475

J. Sep. Sci. 2015, 38, 475–482

Yi-Wei Zhang Ming-Zhe Zhao Jing-Xin Liu Ying-Lin Zhou Xin-Xiang Zhang Beijing National Laboratory for Molecular Sciences (BNLMS), MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry, Peking University, Beijing, China Received September 16, 2014 Revised November 12, 2014 Accepted November 13, 2014

Research Article

Double-layer poly(vinyl alcohol)-coated capillary for highly sensitive and stable capillary electrophoresis and capillary electrophoresis with mass spectrometry glycan analysis Glycosylation plays an important role in protein conformations and functions as well as many biological activities. Capillary electrophoresis combined with various detection methods provided remarkable developments for high-sensitivity glycan profiling. The coating of the capillary is needed for highly polar molecules from complex biosamples. A poly(vinyl alcohol)-coated capillary is commonly utilized in the capillary electrophoresis separation of saccharides sample due to the high-hydrophilicity properties. A modified facile coating workflow was carried out to acquire a novel multiple-layer poly(vinyl alcohol)-coated capillary for highly sensitive and stable analysis of glycans. The migration time fluctuation was used as index in the optimization of layers and a double layer was finally chosen, considering both the effects and simplicity in fabrication. With migration time relative standard deviation less than 1% and theoretical plates kept stable during 100 consecutive separations, the method was presented to be suitable for the analysis of glycosylation with wide linear dynamic range and good reproducibility. The glycan profiling of enzymatically released N-glycans from human serum was obtained by the presented capillary electrophoresis method combined with mass spectrometry detection with acceptable results. Keywords: Capillary electrophoresis / Glycan analysis / Mass spectrometry / Poly(vinyl alcohol) DOI 10.1002/jssc.201401025



Additional supporting information may be found in the online version of this article at the publisher’s web-site

1 Introduction The biological activity of a mature protein can be fully expressed only after diverse post-translational modifications (PTMs) please add to abbreviations [1]. Glycosylation plays an essential role as one of the most remarkable PTM forms in numerous biological processes such as immune system function and immune response [2, 3], protein folding and binding [4], interactions between recognition molecules [5], and diagnosis of many diseases [6, 7]. The research of glycobiology is becoming a far-reaching and attractive field [8]. Especially suitable for high-polarity and Correspondence: Professor Xin-Xiang Zhang, College of Chemistry, Peking University, Beijing, 100871, China E-mail: [email protected] Fax: +86-10-62754680

Abbreviations: G5, maltopentaose; G6, maltohexaose; G7, maltoheptaose; KH-550, 3-aminopropyltriethoxysilane; Meladrazine, N2 ,N2 ,N4 ,N4 -tetraethyl-6-hydrazinyl-1,3,5triazine-2,4-diamine; PGC, porous graphitized carbon; PVA, poly(vinyl alcohol)  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ionic aqueous samples’ analysis, CE is a promising tool for oligosaccharides’ analysis [9]. With the combination of LIF, CE provided significantly progress for derivative glycan analysis in the last two decades [10, 11]. Different fluorescent labeling reagents have been developed since the introduction of 8-aminopyrene-1,3,6-trisulfonic acid by Guttman et al. in 1996 [12, 13]. But the highly sensitive and reliable CE–LIF methods provided limited structure information that was significant in glycomics research. Hence MS-based methods had been developed in different modes that dramatically boosted the structure identification and characterization of complex and heterogeneity glycans [14–16]. The coupling of high-efficiency separation techniques such as CE with strong structure-identification capability detectors such as MS would achieve low sample consumption but highsensitivity analysis toward glycosylation, and even rapid, high-throughput glycoanalysis [17]. A number of targeted MS labeling strategies have been reported to accomplish relative quantification of glycans by ESI-MS [18–20] or MALDIMS [21] and structural analysis [22]. It would be very helpful to glycomics to design a suitable labeling reagent for CE–MS glycan analysis that could meet both requirements of CE www.jss-journal.com

476

J. Sep. Sci. 2015, 38, 475–482

Y.-W. Zhang et al.

separation and MS detection. A new labeling reagent for CE–UV and CE–MS glycan analysis was developed in our group that improved the CE separation, ionization efficiency, and sensitivity of glycans successfully [23]. The triazinebased compound N2 ,N2 ,N4 ,N4 -tetraethyl-6-hydrazinyl-1,3,5triazine-2,4-diamine (Meladrazine) provided multi-charges for CE separation and enhanced sensitivities especially for large glycans during ionization while the conjugated skeleton containing chromophore could be detected at 254 nm. Besides the improvement lead by labeling methods, the capillary used in CE experiment also plays an important role in separation efficiency and detection sensitivity. Coated capillaries have been widely used in CE for the suppression of EOF and for the control of interactions between analytes and capillary inner walls [24, 25]. As for glycans’ separation, various types of coated capillaries have been studied in the previous research. Mechref et al. [6] demonstrated a linear poly(acrylamide)-coated capillary in microchip electrophoresis of N-glycans obtained from serum aliquots to minimize EOF and prevent adsorption. Some commercial capillaries such as N-CHO capillary and eCAP neutral capillary were reported in CE–MS glycan analysis also [23, 26]. Poly(vinyl alcohol) (PVA) is considered as the most hydrophilic polymer among the polymers used in capillary coating strategies. Twenty years ago, the PVA polymer around 50 000 Da was thermally immobilized to silica surfaces after dynamic adsorption for the first time [27]. The permanent PVA coatings proved to be stable for series of separations under a wide range of pH. Belder et al. [28] ameliorated the coating procedure by achieving a cross-linked permanent coating through a solution of glutaraldehyde as cross-linking agent later. Both the covalently linked and dynamic-coated PVA capillary could exhibit wonderful anti-protein folding properties and inhibition of EOF function while they stayed stable over a wide range of pH [29]. These processes have been applied for separations of proteins [30], fatty acids [31], and even quaternary ammonium diastereomeric oligomers [32]. Since saccharides are compounds with strong polarity due to the multihydroxyl groups, it is obvious that PVA might provide huge potential in assisting separation [33, 34]. A modified facile coating procedure was applied to obtain a novel multiple-layer PVA-coated capillary for highly sensitive and stable analysis of glycans in this study. By multicycles of coating, the polymer distributed on the inner wall of capillary reached a more homogeneous state, thus formed a compact thin film along the surface of the inner wall. The layer-by-layer PVA-coated capillaries performed better in both separation efficiency and detection sensitivity than traditional PVA capillaries. A double-layer PVA-coated procedure was finally chosen considering both the effects and simplicity in fabrication. The stabilities and method validation of the double-layer PVA-coated capillary were demonstrated. N-glycans enzymatically released from human serum were analyzed by CE–MS techniques using the novel capillary. It is satisfactory to accomplish N-glycan profiling by the easily homemade capillaries coated layer by layer.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2 Materials and methods 2.1 Chemicals and materials N-glycosidase F (500 000 U/mL) together with denaturing buffer was purchased from New England Biolabs (Ipswich, MA, USA). Maltopentaose (G5), maltohexaose (G6), maltoheptaose (G7), dextran 1000 standard, ammonium acetate, and acetic acid were from Sigma–Aldrich (St. Louis, MO, USA). 3-Aminopropyltriethoxysilane (KH-550) was obtained from Alfa Aesar China Chemical (Tianjin, China). HPLCgrade methanol, isopropanol, and acetonitrile were from Fisher Scientific (Fair Lawn, NJ, USA). Water was purified by a Milli-Q pure water system. The bare fused-silica capillaries (365 ␮m od × 50 ␮m id) were purchased from Sino Sumtech (Hebei, China). C18 Sep-Pak cartridges were purchased from Waters (Milford, MA, USA); porous graphitized carbon (PGC) columns were from Grace (Columbia, MD, USA).

2.2 Coating procedure A fused-silica capillary of 50 ␮m id and 365 ␮m od was coated according to a standard procedure described in the literature with a little modification [28, 35, 36]. All the solutions were pumped into the capillary under controlled nitrogen pressure by a custom-made pressure control stainless-steel autoclave. The coating procedures of one-layer PVA-coated capillary were as follows: the capillary was treated with 5 M HCl for 10 min, pure water for 10 min, 1 M NaOH for 30 min, pure water for 30 min, MeOH for 15 min, and dried by nitrogen. Then KH-550 dissolved in acetone (1:1 v/v) was pumped into the capillary and reacted at 60⬚C for 12 h. After silanization, the capillary was washed with acetone for 15 min and dried by nitrogen. Glutaraldehyde was introduced between silanization reagent and polymer to accomplish covalent linkage. In detail, 10% glutaraldehyde dissolved in 50 mM borate buffer at pH 9.18 was pumped into the capillary for 1 h and rinsed with water for 15 min. The acidified PVA solution (3% aqueous PVA mixed with 5 M HCl in proportions of 9:1 v/v) was pumped for 1 h at 0.5 MPa for coating, and then washed with water for 30 min. For double and more layers of PVA coating for capillary, the glutaraldehyde solution and acidified PVA solution steps were repeated to get multi-layer PVA films, as shown in Scheme 1.

2.3 Sample pretreatment and labeling The human serum was enzymatically treated by PNGase F at 37⬚C for 18 h as the reported protocol without any pretreatment. The released N-glycans were purified with C18 SPE cartridge and PGC SPE cartridge. The purified glycans were collected and dried with SpeedVac (Thermo Fisher, MA, USA). www.jss-journal.com

Electrodriven Separations

J. Sep. Sci. 2015, 38, 475–482

477

Scheme 1. The coating procedures of multiple-layer PVA-coated capillary.

The labeling procedures using our new labeling reagent Meladrazine were carried out according to a previously reported method [23], with some modifications. The mixture of glycan samples was labeled by Meladrazine reagent T in 60% isopropanol solution containing 1% acetic acid with reaction molar ratio of 1:200 (glycans:Meladrazine). The labeling procedures of Meladrazine’s stable isotope compound Meladrazine(d20 ) were the same as those of Meladrazine. All labeled samples were extracted by dichloromethane before injection to remove the excess labeling reagent, which might affect the separation.

2.4 CE–UV measurements CE experiments were performed on a P/ACE MDQ CE system (Beckman Coulter, Brea, CA, USA) equipped with UV detector. Data were collected and processed by 32 Karat Software (Beckman Coulter). Both bare fused-silica capillary and PVA-coated capillary were 365 ␮m od × 50 ␮m id. For the separation of three maltooligosaccharide standards and dextran 1000, 28 cm total length of capillary and 30 kV voltages were applied, while ammonium acetate solution (30 mM, pH 4.8) served as a separation BGE. The hydrodynamic injection was set at 0.5 psi for 10 s. The detection wavelength was 254 nm.

2.5 CE–MS measurements CE–MS experiments were performed on a Beckman PA800 plus CE system and Agilent 6320 ion-trap mass spectrometer (Palo Alto, CA, USA) with sheath liquid interface provided by Agilent Technology. For the three maltooligosaccharide standards and dextran 1000, 65 cm total length double-layer PVA-coated capillary with 365 ␮m od × 50 ␮m id was applied. For N-glycans enzymatically released from human serum, 60 cm total length capillary was applied. The separation BGE was ammonium acetate solution (30 mM, pH 4.8), while 0.1% formic acid in 50% methanol served as sheath liquid with a flow rate of 6 ␮L/min. After the 10 s injection under 0.5 psi, 30 kV voltage was applied to accomplish the separation. The  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

MS detection range was from 500 to 2200 m/z. Spray voltage was 4100 V. The nebulizer gas was 5.0 psi. The capillary temperature was 250⬚C.

3 Results and discussion 3.1 Coating procedure It is difficult to achieve homogenous modifications when immobilizing PVA to the inner wall of a capillary due to the particular properties of PVA. The water-soluble polymer is not suitable for some chemical reactions when fixing the polymer to the wall. The high hydrophilicity may also lead to inhomogeneities during the reactions. Properly choosing the silanization reagent and linking groups is important for PVA coating. KH-550 was chosen as the silanization reagent. According to aldehyde reaction rules, aldehydes can react with alcohols in the presence of acid catalysts to form acetals [37]. In the study, glutaraldehyde, which took along two aldehyde groups to react with amino groups on KH-550 and hydroxyl groups on PVA, acted as the linking reagent. By repetitive cycles of coating with glutaradehyde and acidified PVA solution, double or even multiple layers of PVA coating could be attached on the capillary inner wall. As shown in Scheme 1, the multiple layers of PVA provided a much more firm and dense cover layer on the surface of capillary inner wall. Unlike traditional PVA-coated capillaries, the multilayer coating had less exposed sites of silicon hydroxyl, unreacted silanization reagent, and connection reagent, which as a consequence achieved a more homogeneous state on the inner wall. It would no doubt suppress the EOF as much as possible and improve the capillary stability during separations.

3.2 Separation comparison using a bare fused-silica capillary and PVA-coated capillary Glycan is a strongly polar compound because of the abundant hydroxyl groups in the structures. The separation of the threemaltooligosaccharide standard mixture (G5, G6, and G7) in a bare fused-silica capillary showed an unsatisfactory result www.jss-journal.com

478

Y.-W. Zhang et al.

J. Sep. Sci. 2015, 38, 475–482

Figure 1. Separation of the three maltooligosaccharide standards using uncoated capillary (A) and double-layer PVA-coated capillary (B). Capillary: 28 cm total length with 18 cm effective length; voltage: 30 kV; BGE: ammonium acetate solution (30 mM, pH 4.8); injection: 0.5 psi, 10 s; sample: mixture of G5, G6, and G7 with concentration of 20 ␮g/mL each.

Figure 2. (A) Migration time RSD (n = 10) of different-layer PVA-coated capillary. (B) Electropherogram for dextran 1000 sample using double-layer PVA-coated capillary. Capillary: 28 cm total length with 18 cm effective length; voltage: 30 kV; BGE: ammonium acetate solution (30 mM, pH 4.8); injection: 0.5 psi, 10 s; sample: dextran 1000 with total concentration of 500 ␮g/mL; G2 to G11 represent the degree of the polymerization of glucose units in dextran 1000.

with bad resolution and low signal intensity (Fig. 1A). On the contrary, the separation in the same conditions using a homemade PVA-coated capillary presented good separation efficiency and detection sensitivity (Fig. 1B). This was mainly due to the EOF suppression effect of PVA coating that greatly decreased the absorption of maltooligosaccharides.

3.3 Performance of different PVA-layered capillaries Since it is difficult to characterize the coating on the capillary inner wall by SEM or TEM, the RSD of the separation migration time was chosen as the main index to optimize the layers. The separations of Meladrazine-labeled dextran 1000 in different layer-coated capillaries were studied. As Fig. 2A shows,  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

two-layers PVA-coated capillary had a significant decrease in migration time RSD (n = 10), which was only around 0.6% in contrast to traditional PVA-coated capillary with only onelayer coating. However, three layers show little improvement than the two-layer modification. The excellent stability of migration time provided reliable premise for quantitative CE analysis of glycan samples. Figure 2B represented the typical separation of dextran 1000 sample using the optimized two layers coated capillary. Fifteen components were well separated and detected within 10 min. Dextran 1000 has a linear backbone of D-glucopyranosyl repeating units, which is a mixture of maltooligosaccharides with different polymerization of glucose units. Under the assumption that absorption coefficient was a constant for all Meladrazine-labeled glycans, the concentration of each maltooligosaccharide in dextran could www.jss-journal.com

Electrodriven Separations

J. Sep. Sci. 2015, 38, 475–482

479

Table 1. Estimated concentrations of each maltooligosaccharide in dextran 1000 standard solution

Glycan

Mol. wt.

Average peak area (n = 3)

Concentration (␮g/mL)

G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15

342.1 504.2 666.2 828.3 990.3 1152.4 1314.4 1476.5 1638.5 1800.6 1962.6 2124.7 2286.8 2448.8

1423 16 523 30 193 33 078 28 590 21 572 17 198 12 759 7312 4454 2734 1912 1442 545

1.0 16.6 40.2 54.7 56.5 49.6 45.1 37.6 23.9 16.0 10.7 8.1 6.6 2.7

Experimental conditions: double-layer PVA-coated capillary (28 cm total length with 18 cm effective length); 30 kV separation voltage; ammonium acetate solution (30 mM, pH 4.8) as BGE; 0.5 psi, 10 s injection; dextran 1000 with total concentration of 500 ␮g/mL; G2 to G11 represent the degree of the polymerization of glucose units in dextran 1000.

be estimated by the Lambert–Beer law according to the known concentration of G5, G6, and G7 standards. Table 1 contains the estimated concentrations of each maltooligosaccharide in detail.

3.4 Stability of the double-layer PVA coating To confirm the stability of the double-layer coating, 100 continuous separation runs were carried out. Only water and buffer were used to wash the capillary for 2 min before each run. After every 20 runs, buffers were replaced by fresh ones to eliminate the interferences of composition variation caused by electrolysis around the electrodes and Joule heating. Figure 3 illustrates that the reproducibility of migration time and theoretical plates (USP) of three maltooligosaccharides among the 100 runs maintained stable. The results elucidated that the double-layer coating would not cause serious deciduous phenomenon that might affect separation performance.

Figure 3. Reproducibility of the migration time and theoretical plates (USP) of G5, G6, and G7 mixture using double-layer PVAcoated capillary. Capillary: 28 cm total length with 18 cm effective length; voltage: 30 kV; BGE: ammonium acetate solution (30 mM, pH 4.8); injection: 0.5 psi, 10 s; sample concentration: 20 ␮g/mL for each maltooligosaccharide. Table 2. Calibration curve and LODs (S/N = 3) for three glycan standards

Compound

Calibration curve

Relative LOD (␮g/mL)

Absolute LOD (pg)

G5 G6 G7

A = 2.73 × 102 c + 4.19 × 102 A = 2.23 × 102 c + 4.02 × 102 A = 1.93 × 102 c + 4.09 × 102

0.7 0.5 0.5

5 4 4

Their linearities and LODs (S/N = 3) are summarized in Table 2. The LODs were determined as 0.7, 0.5, and 0.5 ␮g/mL for the three maltooligosaccharides at an S/N ratio of 3, respectively. Considering the tiny volume injection of CE, which was only 22 nL, the absolute LODs were as low as 4–5 pg for the three maltooligosaccharides. This result takes into consideration the incomplete reaction during labeling and the extraction loss during sample-pretreatment procedures. The labeling reaction by Meladrazine could not fully complete due to the intrinsic reversible mechanism and each labeled sample was extracted by dichloromethane before injection. The removal of superfluous labeling reagent accompanied the loss of labeled saccharides to some degree. It is potentially to get a lower detection limit by improved sample-pretreatment techniques.

3.5 Method validation

3.6 Comparison of labeling reagent Meladrazine and its stable isotope form Meladrazine (d20 )

The calibration curves were exhibited in Supporting Information Fig. S1 with correlation coefficients (R2 ) for three maltooligosaccharide standards, all between 0.994 and 0.998. Calibration curves for peak area (A) versus analyte concentration (c) were fitted using a linear equation (A) = a (c) + b. Good linearities and a wide dynamic concentration range (1, 2, 5, 10, 20, 50, 100 ␮g/mL) were obtained for the threemaltooligosaccharide mixture under optimized conditions.

Relative quantification by stable isotope labeling provided a possible tool for quantitative variations analysis in glycan profiling. Since the labeling reagent Meladrazine and its stable isotope compound Meladrazine (d20 ) have feasibility in CE–MS glycan profiling, it is important to ensure the uniform behaviors of the two labeling reagents in CE separation. The migration time is the main parameter for qualitative of various saccharides. To investigate the

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.jss-journal.com

480

J. Sep. Sci. 2015, 38, 475–482

Y.-W. Zhang et al.

Figure 4. The migration time (A) and width at 50% height (B) comparison of labeling reagent Meladrazine- and Meladrazine(d20 )-labeled maltooligosaccharide standards mixture. Capillary: 28 cm total length with 18 cm effective length; voltage: 30 kV; BGE: ammonium acetate solution (30 mM, pH 4.8); injection: 0.5 psi, 10 s; sample concentration: 20 ␮g/mL for each maltooligosaccharide and 20 ␮g/mL Quinine.

migration-time difference in CE separation of the new labeling reagent Meladrazine and its stable isotope-labeled samples, standard maltooligosaccharides mixture labeled by two labeling reagents were separated using the double-layer PVA-coated capillary. A total of 20 ␮g/mL quinine was added as an internal standard for migration time correction due to its appropriate migration behavior. Figure 4A shows the ideal performance of the two labeling reagents. The migration time between two reagents was almost the same for each particular glycan with RSD below 0.3% (n = 3). The uniformity validated that the stable isotopic labeling reagent was reliable without obvious variances in migration time compared with Meladrazine in separation process. An additional parameter, width at 50% height, was evaluated, which also presented the same spikes for two reagents (Fig. 4B).

3.7 CE–ESI-MS analysis and glycan profiling of human serum sample CE with sheath liquid ESI-MS using the enhanced coating procedure was carried out to demonstrate the applicability of the double-layer PVA-coated capillary in combination analysis. Standard maltooligosaccharides—including G5, G6, and G7—mixture and dextran 1000 were studied first. Figure 5A was the extracted ion chromatogram of three maltooligosaccharides with concentration of 5 ␮g/mL for each. Since the ionization efficiencies for larger maltooligosaccharides are lower than that of G5, G7 and G6 showed much lower intensity in contrast with G5. However, the baseline separation of three maltooligosaccharides was still obtained with good peak shapes. The total ion chromatogram is shown in Supporting Information Fig. S2. Similarly, the extracted ion chromatogram and total ion chromatogram of dextran 1000 are illustrated in Supporting Information Fig. S3. The identification of each maltooligosaccharide in dextran was  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

achieved from Supporting Information Fig. S3A, which agreed with the results demonstrated in Fig. 2B. The structural information provided by MS made glycan profiling more applicable. The enzymatically released Nglycans from human serum were analyzed by the presented method. A total of 10 ␮L serum was directly treated with PNGase F, and then purified by a C18 column and PGC column in succession to remove the excess proteins, peptides, and salts. The released N-glycans were finally labeled by Meladrazine, and then dissolved in 50 ␮L deionized water for injection after extraction. The extracted ion chromatogram and corresponding total ion chromatogram of N-glycans from human serum were presented in Fig. 5B and Supporting Information Fig. S4. The m/z values and structures of detected N-glycans are listed in the Table 3. These identification results of the complex and heterogeneity glycans from human serum were consistent with reported values [38,39]. Although the dilution effect caused by sheath liquid and siphon resulting from nebulizer gas pressure greatly diminished the sensitivity and separation efficiency, the coated capillary performed well in the CE–ESI-MS test with stable current and satisfied separation. Together with MS, a kind of structural identification capability detector, the novel homemade double-layered PVA capillary proved to be feasible in CE–MS N-glycan profiling.

4 Concluding remarks In the study, an improved facile coating method of multiplelayer PVA-coated capillary for high-sensitivity CE and CE–MS analysis of glycans was introduced. By cycling the coating procedure, stable and compact multiple PVA films were attached to the surface of capillary inner wall, which provided a more homogeneous state and performed better in the separation processes. The double-layer PVA-coated capillary formed by two cycles coating showed the best separation stability of www.jss-journal.com

Electrodriven Separations

J. Sep. Sci. 2015, 38, 475–482

481

Figure 5. Extracted ion chromatograms of G5, G6, and G7 mixture (A) and human serum N-glycans (B) by CE–MS analysis. Capillary: 65 cm (A) and 60 cm (B) total length double-layer PVA-coated capillary with 365 ␮m od × 50 ␮m id; voltage: 30 kV; BGE: ammonium acetate solution (30 mM, pH 4.8); injection: 0.5 psi, 10 s; sheath liquid: 0.1% formic acid in 50% methanol with flow rate of 6 ␮L/min; nebulizer gas: 5.0 psi; sample concentration: 5 ␮g/mL for each maltooligosaccharide. The blue square stands for N-acetylglucosamine; green circle is for mannose; yellow circle is for galactose; red triangle is for fucose.

Table 3. The analysis results of human serum N-glycans

m/z

Glycan

1349.8/[M+H]2+

Hex3 HexNAc3

1470.8/[M+H]2+

Hex5 HexNAc2

1495.8/[M+H]2+

Hex3 HexNAc3 Fuc1

1552.8/[M+H]2+

Hex3 HexNAc4

849.8/[M+2H]2+

Hex3 HexNAc4 Fuc1

1714.8/[M+H]2+

Hex4 HexNAc4

930.0/[M+2H]2+

1012.3/[M+2H]2+

Structure

Hex4 HexNAc4 Fuc1

Hex5 HexNAc4 Fuc1

N-acetylglucosamine Fucose. Mannose Galacotose.

Meladrazine(d20 ) during the separation. With acceptable resolution and well reproducibility, the easily homemade doublelayer PVA-coated capillaries were suitable for CE–ESI-MS analysis as well, which revealed their great potential in Nglycan profiling, and made a contribution to CE-and CE–MSbased glycomics research. This work was supported by National Natural Science Foundation of China (no. 21275009). The authors have declared no conflict of interest.

5 References [1] Alley, W. R., Mann, B. F., Novotny, M. V., Chem. Rev. 2013, 113, 2668–2732. [2] Lowe, J. B., Cell 2001, 104, 809–812. [3] Wang, C.C., Chen, J.R., Tseng, Y.C., Hsu, C.H., Hung, Y. F., Chen, S. W., Chen, C. M., Khoo, K. H., Cheng, T. J., Cheng, Y. S E., Jan, J. T., Wu, C. Y., Ma, C., Wong, C. H., Proc. Natl. Acad. Sci. USA 2009, 106, 18137–18142. [4] Wijeyesakere, S. J., Rizvi, S. M., Raghavan, M., J. Biol. Chem. 2013, 288, 35104–35116. [5] Kleene, R., Schachner, M., Nat. Rev. Neurosci. 2004, 5, 195–208.

migration time and kept in good condition in 100 sequence runs. The capillaries had no distinction for both labeling reagent Meladrazine and its stable isotope form  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

[6] Mitra, I., Zhuang, Z., Zhang, Y., Yu, C. Y., Hammoud, Z. T., Tang, H., Mechref, Y., Jacobson, S. C., Anal. Chem. 2012, 84, 3621–3627.

www.jss-journal.com

482

Y.-W. Zhang et al.

[7] Mitra, I., Alley, W. R., Jr., Goetz, J. A., Vasseur, J. A., Novotny, M. V., Jacobson, S. C., J. Proteome Res. 2013, 12, 4490–4496. [8] Dwek, R. A., Chem. Rev.1996, 96, 683–720. [9] Bakry, R., Huck, C. W., Najam-ul-Haq, M., Rainer, M., Bonn, G. K., J. Sep. Sci. 2007, 30, 192–201. [10] Liu, J. P., Shirota, O., Wiesler, D., Novotny, M., Proc. Natl. Acad. Sci. USA 1991, 88, 2302–2306. [11] Wang, Y., Santos, M., Guttman, A., J. Sep. Sci. 2013, 36, 2862–2867. [12] Guttman, A., Nature 1996, 380, 461–462. [13] Guttman, A., Chen, F. T. A., Evangelista, R. A., Electrophoresis 1996, 17, 412–417. [14] Wuhrer, M., Glycoconj. J. 2013, 30, 11–22. [15] Nakano, M., Higo, D., Arai, E., Nakagawa, T., Kakehi, K., Taniguchi, N. Kondo, A., Glycobiology 2009, 19, 135–143. [16] Gennaro, L. A., Salas-Solano, O., Anal. Chem. 2008, 80, 3838–3845. [17] Lazar, I. M., Lazar, A. C., Cortes, D. F., Kabulski, J. L., Electrophoresis 2011, 32, 3–13. [18] Walker, S. H., Budhathoki-Uprety, J., Novak, B. M., Muddiman, D. C., Anal. Chem. 2011, 83, 6738–6745. [19] Yang, S., Yuan, W., Yang, W. M., Zhou, J. Y., Harlan, R., Edwards, J., Li, S. W., Zhang, H., Anal. Chem. 2013, 85, 8188–8195.

J. Sep. Sci. 2015, 38, 475–482

[23] Tie, C., Zhang, X. X., Anal. Methods 2012, 4, 357–359. [24] Haselberg, R., de Jong, G. J., Somsen, G. W., J. Sep. Sci. 2009, 32, 2408–2415. [25] Kamande, M. W., Fletcher, K. A., Lowry, M., Warner, I. M., J. Sep. Sci. 2005, 28, 710–718. ¨ [26] Bunz, S. C., Rapp, E., Neususs, C., Anal. Chem. 2013, 85, 10218–10224. [27] Gilges, M., Kleemiss, M. H., Schomburg, G., Anal. Chem. 1994, 66, 2038–2046. [28] Belder, D., Deege, A., Husmann, H., Kohler, F., Ludwig, M., Electrophoresis 2001, 22, 3813–3818. [29] Steiner, F., Hassel, M., Electrophoresis 2003, 24, 399–407. [30] Baderia, V. K., Gowri, V. S., Sanghi, S. K., Shukla, A., Singh, D. K., Sanghi, S. B., J. Anal. Chem. 2012, 67, 278– 283. [31] Buglione, L., See, H. H., Hauser, P. C., Electrophoresis 2013, 34, 2072–2077. [32] Zhang, B., Krull, I. S., Cohen, A., Smisek, D. L., Kloss, A., Wang, B., Bourque, A. J., J. Chromatogr. A 2004, 1034, 213–220. [33] Liu, Y., Salas-Solano, O., Gennaro, L. A., Anal. Chem. 2009, 81, 6823–6829. ¨ [34] Jooß, K., Sommer, J., Bunz, S.-C., Neusuß, C., Electrophoresis 2013, 00, 1–8. [35] Xu, L., Dong, X. Y., Sun, Y., J. Chromatogr. A 2009, 1216, 6071–6076.

[20] Wang, C. J., Wu, Z. Y., Yuan, J. B., Wang, B., Zhang, P., Zhang, Y., Wang, Z. F., Huang, L. J., J. Proteome Res. 2013, 13, 372–384.

[36] Xu, L., Dong, X.-Y., Sun, Y., Biochem. Eng. J. 2010, 53, 137–142.

[21] Kaneshiro, K., Watanabe, M., Terasawa, K., Uchimura, H., Fukuyama, Y., Iwamoto, S., Sato, T-A., Shimizu, K., Tsujimoto, G. Tanaka, K., Anal. Chem.2012, 84, 7146– 7151.

[38] Cao, L. W., Zhang, Y., Chen, L. L., Shen, A. J., Zhang, X. W., Ren, S. F., Gu, J. X., Yu, L., Liang, X. M., Analyst 2014, 139, 4538–4546.

[22] Nishikaze, T., Kaneshiro, K., Kawabata, S.-I., Tanaka, K., Anal. Chem. 2012, 84, 9453–9461.

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

[37] Meskens, F. A. J., Synthesis 1981, 7, 501–522.

[39] Gaye, M. M., Valentine, S. J., Hu, Y., Mirjankar, N., Hammoud, Z. T., Mechref, Y., Lavine, B. K., Clemmer, D. E., J. Proteome Res. 2012, 11, 6102–6110.

www.jss-journal.com

Double-layer poly(vinyl alcohol)-coated capillary for highly sensitive and stable capillary electrophoresis and capillary electrophoresis with mass spectrometry glycan analysis.

Glycosylation plays an important role in protein conformations and functions as well as many biological activities. Capillary electrophoresis combined...
4MB Sizes 1 Downloads 8 Views