Release, separation, and recovery of monomeric reducing N-glycans

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Release, separation, and recovery of monomeric reducing N-glycans by Pronase E combined with Fmoc-Cl and glycosylasparaginase Yu Lu, Cheng Li, Ming Wei, Yue Jia, Jingjing Song, Ying Zhang, Chengjian Wang, Linjuan Huang, and Zhongfu Wang Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01224 • Publication Date (Web): 19 Jan 2019 Downloaded from http://pubs.acs.org on January 21, 2019

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Biochemistry

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Release, separation, and recovery of monomeric reducing N-glycans by Pronase E combined with

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Fmoc-Cl and glycosylasparaginase

3 4

1Yu

Lu, 1Cheng Li, 1Ming Wei, 1Yue Jia, 1Jingjing Song, 1Ying Zhang,1,2Chengjian Wang,1,2Linjuan Huang*,

5

1,2Zhongfu

Wang*

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1The

College of Life Sciences, Northwest University, Xi'an 710069, China

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2Glycobiology

and Glycotechnology Research center, College of Food Science and technology, Northwest

University, Xi’an 710069, China

11 12

*Corresponding authors:

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Linjuan Huang and Zhongfu Wang

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Phone: +86 29 88305853

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Fax: +86 29 88303534

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E-mails: [email protected] (Linjuan Huang); [email protected] (Zhongfu Wang)

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Short title: Preparation of monomeric reducing N-glycans

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Subject category: Carbohydrates

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ABSTRACT

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Glycan moiety of glycoproteins play key roles in various biological processes. However, there are few versatile

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methods for releasing, separating, and recovering monomeric reducing N-glycans for further functional analysis.

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In this study, we developed a new method to achieve the release, separation, and recovery of monomeric

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reducing N-glycans using enzyme E (Pronase E) combined with 9-chloromethyl chloroformate (Fmoc-Cl) and

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glycosylasparaginase (GA). Ovalbumin, Ribonuclease B (Ribo B), ginkgo, and pine nuts glycoproteins were

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used as materials and were sequentially enzymatically hydrolyzed with Pronase E, derivatized with Fmoc-Cl,

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and enzymatically hydrolyzed with GA. The products produced by this method were then detected by

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electrospray ionization mass spectrometry (ESI-MS), high-performance liquid chromatography (HPLC), and

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online hydrophilic interaction chromatography (HILIC-MS )separation. The results showed that all N-glycans

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with essentially one amino acid obtained by Pronase E were labeled with Fmoc-Cl and could be efficiently

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separated and detected via HPLC and HILIC-MS). Finally, the isolated Asn-glycan derivatives were digested

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with GA, enabling the recovery of all monomeric reducing N-glycans modified by core α-1,3 fucose. This

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method was simple, low cost, and broadly applicable and could therefore have great importance for analysis of

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the structure-function relationships of glycans.

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KEYWORDS: reducing N-glycans; 9-chloromethyl chloroformate; electrospray ionization mass spectrometry;

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liquid chromatography mass spectrometry

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Biochemistry

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INTRODUCTION

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N-Glycosylation of proteins is an important post-translational modification.1 Glycan moieties of glycoproteins

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have essential roles in a variety of biological processes,2-4 such as protein folding, solubility, half-life,

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antigenicity, and biological activity. Glycans, as direct receptors for extracellular surface signaling molecules,

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are involved in cell recognition, adhesion, signal transduction between cells, immune responses,5-7

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inflammation, autoimmune diseases, aging, abnormal proliferation and metastasis of cancer cells, and pathogen

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infection8,9.

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Due to differences in the physicochemical properties of carbohydrate and protein moieties, in order to

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study the structures of glycans and their structure-activity relationships, it is first necessary to release the

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glycans from the peptide backbone. PNGase F is the most commonly used enzyme for the release of

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N-glycans;10-12 however, the enzyme does not release core α-1,3 fucose-modified N-glycans derived from

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plants, lower animals, and microorganisms. PNGase A13 can be used to release all N-glycans with a

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pentasaccharide core structure, including core α-1,3 fucose.14 Unfortunately, the use of this enzyme is limited by

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its high cost, low enzyme activity, and lack of ability to release acidic glycans. In addition, PNGase A15 does not

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act on glycoproteins, but only on glycopeptide. Therefore, glycoproteins must be hydrolyzed with pepsin or

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trypsin to glycopeptide before using this enzyme, further limiting its range of applications.

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Because of the non-templated nature of glycan synthesis, glycans released from glycoproteins generally

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include a mixture of various glycans, necessitating further separation into single structure glycan components by

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high-performance liquid chromatography (HPLC) for subsequent analysis of the structure-function relationships

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of the glycans. However, these native glycans have no chromophore groups; therefore, separating and detecting

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such glycans by HPLC/HILIC-MS is challenging. Derivatization not only enables the glycans to carry an

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ultraviolet or fluorescent chromophore group for detection, improving the detection sensitivity, but also reduces

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the polarity of the glycans, enhancing the retention of the glycans on the reversed-phase column and facilitating

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their separation. At present, there are many precolumn derivatization methods for the aldehyde group of the

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reducing glycan C-1 terminal,16 and the reductive amination method is commonly used17,18 for direct labeling of

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the reducing glycans. This method produces derivatives with an open-ring structure,19 resulting in partial loss of

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biological information of the glycans and affecting some of the activities of the glycans. In addition, recovery of

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the reducing glycans is hindered after separation of the labeled glycans, limiting further research applications,

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such as use of glycan and protein carrier bonding to prepare antibodies for antigens or immobilization of glycans

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onto chips to construct a glycan microarray.21

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In a previous study, Song et al.19 used Pronase E to enzymatically digest the polypeptide, obtaining

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Asn-glycans with different lengths, and the amino groups were then derivatized and, applied for glycan

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microarray analysis. Moreover, An et al.22 used Pronase E to obtain Asn-glycans with multiple amino acids,

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investigating the glycosylation sites of N-glycans by comparison with the glycans released by PNGase F.

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However, these methods yielded Asn-glycans with different lengths, affecting further glycan analysis.

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Subsequently, our laboratory23 succeeded in obtaining an N-glycan with only one asparagine residue by

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optimizing the enzymatic hydrolysis conditions for Pronase E, retaining the native closed-ring structure of the

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glycans, and avoiding the complicated problems caused by the heterogeneity of the glycopeptide in the presence

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of insufficient amounts of enzyme. The Asn-glycan molecules produced under enzymatic conditions were

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labeled with methyl 9-chloroformate (Fmoc-Cl). However, further recovery of reducing N-glycans was not

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carried out.

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Currently, the main method for recovering monomeric reducing N-glycans is chemical approach.24-26

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Biochemistry

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Takahashi et al.26 reported the conversion of pyridine-derived glycans to reducing glycans by a two-step

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reaction. Additionally, Suzuki et al.25 found that glycans could be derivatized by reductive amination and then

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incubated with hydrogen peroxide solution to recover the reducing glycans. However, both of these methods

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involved harsh reaction conditions and resulted in low glycan recovery. Hence, Song et al.24 developed a method

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through which aromatic amine reagent-labeled glycans could react with N-bromosuccinimide, yielding reducing

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glycans. The reaction conditions for method are mild; however, many by-products are produced. Therefore, it is

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particularly important to establish an effective method for releasing, separating, and recovering reducing

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N-glycans.

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In this study, we developed a new method for the release, separation, and recovery of reducing glycans

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including core α-1,3 fucose-modified ones using Pronase E digestion, derivatization with Fmoc-Cl carrying a

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ultraviolet/fluorescent chromophore, and fragmentation of Fmoc-labeled Asn-glycans using the lysosomal

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hydrolase glycosylasparaginase (GA), producing monomeric reducing N-glycans.27 Our results provided

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important insights into analysis of the structure-function relationships of glycans and may facilitate the

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construction of glycan libraries.

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MATERIALS AND METHODS

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Materials. Pronase E was purchased from Beijing BioDee Technology Co. Ltd (CN), ovalbumin from chicken

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egg white (ovalbumin), Fmoc-Cl, and microcrystalline cellulose filler (microcrystalline) powder were purchased

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from Sigma (St. Louis, MO, USA). Burdock Ribo B was purchased from Worthington, and the Sep-Pak C18

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columns and graphite carbon columns (GCCs) were purchased from Waters. Other reagents were analytical

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pure.

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Extraction of total glycoprotein. For isolation and purification of total protein from ginkgo, ginkgo seeds were

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hulled into powder, which was then defatted with petroleum ether. Next, 0.15 M Tris-HCl (pH = 8.0) buffer was

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added at a w/v (ginkgo defatted powder/buffer) ratio of 1:20. Leaching was performed for 4 h, and samples were

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then centrifuged at 4°C and 11000 rpm for 30 min. The supernatant was then carefully removed from the

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beaker, ammonium sulfate was slowly added until the saturation was 35%, and samples were centrifuged at 4°C

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and 11000 rpm for 15 min. The supernatant was then removed, and ammonium sulfate was further added to a

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saturation of 90%. The mixture was centrifuged under the same conditions, and the precipitate was collected and

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redissolved in deionized water. The pH was adjusted to neutral, and the samples were dialyzed and freeze-dried

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for use.28

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Isolation and purification of total pine proteins were carried out as described for total protein above.

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Release of Asn-glycans. For Pronase E enzymatic hydrolysis, 5 mg Pronase E was dissolved in 1 mL of 1 M

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NH4HCO3 buffer (pH = 8) and preheated at 60°C for 30 min. Next, 5 mg protein was added to the above

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solution and incubated at 37°C for 24 h. After denaturation at 100°C for 5 min, the Asn-glycans were

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concentrated using a nitrogen blow dryer.23

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Purification of glycans was carried out as follows. First, a large amount of protein and a small amount of

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salt in the reaction sample were removed with microcrystalline cellulose. The column was eluted with a large

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amount of Mill-Q water (about 100 times the column volume), removing some of the oligosaccharides

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contained in the filler itself, and then two times the volume of the protein eluate (n-butanol:methanol:water =

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4:1:1) was used to equilibrate the solution. After enzymatic hydrolysis, the sample was dissolved in a protein

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Biochemistry

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eluate, and the impurities were eluted with about 50 column volumes of protein eluate. Finally, the glycans were

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eluted with 2 mL Milli-Q water and collected in the tubes. The organic solvent was removed by a nitrogen blow

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dryer and concentrated for use in subsequent derivatization.29-31

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Derivatization of Asn-glycans. For Fmoc-Cl derivatization, the obtained Asn-glycans were dissolved in 200 μL

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double distilled water, and the Fmoc-Cl was dissolved in tetrahydrofuran. Next, an equal volume of 50 M

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sodium bicarbonate solution, an equal volume of water, and an equal volume of 20 M Fmoc-Cl solution were

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added to the Asn-glycans, and the mixture was shaken for 2 h and extracted with ethyl acetate three times. The

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upper layer was discarded, and the aqueous layer was left. The sample was then concentrated and dried.

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For purification of glycans, a C18 solid-phase extraction column was used. The procedure was as follows.

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First, the column was activated with 3 mL acetonitrile and then equilibrated with 10 mL double distilled water.

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Next, the sample was repeatedly applied three times, washed with 12 mL water, eluted with 25% acetonitrile,

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collected, and concentrated for use in subsequent HPLC separation and online HILIC-MS separation analysis.

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Recovery of reducing glycans. GA enzymatic hydrolysis was carried out as follows. First, Fmoc-labeled

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Asn-glycans were dissolved in 200 μL of 1 M NH4HCO3 buffer (pH = 8), and 5 μL of GA enzyme was added

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and reacted at 37°C for 24 h. Then, the tubes were denatured at 100°C for 5 min and dried by a nitrogen blow

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dryer.33

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Purification of glycans was performed using a C18 solid-phase extraction column and GCC. The

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purification process for the C18 solid phase extraction column was as follows. First, the column was activated

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with 3 mL acetonitrile and equilibrated with 10 mL double distilled water, and the sample was then repeatedly

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applied three times. Finally, the column was washed with 20 mL water, and the samples were collected with

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tubes. Next, the samples were further purified by GCC. The column was activated with 3 mL acetonitrile and

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equilibrated with 10 mL double distilled water. Then, samples were loaded sequentially, and 10 mL water was

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used to remove the salt. Finally, the samples were eluted and collected with 25% acetonitrile for MS.

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ESI-MS conditions. The MS analysis was carried out with a Thermo Scientific LTQ-XL ion-trap mass

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spectrometer under the following conditions: set spray voltage, auxiliary gas flow rate, sheath gas flow rate,

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heated capillary temperature and voltage, and the tube lens voltage were set to 5 kV, 5.0 arb, 40.0 arb, 375°C

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and 37 V, and 250 V, respectively. The MS data were recorded using LTQ Tune software.

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HPLC conditions. HPLC analysis was carried out by employing a Shimazu LC-2010A HT system using a 4.6

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mm × 250 mm TSK-GEL Amide-80 column at room temperature. The detection wavelength was set to 254 nm,

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and the flow rate was set to 1.0 mL min-1. The injection volume was 20 µL. Solvents A, B, and C were

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acetonitrile, 100 mM ammonium acetate (pH = 6.0),34 and Milli-Q water, respectively. The sample separation

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gradient was as follows: t = 0 min, 75% A, 25% B; t = 120 min, 55% A, 45% B.

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Online LC-MS conditions. Online LC-UV-ESI-MS/MS analysis was performed on a Thermo Scientific

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HPLC-ESI-MS system using a 4.6 mm × 250 mm TSK-GEL Amide-80 column at room temperature. The UV

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detection wavelength was set to 254 nm, the flow rate was set to 1.0 mL min-1, and the injection volume was 20

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μL. Solvents A, B, and C were acetonitrile, 100 mM ammonium acetate (pH = 6.0), and Milli-Q water,

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respectively. The separation gradient was as follows: t = 0 min, 75% A, 25% B; t = 120 min, 55% A, 45% B.

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Biochemistry

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The fractions eluted from chromatographic columns were directly imported into the ESI-MS system for

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detection. For the ESI-MS system, a rapid alteration mode between the segments of positive-form MS and data

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dependent acquisition (DDA)-based MS/MS was adopted. For the DDA-based MS/MS, one top ion was chosen,

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the normalized collision energy was set to 30, and the lowest signal intensity was set to 500. Other parameters

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used for MS and MS-dependent MS/MS were the same as those described above. Data acquisition was

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performed using Xcalibur software (Thermo). The obtained data were manually interpreted, and the proposed

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glycan compositions and sequences were checked using GlycoWorkbench software.

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RESULTS AND DISCUSSION

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Release, separation and recovery of reducing N-glycans from glycoproteins

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We used Pronase E in combination with Fmoc-Cl and GA enzymes to release, isolate, and recover all reducing

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N-glycans containing α-1,3 fucose modifications. The basic principle of the reaction was described (Figure 1).

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Pronase E, can randomly act on any site on the polypeptide to release glycoproteins into a glycopeptide with

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amino acids. In our previous study, glycopeptides with only one amino acid were basically obtained.23

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Fmoc-Cl is an aromatic chromogenic reagent that can be used for chromatographic separation. The

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principle of the derivatization reaction is as follows. The amino group of Asn-glycan was involved in the

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nucleophilic-substituted reaction with Fmoc-Cl, and the Fmoc-labeled glycans were further separated and

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analyzed via HPLC and online HILIC-MS.

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The GA enzyme belongs to a lysosomal hydrolase that can be obtained from different species by chemical

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and physical methods27 and can be cloned and expressed in vitro. Moreover, GA can specifically cleave the

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β-N-aspartylglucosylamine bond in the aspartate-linked glycoprotein.

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Fig. 1. Scheme of the strategy for the release, separation, and recovery of monomeric reducing N-glycans by

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Pronase E combined with Fmoc-Cl and GA.

191 192

Pronase E enzyme combined with Fmoc-Cl and GA facilitate the release, separation, and recovery of

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reducing N-glycans, as demonstrated by MS

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We used the glycoprotein ovalbumin, Ribo B, ginkgo, and pine nuts as materials to perform Pronase E

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enzymatic hydrolysis, Fmoc-Cl derivatization, and GA digestion, respectively (Figures 2-3 and Supplemental

196

Figure S1-S2).

197

First, the ESI-MS spectra of the Asn-glycans obtained by Pronase E were analyzed via ESI-MS in the

198

positive ion mode. 24 Asn-glycans were obtained from ovalbumin (Figure 2a). The relative molecular mass of

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each Fmoc-labeled N-glycan was 114 Da larger than the corresponding native form, respectively (m/z: 1021.88,

200

1047.33, 1061.08, 1082.50, 1123.08, 1145.33, 1184.33, 1205.33, 1209.17, 1250.25, 1286.33, 1371.25, 1412.25, 10

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Biochemistry

201

1453.25, 1534.25, 1574.25, 1615.25, 1654.17, 1759.33, 1777.33, 1818.33, 1859.33, 1939.25, and 1980.33). We

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got the ESI-MS spectrum of the Fmoc-labeled Asn-glycan derivatives (Figure 2b). There were 24 Fmoc-labeled

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Asn-glycans with a molecular weight increase of 222 Da based on the native Asn-glycans. The results indicated

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that the derivatization reaction was complete. The ESI-MS spectra of the reducing N-glycans were obtained by

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hydrolysis with GA; there were 24 reducing N-glycans, of which five glycans existed in the form of a double

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charge (m/z: 1067.75, 1087.33, 1116.08, 1148.33, and 1218.33) (Figure 2c). These results are consistent with

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the literature.

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Fig. 2. ESI-MS profiles of Asn-glycans of ovalbumin (a). ESI-MS profiles of Fmoc-Asn-glycans of ovalbumin

210

(b). ESI-MS profiles of reducing N-glycans of ovalbumin (c).

211

galactose.

, N-acetylglucosamine;

, mannose;

,

212 213

The ESI-MS spectrum of Asn-glycans from ginkgo protein obtained using Pronase E indicated that there

214

were 13 glycans, of which 11 had an amino acid (Figure 3a). The relative molecular mass of each Fmoc-labeled 12

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Biochemistry

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Asn-glycan was 114 Da larger than the corresponding native form (m/z: 1179.33, 1325.33, 1371.08, 1382.42,

216

1511.33, 1528.33, 1584.42, 1656.33, 1736.33, 1819.33 and 1922.17). There were two Asn-glycans with one

217

asparagine (Asn) and arginine (Arg), increasing the molecular weight by 227 Da (m/z: 1438.33 and 1483.42).

218

Five glycans modified with core α1,3-fucose were also observed (m/z: 1325.33, 1438.33, 1528.33, 1736.33 and

219

1922.17),the ESI-MS spectra of the Fmoc-labeled Asn-glycans indicated that there were four Asn-glycans

220

modified by xylose (m/z: 1179.33, 1325.33, 1382.42 and 1528.33) (Figure 3b). In total, 13 glycans showed

221

increases in molecular weights by 222 Da on the basis of the pro-Asn-glycans. Moreover, no native Asn-glycans

222

were found in the profiles, suggesting that Asn-glycans were completely transformed to Fmoc-derivatives, and

223

there were doubly charged glycans (m/z: 1047.33 and 1092.50). Figure 3c shows the ESI-MS spectra of the

224

reducing N-glycans, which were obtained by GA. In total, there were 11 reducing N-glycans, of which four

225

were modified by fucose (m/z: 1211.25, 1414.25, 1584.33 and 1809.33). There were five glycans modified by

226

xylose (m/z: 1065.25, 1211.25, 1268.33, 1414.25, and 1471.08). We found that the number of Asn-glycans after

227

enzymatic hydrolysis Pronase E was two more than that of the reducing glycans obtained after GA digestion,

228

The reason for this phenomenon was that the total Asn-glycans after enzymatic hydrolysis of Pronase E

229

contained two glycans with one or two amino acids. The Asn-glycans harbored an asparagine (m/z: 1325.33 and

230

1371.08). The Asn-Arg-glycans carried an asparagine and arginine acid (m/z: 1438.33 and 1483.42). However,

231

after digestion with GA, m/z 1325.33 and 1438.33 were the same reducing glycans, and m/z 1371.08 and

232

1483.42 were the same reducing glycans. Therefore, the ESI-MS spectrum in Figure 3a contained two more

233

glycans than that in Figure 3c.

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Fig. 3. ESI-MS profiles of Asn-glycans of ginkgo protein (a). ESI-MS profiles of Fmoc--Asn-glycans of ginkgo

236

protein(b). ESI-MS profiles of reducing N-glycans of ginkgo protein(c).

237

mannose;

, galactose;

, N-acetylglucosamine;

,

, fucose.

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Biochemistry

In addition, the ESI-MS spectrum of the Asn-glycan, Fmoc-labeled Asn-glycans and reducing N-glycans from Ribo B and Pine nuts, respectively, which were described in Supplemental Figure S1-S2.

241 242

HPLC separation and analysis of Fmoc-labeled Asn-glycans

243

To recover monomeric reducing N-glycans, the Fmoc-labeled Asn-glycans obtained above were separated by a

244

4.6 mm × 250 mm TSK-GEL Amide-80 column. We got the HPLC profile of the Fmoc-labeled Asn-glycans

245

derived from ovalbumin (Figures 4) and the ESI-MS profile of each peak after separation and purification and

246

the monomeric reducing glycans obtained via GA (Figure 4b and Figure 4c), respectively. As shown in Figure

247

4a, 18 peaks were obtained, and each of the recovered peaks was detected by ESI-MS (Figure 4b). Two peaks

248

(peaks 2 and 3) were a pair of isomers (m/z 1431.33). Three peaks (4–6) were isomer peaks with m/z 1472.45,

249

and peaks 14 and 15 were absorption peaks with m/z 1011.58, 1154.58, and 1754.83. We found that the

250

separation of three peaks was not good, and subsequent two-dimensional liquid phase technology was used for

251

separation. Peaks 17 and 18 did not show signals by ESI-MS. The ESI-MS profiles of the monomeric reducing

252

N-glycans from each peak component obtained by GA indicated that the reaction was complete (Figure 4c). The

253

HPLC profile of Fmoc-labeled Asn-glycans derived from Ribo B were described in Supplemental Figure S3.

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254 255

Fig. 4. UV chromatograms of Fmoc-Asn-glycans derived from ovalbumin obtained by HILIC separation (a).

256

ESI-MS profiles of each Fmoc-Asn-glycan derived from ovalbumin (b). ESI-MS profiles of monomeric

257

reducing N-glycans derived from ovalbumin (c). 16

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258 259

The method was then applied to complex biological samples. The HPLC profiles of Fmoc-labeled

260

Asn-glycans derived from ginkgo showed there were five peaks in total (Figure 5a) , and the ESI-MS profiles of

261

each peak detected by ESI-MS were discribed ( m/z 1401.33, 1543.33, 1822.42, and 1942.83) (Figure 5b). Peak

262

5 was not detected by ESI-MS. The ESI-MS profiles of the monomeric reducing glycans of each peak obtained

263

by GA were discribed (m/z: 1065.42, 1211.67, 1052.08, and 1584.92) (Figure 5c); isomers were not found. The

264

HPLC profile of the Fmoc-labeled Asn-glycans derived from pine nuts were described in Supplemental Figure

265

S4.

266 267

Fig. 5. UV chromatograms of Fmoc-Asn-glycans derived from ginkgo protein obtained by HILIC separation (a).

268

ESI-MS profiles of each Fmoc-Asn-glycan derived from ginkgo protein (b). ESI-MS profiles of monomeric

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reducing N-glycans derived from ginkgo protein (c).

270 271

Separation and analysis of Fmoc-labeled Asn-glycans by online HILIC-MS

272

Because the detection sensitivity of MS is higher than that of UV, the structures of the glycans can be further

273

identified. Therefore, an 4.6 mm × 250 mm TSK-GEL Amide-80 column was selected to separate and analyze

274

the Fmoc-labeled Asn-glycans derived from sample by online HILIC-MS, and the isomers of glycans were

275

determined.

276

First, standard glycoproteins, such as ovalbumin and Ribo B, were separated and analyzed using online

277

HILIC-MS in the positive ion mode. The obtained UV profiles of the Fmoc-labeled Asn-glycans derived from

278

ovalbumin were discribed (Figure 6a), and Figure 6b presents extracted ion chromatograms (EICs) of the

279

Fmoc-labeled Asn-glycans derived from ovalbumin. In total, 35 Fmoc-labeled Asn-glycans of ovalbumin were

280

observed, of which five had two isomers (m/z: 1134.08, 1431.33, 1472.33, 1634.50 and 1837.33), and two of

281

which had two isomers (m/z: 1064.33 and 1999.33). Compared with HPLC detection, 15 Fmoc-labeled

282

Asn-glycans were detected (m/z: 1039.83, 1064.33, 1104.33, 1134.08, 1194.33, 1234.83, 1256.33, 1295.83,

283

1316.33, 1337.08, 1397.33, 1675.83, 1728.83, 1878.33, and 1917.08). In addition, we found that there were

284

seven glycans with isomers (m/z: 1431.33, 1472.33, 1634.50, 1837.33, 1999.33, 1064.33, and 1134.08), and no

285

isomers were found in five glycans detected by HPLC (m/z: 1634.50, 1837.33, 1999.33, 1064.33, and 1134.08).

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The Fmoc-labeled N-glycans were analyzed using online HILIC-MS in the positive ion mode. Supplemental

287

Figure S5a shows the UV profiles of the Fmoc-labeled N-glycans derived from Ribo B. The EICs of the labeled

288

N-glycans derived from Ribo B were discribed (Supplemental Figure S5b).

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289 290

Fig. 6. Online HILIC-MS/MS UV analysis of Fmoc-labeled Asn-glycans from ovalbumin. UV profiles of

291

Fmoc-Asn-glycans from ovalbumin (a). Extracted ion chromatograms (EICs) of Fmoc-Asn-glycans from

292

ovalbumin (b).

293 294

Next, this method was applied to the separation and analysis of Fmoc-labeled Asn-glycans derived from

295

complex biological samples. The UV profiles and EICs of the Fmoc-labeled Asn-glycans derived from ginkgo

296

are shown in Figure 7a and 7b. Overall, 14 Fmoc-labeled Asn-glycans were discovered, of which two glycans

297

had two isomers. Additionally, we found that the online HILIC-MS technology detected ten glycans more than

298

HPLC. Figure 7c shows the UV profiles of Fmoc-labeled Asn-glycans derived from pine nuts proteins, and

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Figure 7d shows the EICs of Fmoc-labeled Asn-glycans derived from pine nuts proteins. Overall, 20 glycans

300

were detected, and 18 glycans were detected more than HPLC, among which two glycans had two isomers (m/z:

301

1643.33 and 1620.33).

302 303

Fig. 7. Online HILIC-MS/MS UV analysis of Fmoc-labeled Asn-glycans from ginkgo and pine nuts. UV

304

profiles of Fmoc-Asn-glycans from ginkgo (a) and pine nuts (c). Extracted ion chromatograms (EICs) of

305

Fmoc-Asn-glycans from ginkgo (b) and pine nuts (d).

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In summary, compared with HPLC, more absorption peaks and isomers were detected via online

308

HILIC-MS. This phenomenon can be explained by the observation that HILIC-MS had higher detection

309

sensitivity, resulting in detection of some low-level glycans.

310 311

CONCLUSIONS

312

In this study, we established a new method to release, separate, and recover monomeric reducing glyans of core

313

α-1,3 fucose modification by ESI-MS, HPLC, and online HILIC-MS. First, Asn-glycans with one amino acid

314

were obtained by Pronase E and then derivatized with Fmoc-Cl for HPLC and online HILIC-MS detection.

315

Finally, the Fmoc-labeled Asn-glycans were digested with GA, yielding the monomeric reducing glycans. This

316

approach could be used for the standard glycoproteins and some plant-derived samples (such as ginkgo and pine

317

nuts). Particularly in plant samples, some N-glycans with core α-1,3 fucose could not released by PNGase F.

318

Therefore, this method is simpler to operate, lower in cost, and wider in scope and can be used for the

319

subsequent release of more N-glycans (including N-glycans containing α1,3-fucose) in plants. Moreover, this

320

method will facilitate the isolation and recovery of monomeric reducing N-glycans for subsequently

321

structure-function analysis.

322

Notably, the Asn-glycans derived from some samples obtained via Pronase E, such as ginkgo protein,

323

released some glycans with asparagine and will also release a few glycans with one more amino acid redsidue;

324

however, these Asn-glycans can be successfully derived with Fmoc-Cl for HPLC and online HILIC-MS

325

analysis, and this approach will not affect the subsequent GA process as the enzyme cleavage site is the peptide

326

bond between glycans and the first amino acid. Accordingly, the recovery of the monomeric reducing N-glycans

327

will also not be affected. In addition, we also found that the absorption peaks of Fmoc-labeled Asn-glycnas were

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not detected in some samples by HPLC, and no isomers were found. In contrast, more Fmoc-labeled

329

Asn-glycans and corresponding isomers were detected by online HILIC-MS analysis, including acid

330

Fmoc-derivative absorption peaks and isomers. This phenomenon was related to the fact that some

331

low-concentration glycans were detected owing to the higher sensitivity of MS compared with UV, resulting in

332

greater detection of glycan derivatives and isomers. We also showed that the recovery Fmoc-labeled derivatives

333

of monomeric absorption peaks was mixed during HPLC and online HILIC-MS analysis, resulting in increased

334

detection of signals by ESI-MS. For example, peak 11 was from a mixture of two Asn-glycan derivatives, and

335

peaks 14 and 15 were from a mixture of three Asn-glycan derivatives in the HPLC separation profiles of the

336

Fmoc-labeled Asn-glycans derived from ovalbumin, yielding the reducing N-glycan mixture obtained by GA.

337

The reducing N-glycans obtained by enzymatic hydrolysis were not individual. These results may be related to

338

the use of a one-dimensional normal phase chromatography separation column during chromatographic

339

separation; further separation by multidimensional chromatography may be necessary.

340

The core α-1,3 fucose-modified N-glycans exist in plants and some insects. Therefore, this method was initially

341

verified in silkworm egg proteins (Supplemental Figure S6), demonstrating release of all N-glycans containing

342

the core α-1,3 fucose-modified N-glycans. The results indicated that this method could also be applicable to

343

insect proteins. Further studies are required to verify these findings to achieve optimal release, separation, and

344

recovery of all reducing N-glycans derived from insects. Moreover, we predict that the method shows potential

345

for the release, separation and recovery of reducing N-glycans modified with sialic acid as the reaction is carried

346

out under mild condition. In future work, we will validate the release, separation and recovery of reducing

347

N-glycan from sialylated species by our method. In addition, such studies may also broaden the scope of

348

application for more complex biological samples, such as IgG and human serum, laying the foundation for

349

structure-function analysis.

350

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Biochemistry

351

SUPPORTING INFORMATION

352

Figures S1−S6. This material is available free of charge via the Internet at http://pubs.acs.org.

353 354

ACKNOWLEDGEMENTS

355

This work was supported by the National Natural Science Foundation of China (grant nos. 31670808,

356

31870798, 31600647), The Scientific Research Program Fund for Shanxi Province Key Laboratory (grant nos.

357

16JS109, 14JS101).

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TOC figure

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