, which are detected at the anodic end. If the terminal monosaccharides of
41
APTS-labeled oligosaccharides are released by the action of an exoglycosidase, the migration times of the
42
oligosaccharides shift to those of digested oligosaccharides. We examined β-galactosidase,
43
α-mannosidase, β-N-acetylhexosaminidase, α-neuraminidase, and α-fucosidase, and found only
44
β-galactosidase and α-neuraminidase showed good reactivity toward APTS-labeled oligosaccharides; the
45
reaction was completed by injecting a 3.6 cm long plug of 200 and 50 mU/mL concentration of
46
exoglycosidases. In contrast, other exoglycosidases could not react with APTS labeled oligosaccharides at
47
a concentration up to 5 U/mL. The β-N-acetylhexosaminidase reaction was successively followed by the
48
electrophoretic mobility of APTS oligosaccharides and stopped for 10 min when saccharide derivatives
49
were achieved in the enzyme plug. The reaction of α-fucosidase and α-mannosidase was completed by
50
decreasing the electrophoretic voltage to –2 kV when the APTS oligosaccharides were passing through an
51
exoglycosidase plug. We established the CE conditions for all of the glycosidic linkage analysis of
52
glycoprotein glycans.
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2 Page 2 of 22
53 1. Introduction
55
Glycosylation of proteins is a typical post-translational modification. Oligosaccharides in glycoproteins
56
are synthesized by the combined actions of glycosyltransferases and glycosidases, which creates several
57
populations of glycans that may control the diverse functions of proteins [1]. Some of these play a pivotal
58
role in many biological processes, including protein folding [2,3], physicochemical stability [4,5],
59
immune response [6-11], and inflammation [12]. Some oligosaccharides function as biomarkers
60
indicating malignancy and physiological state of specific diseases. Occupancy of a glycosylation site and
61
the particular carbohydrate sequences and linkages at each site can vary considerably and contribute to
62
heterogeneity [13,14]. The structures of saccharide chains have been determined by comprehensive
63
studies using high resolution separation methods combined with mass spectrometry and chemical and
64
biological reactions. However, the poor sensitivity of MS towards carbohydrates reduces the reliability of
65
oligosaccharide analysis, which requires more convenient method for highly sensitive and quantitative
66
determination of glycan profiles.
Ac ce pt e
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54
67
CE has been emerged as a powerful and versatile separation tool for the study of minute quantities of
68
samples with short analysis time. The most important feature of CE is that the high resolution can be
69
obtained under physiological conditions, such as in a high ionic strength solution at neutral pH. This
70
feature enables a biological assay called electrophoretically mediated microanalysis [15], in which the
71
capillary is used as a microbioreactor and for the separation of substrates and products. Since then many
72
reports concerned on in-capillary bioassay CE have been published and they are summarized in some
73
reviews [16, 17]. Some of them were concerned with the field of carbohydrate analysis [18]. There are
74
two ways to mix the reaction components in a capillary. One is the continuous mode, in which the
75
capillary is initially completely filled with one of the reactants, and the second reactant is introduced as a
76
plug or continuously from the inlet vial. Application of this mode includes lectin affinity CE of milk
3 Page 3 of 22
77
oligosaccharides [19] and glycoproteins [20], quantitative analysis of disaccharides derived from
78
chondroitin sulfates by in-capillary enzymatic digestion [21], lectin affinity CE of glycoprotein glycans
79
on a microfluidic chip [22]. Another mode of bioassay CE is called plug-plug kinetic CE (ppkCE), in which substrates and an
81
enzyme are introduced into the capillary as different plugs and the reaction products generated in the
82
enzyme plug are separated at the remaining part of the capillary [23]. Lectins specifically recognizes a
83
wide variety of glycans, but the mixing of lectins causes aggregation because the most of lectins are
84
glycoproteins [24]. Exoglycosidases are commonly used for sequence analysis of complex
85
oligosaccharides. Serial injection of a series of exoglycosidase could directly indicates the sequence of
86
oligosaccharides. Therefore ppkCE could be preferable for exoglycosidase and lectin CE. Accordingly,
87
we previously applied the method to various purpose; the ppkCE analysis of glycoprotein glycans with a
88
series of lectins enables simultaneous determination of binding constants of a series of closely related
89
oligosaccharides [25], a sequential injection of four lectins to remove all peaks of complex type
90
oligosaccharides from a mixture of high-mannose and complex type oligosaccharides derived from
91
thyroglobulin, and a large volume sample stacking ppkCE for 800 times enhancement of the sensitivity in
92
lectin ppkCE [26]. Archer-Hartmann et al. applied lectin and exoglycosidase ppkCE to profile
93
glycoproteins in MCF7 cells [27], and therapeutic IgGs [28]. ppkCE was also applied for the detection of
94
two types of epitopes, N-glycolylneuraminic acid and α-galactose (α-Gal) residues in IgG
95
pharmaceuticals [29]. However, previously published data are limited to specific exoglycosidases or
96
incomplete digestion. This will be due to slow kinetics of some exoglycosidases in ppkCE analysis.
97
Ac ce pt e
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80
Here, we have focused on the reactivity in ppkCE of some exoglycosidases commonly used for
98
structural analysis of glycoprotein glycans,
99
β-N-acetylhexosaminidase, α-neuraminidase, and α-fucosidase, to establish the method for sequence
100
which includes β-galactosidase,
α-mannosidase,
analysis of glycoprotein glycans.
4 Page 4 of 22
101 2. Materials and Methods
103
2.1. Chemicals
104
8-Aminopyrene-1,3,6-trisulfonic acid (APTS), α-fucosidase (bovine kidney), bovine pancreas
105
ribonuclease B, human α1-acid glycoprotein, human transferrin, and human IgG were obtained from
106
Sigma-Aldrich Japan (Tokyo, Japan). Peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F
107
(PNGase F, EC 3.5.1.52) was from Roche Applied Science (Tokyo, Japan). Neuraminidase (Arthrobacter
108
ureafaciens) was from Nacalai Tesque (Tokyo, Japan). β-Galactosidase, α-mannosidase, and
109
β-N-acetylhexosaminidase from Jack bean meal were from Prozyme, Inc. (CA, USA). Sodium
110
cyanoborohydride and iodoacetamide were obtained from Wako Pure Chemical Industries Limited
111
(Osaka, Japan). The NAP5 column (Sephadex 25-packed short column) was from GE Health Care Japan
112
(Hino, Japan). Other reagents and solvents were of the highest commercial grade.
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2.2. Preparation of APTS-labeled oligosaccharides [29]
115
N-Linked oligosaccharides were prepared from 0.1 mg of lyophilized glycoprotein. A sample was
116
dissolved in 50 µL of 50 mM phosphate buffer (pH 7.9) containing 0.1 w/v% sodium dodecyl sulfate and
117
2% 2-mercaptoethanol. The solution was heated at 100 °C for 5 min. After cooling, the solution was
118
mixed with 5 µL of 7.5 w/v% NP-40 and 2 mU (2 µL) of PNGase F and the reaction mixture incubated
119
for 2 h at 37 °C. Deglycosylated proteins were precipitated by the addition of 180 µL of ice-cold ethanol
120
and removed by centrifugation at 10,000 rpm for 5 min. The released oligosaccharides in the supernatant
121
were transferred to another tube and dried in a centrifugal vacuum evaporator. To the resultant residue,
122
each 2 µL of 0.2 M APTS in 15 v/v% acetic acid and 2 µL of 1 M NaBH3CN in tetrahydrofuran was
123
added, and the mixture was heated for 1 h at 80 °C for neutral oligosaccharides or 1.5 h at 55 °C for
124
sialylated oligosaccharides. The solution was then diluted with 85 µL of water and APTS-
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125
oligosaccharides were separated from excess APTS using a NAP5 column by collecting 0.6–1.3 mL of
126
eluate. The eluate was evaporated to dryness by a centrifugal evaporator. The residue was dissolved in
127
500 µL of water and used for analysis.
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128 2.3. ppkCE of APTS-labeled oligosaccharides
130
In all CE separations, polydimethylsiloxane (PDMS)-coated capillaries (InertCap I®; GL Sciences Inc.,
131
Tokyo, Japan) of 50 µm i.d. with an effective length of 40 cm (50 cm in total) was used with 50 mM
132
Tris-acetate buffer containing 0.5 w/v% hydroxypropylcellulose as electrophoresis buffers, whose pHs
133
were adjusted to optimum pH of each exoglycosidase : pH 7.0 for β-galactosidase and neuraminidase, pH
134
6.0 for α-fucosidase and β-N-acetylhexosaminidase, and pH 4.5 for α-mannosidase. A P/ACE MDQ
135
(Beckman Coulter, Brea, CA, USA) equipped with an argon laser-induced fluorimetric detection system
136
with a band path filter for fluorescein was used for the CE analysis. The capillary was thermostated at
137
25 °C. An exoglycosidase solution was injected to the capillary by applying a pressure of 3.45 kPa (0.5
138
psi) for 60 s followed by an intermediate buffer plug for 2 s at 3.45 kPa and APTS-oligosaccharides
139
solution for 5 s at 3.45 kPa. The APTS- oligosaccharides were separated at –15 kV. Three mixing modes
140
in exoglycosidase phases were applied to provide maximum recovery for each exoglycosidase:
141
β-Galactosidase and neuraminidase digestion were conducted by applying a separation voltage of –15 kV.
142
β-N-Acetylhexosaminidase digestion was conducted by stopping the voltage after migrated for 1 min at
143
–15 kV; after 10 min the separation was resumed. Capillary digestion using α-fucosidase and
144
α-mannosidase was completed by applying –2 kV for 10 min, then applying –15 kV for separation of
145
reaction products. After each run, an enzyme solution in the capillary was removed by introducing buffer
146
solution from the outlet by pressure application (207 kPa, 5 min) and conditioned by rinsing with 0.1 M
147
NaOH solution. The volume of enzyme solution injected in a capillary was calculated using CE Expert
148
supplied by Beckman Coulter according to the following equation.
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6 Page 6 of 22
149
V= Where V is the volume of delivered solution across the capillary (liter), ∆P denotes the pressure drop
151
across the capillary (in Pascals), d signifies the internal diameter of the capillary (meters), t is the duration
152
of pressure application (seconds), η represents the buffer viscosity (Pascal seconds), and L is the total
153
capillary length (meters). Enzymatic activity was estimated from the concentration and the
154
passing-through time of APTS-labeled oligosaccharides and the unit of an exoglycosidase in enzyme
155
plug.
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156 3. Results and Discussion
158
3.1. Principle of exoglycosidase digestion ppkCE
159
The overall scheme of exoglycosidase digestion ppkCE is shown in Fig. 1. In this method, solutions of an
160
exoglycosidase and a mixture of APTS-labeled oligosaccharides are introduced to the PDMS-coated
161
capillary filled with an electrophoresis buffer in this order. Both ends of the capillary are then immersed
162
in electrophoresis buffer and the separation voltage is applied. Because electroosmotic flow (EOF) in the
163
PDMS-coated capillary is negligible, the enzyme plug could stay near the introduced position in the
164
electric field. Whereas APTS-labeled oligosaccharides, which carry three negatively charged sulfonate
165
groups, migrate faster toward the anode and contact with exoglycosidase. In this step, any
166
oligosaccharides recognized by the glycosidase are converted to product oligosaccharides, which indicate
167
the mobilities of the products according to size and charge ratio differences. By comparing separation
168
profiles under the presence and absence of the glycosidase plug, we can find the presence or absence of
169
terminal monosaccharide residues on each saccharide peak.
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170
PDMS-coated capillary and hydroxypropylcellulose in electrophoresis buffer yielded a good
171
separation by suppressing EOF and preventing adsorption of glycosidases as well as APTS-labeled
172
oligosaccharides onto the inner surface of the capillary. EOF generation was significantly suppressed by
7 Page 7 of 22
an EOF velocity of 6.3 × 10-5 cm2V-1s-1 from anode to cathode in 100 mM Tris/acetate, pH 7.0 containing
174
0.5 w/v% hydroxypropylcellulose as electrophoresis buffer. Electrophoretic mobility of APTS
175
oligosaccharides in the same buffer ranges between –2.5 × 10-3 and –1.5 × 10-4 cm2V-1s-1. Therefore, all of
176
the APTS oligosaccharides moved and passed through the exoglycosidase plug in the ppkCE mode.
177
Typically, 3.45 kPa for 5 s was applied for hydrodynamic injections of APTS-labeled saccharide mixture
178
and 3.45 kPa for 1 min of exoglycosidase solution. The injection volumes calculated by CE Expert are
179
5.94 nL and 71.3 nL, respectively, and their plug length of the enzyme is 3 mm and 36 mm, respectively,
180
which induces ranges of contact time of APTS-labeled oligosaccharides between 1.0 min and 2.5 min at
181
–15 kV.
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182
3.2. Optimization of the ppkCE digestion using α-neuraminidase and transferrin derived glycans as a
184
model
185
The efficiency of exoglycosidase digestion ppkCE was examined using α-neuraminidase and
186
APTS-labeled oligosaccharides derived from human transferrin as a model system. Transferrin mainly
187
contains biantennary complex-type oligosaccharides with two terminal α2,6-linked NeuAc residues [30].
188
α-Neuraminidase action on the transferrin-derived oligosaccharides induces the removal of NeuAc and
189
produces neutral biantennary oligosaccharides via monosialylated oligosaccharides. Transferrin has a
190
molecular weight of around 80 kDa and contains two glycosylation sites [31]. Injected amount of
191
oligosaccharides were estimated to be 28 fmol in total. Amount of neuraminidase (25 mU/mL injected for
192
1 min) was calculated to be 1.8 µU/71 nL. The disialylated oligosaccharide may pass through the
193
neuraminidase plug for 1.1 min, which means 2.0 pmol of sialic acid linkages are hydrolyzed.
Ac ce pt e
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194
Fig. 2A shows selected ppkCE analysis of neuraminidase digestion of 28 fmol of a transferrin-derived
195
oligosaccharide mixture. Under the absence of neuraminidase (trace R), the transferrin glycan mixture
196
indicated disialylated biantennary oligosaccharides at 12.2 min with small peaks of monosialylated
8 Page 8 of 22
oligosaccharides at 13.9 min. A small peak at 12.3 min was identified as a core fucosylated disialylated
198
saccharide. Injection of 25 mU/mL of neuraminidase for 5 s induced complete dissipation of the
199
disialylated peak (12.2 min) and its conversion to a monosialylated saccharide (13.9 min) and an
200
asialylated saccharide at 16.2 min (trace a). With the increase of injection time of neuraminidase, the
201
asialylated saccharide peak (16.2 min) was increased, but the monosialylated peak remained. The
202
completed conversion to asialylated oligosaccharides requires 30 s injection of 50 mU/mL of
203
neuraminidase (trace f).
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Fig. 2B shows the effect of the concentration of neuraminidase injected for 1 min on the removal of
205
sialic acids from transferrin-derived oligosaccharides. Disialylated oligosaccharide (28 fmol) can rapidly
206
disappear by injecting 5 mU/mL of neuraminidase (trace j) and mainly converting to monosialylated
207
oligosaccharides (13.9 min). In contrast, monosialylated oligosaccharides were somewhat slowly
208
converted to neutral oligosaccharides (16.2 min) with increasing neuraminidase concentration, while the
209
complete removal of sialic acids of 28 fmol of biantennary oligosaccharides requires more than 25
210
mU/mL of neuraminidase (trace m).
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211
Fig. 2C shows the reaction varying the amount of saccharide within the 56–896 fmol range.
212
Surprisingly, 25 mU/mL (148 nU) could hydrolyze more than 896 fmol of disialylated oligosaccharides
213
(trace r) and converted to monosialylated oligosaccharides. In contrast, the monosialylated biantennary
214
oligosaccharides
215
oligosaccharides with low efficiency, and monosialylated oligosaccharides remained at amount of 56 fmol
216
of sample (trace n).
resisted
neuraminidase
digestion
and
could
be
hydrolyzed
to
asialylated
217
As a result, the neuraminidase reaction on disialylated oligosaccharides proceeded with two times
218
higher reactivity estimated from the unit definition. High enzymatic activity of neuraminidase may be due
219
to the effective mixing of substrate oligosaccharides and neuraminidase in the electric field. However,
220
monosialylated oligosaccharides indicated low reactivity toward neuraminidase digestion and only 10%
9 Page 9 of 22
of the reaction efficiency. We examined the effect of capillary temperature on hydrolysis efficiency, but
222
the recovery of glycosidase digestion did not change between 25 °C to 35 °C (see Figure S1). This will be
223
due to higher temperatures inside the capillary from Joule heating. Previously Evenhuis et al. reported
224
that the temperature of capillary insides with 75 µm i.d. is up to ca. 20°C higher than external temperature
225
[32]. The low reactivity to NeuAc in monosialylated biantennary oligosaccharides may be due to
226
substrate specificity.
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227
We also examined hydrolysis efficiency of other exoglycosidases commonly used for the analysis of
229
glycoprotein-derived oligosaccharides and found that α- and β-galactosidase indicated only a few percent
230
of the efficiency expected from enzyme unit definition, and the digestion of ca. 50 fmol of IgG
231
oligosaccharides required 200 mU/mL of galactosidase [29]. The other glycosidases i.e., α-mannosidase,
232
α-fucosidase, and β-N-acetylhexosaminidase, indicated very low reaction efficiency; the hydrolytic
233
reaction could not be completed by using 5 U/mL of glycosidases. Increasing the concentration and/or
234
injection time of glycosidases impaired the high resolution of oligosaccharides. To enhance the online
235
incubation time, we examined two approaches; “zero potential amplification” [15] and the “low mixing
236
voltage method” [33] for increasing the contact time of oligosaccharides with the exoglycosidases.
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228
238
3.3. Application of zero potential amplification for β-N-acetylhexosaminidase digestion of IgG glycans
239
β-N-Acetylhexosaminidase from Jack bean meal is commonly used for sequence analysis of glycoprotein
240
glycans, which can release both of the terminal β-linked GlcNAc and GalNAc, but the optimal pH
241
differed for two saccharides and reported to be pH 5.0 to 6.0 for releasing GlcNAc and pH 3.5 to 4.0 for
242
GalNAc [34]. Most N-linked oligosaccharides found in glycoproteins commonly possess β-linked
243
GlcNAc. Therefore, we chose pH 6.0 buffer for the ppkCE analysis.
244
The efficiency of acetylhexosaminidase ppkCE was examined using IgG-derived oligosaccharides.
10 Page 10 of 22
IgG oligosaccharides mainly consist of a series of biantennary core-fucosylated complex-type
246
oligosaccharides terminated with zero, one, and two β-linked Gal residues, which means two GlcNAc
247
residues are exposed in their non-reducing end of the agalactosylated saccharide and one GlcNAc residue
248
on two monogalactosylated oligosaccharides. Therefore, β-N-acetylhexosaminidase digestion induces
249
conversion of three peaks corresponding to two monogalactosylated to monoantennary oligosaccharides,
250
and an agalactosylated to trimannosyl (Man3GlcNAc2Fuc1) oligosaccharides.
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As described in the previous section, enzymatic activity of β-N-acetylhexosaminidase is not high
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245
enough for complete digestion of saccharides when using 5 U/mL of this enzyme. Therefore, we applied a
253
zero potential amplification method for ppkCE digestion of GlcNAc residues. Fig. 3 shows the results.
254
We used somewhat increased concentrations of β-N-acetylhexosaminidase (5 U/mL). In-capillary
255
digestion was conducted by applying –15 kV for 1 min to overlay APTS oligosaccharides near the end of
256
the enzyme plug, then stopping applied voltage for 2 min (b) to 10 min (d), after which we again applied
257
separation voltage. As shown in trace a, no application of zero potential indicated insufficient hydrolysis
258
and 50% of agalactosylated, while 40% and 20% of two monogalactosylated peaks remain without
259
digestion. Further, two isomeric oligosaccharides containing one GlcNAc residue derived from the
260
agalactosylated oligosaccharide appeared at 17.3 and 17.5 min with a small peak of Man3GlcNAc2Fuc1 at
261
15.9 min. Applying zero potential as the incubation time decreased undigested and partially digested
262
oligosaccharide peaks. Undigested peaks almost disappeared following 10 min incubation (trace d).
263
Further extension of stopping time causes broadening of oligosaccharide peaks. From these results, zero
264
potential application for 10 min seems optimal for β-N-acetylhexosaminidase digestion.
Ac ce pt e
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265 266
3.4. Application of low mixing voltage method
267
3.4.1. α-Fucosidase digestion of asialylated oligosaccharides from α1-acid glycoproteins
268
α-Fucosidase from bovine kidney is commonly used for releasing both α1,6-linked fucose (Fuc) bound to
11 Page 11 of 22
β-GlcNAc residues in reducing termini and α1,3-linked Fuc bound to lactosamine branches of complex
270
type oligosaccharides. Optimal pH of this enzyme is 6.0. Therefore, an electrophoresis buffer of pH 6.0
271
was used for in-capillary digestion with α-fucosidase and asialylated oligosaccharides derived from
272
human α1–acid glycoprotein. We increased concentration of α-fucosidase to 5 U/mL in the ordinary
273
plug-plug kinetic mode, but only ~1 fmol of Fuc residues were released from oligosaccharides (see Figure
274
S2). Here, low mixing voltage method was applied to further enhance enzymatic digestion.
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269
Fig. 4 shows the effect of the mixing voltage until the APTS-oligosaccharides completely passed
276
through the α-fucosidase plug; it takes about 9.6 min for –5kV and 48 min for –1kV. Bottom
277
electropherogram (trace R) indicates the separation of asialylated oligosaccharides from α1-acid
278
glycoproteins without injecting fucosidase. Biantennary, triantennary, fucosylated triantennary,
279
tetraantennary, and fucosylated tetraantennary oligosaccharides were completely separated. α-Fucosidase
280
digestion of the saccharides decreased fucosylated oligosaccharides appearing at 23.3 min and 26 min and
281
increased peak areas at 22.7 min and 25.1 min, respectively. As shown in Fig. 4, two peaks of fucosylated
282
oligosaccharides at 23.3 min and 26 min were drastically reduced by decreasing voltage and disappeared
283
by applying voltage lower than –2 kV. Further decrease of voltage (–1 kV) induced broadening of peaks
284
as shown in trace d; –2 kV seems most suitable for potentially applying the low mixing voltage method.
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275
286
3.4.2. α-Mannosidase digestion of high-mannose type glycans from ribonuclease B
287
Jack bean α-mannosidase can release all α-mannose residues from oligosaccharides in their non-reducing
288
termini, but the specificity is somewhat complicated. This enzyme can hydrolyze α1,2- and α1,6-linked
289
mannose (Man) more quickly than α1,3-linked Man residues. The enzyme cannot release α1,6-linked
290
Man from Manα1-6Manβ1-4GlcNAcβ1-4GlcNAc, but can release Man from α1,6-linked Man from
291
Manα1-3Manβ1-4GlcNAcβ1-4GlcNAc [35]. This means the digestion of high-mannose oligosaccharides
12 Page 12 of 22
292
by this enzyme gave two products; Manα1-6Manβ1-4GlcNAcβ1-4GlcNAc and
293
Manβ1-4GlcNAcβ1-4GlcNAc. As shown in Fig. 5, overnight digestion of APTS-labeled, high-mannose type oligosaccharides with
295
α-mannosidase mainly produced a completely digested product (Manβ1-4GlcNAcβ1-4GlcNAc) and
296
minute
297
Manα1-6Manβ1-4GlcNAcβ1-4GlcNAc from the enzyme specificity. However, application of zero
298
potential for 1.5 min indicated Man2 to Man4 oligosaccharides (trace b), while the application of low
299
mixing voltage at –2 kV for 10 min for digestion indicated mainly single peaks corresponding to
300
Manβ1-4GlcNAcβ1-4GlcNAc (trace c). The difference in the content of Man2GlcNAc2 by overnight
301
digestion and in-capillary digestion is not certain, but the complete digestion in this method may be of use
302
in the analysis of oligosaccharide structure.
of
Man2GlcNAc2
(trace
a),
which
was
estimated
to
be
M
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amounts
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294
303 4. Conclusion
305
We have optimized the conditions for online exoglycosidase digestion ppkCE for analyzing the
306
saccharide structures derived from glycoproteins. Neuraminidase digestion were completed with the plug
307
length of 36 mm (60 s injection) and applied to the separation voltage. Other exoglycosidases required
308
further optimization of other parameters to increase reaction time and produce an accurate overlap of
309
oligosaccharides with the enzyme plug. Full digestion could be obtained by zero potential amplification
310
for β-N-acetylhexosaminidase digestion and low mixing voltage application for α-fucosidase and
311
α-mannosidase digestion. Our method can be applied to other glycoprotein glycans. We believe the
312
information obtained by this method, as well as with lectin affinity ppkCE [24 -28], are useful for
313
profiling glycans in a glycoprotein. Low consumption (~10 nL) of sample solution in CE enables a series
314
of ppkCE analyses for online incubation with a series of exoglycosidases and lectins, which appears
315
straightforward in glycan analysis for minute amounts of glycoproteins.
Ac ce pt e
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13 Page 13 of 22
316 317 318
Acknowledgements This work was supported by JSPS KAKENHI Grant Number 15K18852 and 16K08209. This work
319
also
320
Technology)-supported Program for the Strategic Research Foundation at Private Universities, 2014-2018
321
(S1411037)
MEXT
(Ministry
of
Education,
Culture,
Sports,
Science
and
ip t
by
cr
supported
Ac ce pt e
d
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partly
14 Page 14 of 22
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electrophoresis
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oligosaccharides
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capillary
[26] S. Gattu, C.L. Crihfield, L.A. Holland. Microscale measurements of Michaelis-Menten constants of
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neuraminidase with Nanogel capillary electrophoresis for the determination of the sialic acid Linkage,
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Anal. Chem. 89 (2017) 929-936.
an
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[27] E. Fukushima, Y. Yagi, S. Yamamoto, Y. Nakatani, K. Kakehi, T. Hayakawa, S. Suzuki, Partial filling
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affinity capillary electrophoresis using large-volume sample stacking with an electroosmotic flow
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pump for sensitive profiling of glycoprotein-derived oligosaccharides, J. Chromatogr. A 1246 (2012)
398
84-89.
d
M
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[28] S.A. Archer-Hartmann, L.M. Sargent, D.T. Lowry, L.S. Holland, Microscale exoglycosidase
400
processing and lectin capture of glycans with phospholipid assisted capillary electrophoresis
401
separations, Anal. Chem. 83 (2011) 2740-2747.
Ac ce pt e
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[29] S.A. Archer-Hartmann, C.L. Crihfield, L.A. Holland, Online enzymatic sequencing of glycans
403
from Trastuzumab by phospholipid-assisted capillary electrophoresis, Electrophoresis 32 (2011)
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3491-3498.
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[30] Y. Yagi, S. Yamamoto, K. Kakehi, T. Hayakawa, Y. Ohyama, S. Suzuki, Application of partial-filling
406
capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in
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a therapeutic antibody, Electrophoresis 32 (2011) 2979-2985.
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[31] Y. Satomi, Y. Shimonishi, T. Hase, T. Takao, Site-specific carbohydrate profiling of human
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transferrin by nano-flow liquid chromatography/electrospray ionization mass spectrometry, Rapid
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commun. Mass spectrom. 18 (2004) 2983-2988.
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[32] G. Spik, B. Bayard, B. Fournet, G. Strecker, S. Bouquelet, J. Montreuil, Studies on glycoconjugates.
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LXIV. Complete structure of two carbohyrate units of human serotransferrin, J. FEBS Lett. 50 (1975)
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dissipation in capillary electrophoresis. Anal. Chem. 78 (2006) 2684-2693. [34] A.E. Jones, E. Grushka, Nature of temperature gradients in capillary zone electrophoresis, J. Chromatogr. 466 (1989) 219-225.
ip t
415
[33] C.J. Evenhuis, R.M. Guijt, M. Macka, P.J. Marriott, P.R. Haddad, Temperature profiles and heat
[35] C.H. Wu, R.J. Yang, Improvements on the electrokinetic injection technique for microfluidic chips, Electrophoresis 27 (2006) 4970-4981.
cr
414
296–299.
[36] S.-C. Li, Y.-T. Li, Studies on the glycosidases of Jack bean meal III. Crystallization and properties of β-N-acetylhexosaminidase, J. Biol. Chem. 245 (1970) 5135-5160.
us
413
[37] H. Oku, S. Hase, T. Ikenaka, Separation of oligomannose-type sugar chains having one to five
423
mannose residues by high-performance liquid chromatography as their pyridylamino derivatives, Anal.
424
Biochem. 186 (1990) 331-334.
427 428
M
426
[38] Z.Szabo, A. Guttman, T. Rejtar, B.L. Karger, Improved sample preparation method for glycan analysis of glycoproteins by CE-LIF and CE-MS, Electrophoresis 31 (2010) 1389-1395. [39] L.A. Gennaro, O. Salas-Solano, S. Ma, Capillary electrophoresis-mass spectrometry as a characterization tool for therapeutic proteins, Anal. Biochem. 355 (2006) 249-258.
d
425
an
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[40] F. Dang, K. Kakehi, J. Cheng, O. Tabata, M. Kurokawa, K. Nakajima, M. Ishikawa, Y. Baba, Hybrid
430
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432 433 434
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[41] Z. Zhuang, J.A. Starkey, Y. Mechref, M.V. Novotny, S.C. Jacobson, Electrophoretic analysis of N-glycans on microfluidic devices, Anal. Chem. 79 (2007) 7170-7175.
18 Page 18 of 22
434 435
Figure captions
436
Fig. 1. An image of ppkCE for in-capillary digestion with exoglycosidases. Hydrodynamic injection for 1
437
min at 3.45 kPa introduces 71.26 nL of solution which occupies 36.3 mm of the capillary.
ip t
438 Fig. 2. α-Neuraminidase digestion ppkCE of transferrin-derived oligosaccharides. (A) Oligosaccharides
440
(28 fmol) were treated with 25 mU/mL of neuraminidase injected for 5 s (a), 10 s (b), and 20 s (c), or 50
441
mU/mL injected for 5 s (d), 15 s (e), and 30 s (f). (B) Neuraminidase was injected for 1 min at
442
concentrations of 0.5 mU/mL (g), 1 mU/mL (h), 2.5 mU/mL (i), 5 mU/mL (j), 7.5 mU/mL (k), 12.5
443
mU/mL (l), or 25 mU/mL (m). (C) Neuraminidase (25 mU/mL) was injected for 1 min for the digestion
444
of 56 fmol (n), 112 fmol (o), 224 fmol (p), 448fmol (q), and 896 fmol (r) of oligosaccharides. Peaks were
445
identified according to the reference [38].
us
cr
439
an
446
Fig. 3. β-N-Acetylhexosaminidase digestion ppkCE of IgG glycans by the zero potential method. 5 U/mL
448
solution of β-N-acetylhexosaminidase was used. Application of voltage was stopped at 1 min after
449
injection of APTS oligosaccharides for 0 min (a), 2 min (b), 5 min (c), and 10 min (d). ppkCE data were
450
shifted +1.5 min to align with the reference data indicated on the bottom trace. Electropherograms a to d
451
were shifted + 1 min to align them to reference data. Peaks were identified according to the reference
452
[39].
454
Fig. 4. α-Fucosidase digestion ppkCE analysis of asialo-oligosaccharides from human α1-acid
455
glycoprotein by applying a low mixing voltage at –5 kV for 9.6 min (a), –3 kV for 16 min (b), –2 kV for
456
24 min (c), and –1 kV for 48 min (d). α-Fucosidase was dissolved in 100 mM Tris acetate (pH 6.0) at a
457
concentration of 5 U/mL. Oligosaccharides containing terminal Fuc residue (▲) were reduced and
458
disappeared by decreasing mixing voltage. Electropherograms a to d were shifted + 3.2 min to align them
459
to reference data. Peaks were identified according to the reference [40].
460
Ac ce pt e
453
d
M
447
461
Fig. 5. α-Mannosidase digestion ppkCE analysis of oligosaccharides from ribonuclease B. From bottom,
462
R, separation of ribonuclease B-derived oligosaccharides as reference; a, overnight digestion with 150
463
mU of α-mannosidase; b, zero potential was applied for 10 min with 5 U/mL α-mannosidase; c,
464
application of mixing voltage at –2 kV for 10 min with 5 U/mL of α-mannosidase. Electropherograms b
465
and c were shifted +1 min and +1.3 min to align them to reference data. Peaks were identified according
466
to the reference [41].
19 Page 19 of 22
467 468
Fig. 1.
ip t
469
Fig. 2.
A
us
471
cr
470
60000
B
50000
40000
f
l
e
k
d
j
20000
c
10000
a
0
R
11 12 13 14 15 16 17
474 475 476 477
20000
h
10000
g
0
30000
r q
i
Ac ce pt e
b
M
20000
10000
472 473
40000
30000
d
Ffluorescence intensity
m 30000
C
an
40000
p o n
R
0
11 12 13 14 15 16 17
R 11 12 13 14 15 16 17
Migration time (min)
20 Page 20 of 22
477 478
Fig. 3.
2500000
ip t
2000000
c 1500000
cr
b 1000000
us
a 500000
R
0
16
18
20
22
Migration time (min)
M
479 480 481 482
an
Fluorescence intensity
d
Fig. 4.
d
2000000
1600000
d
1400000
c
Ac ce pt e
Fluorescence intensity
1800000
1200000
b
1000000
800000
a
600000 400000 200000
R
0
16 483 484
18
20
22
24
26
28
30
Migration time (min)
21 Page 21 of 22
484 Fig. 5
ip t
b
cr
400000
c
us
600000
a
200000
an
Fluorescence intensity
485
R
0
486
10
12
14
16
18
M
8
20
Migration time (min)
Ac ce pt e
488
d
487
22 Page 22 of 22