Showcasing research on purification of human serum N-glycans by using a highly hydrophilic zwitterionic-type material named Click TE-Cys from the laboratory of Professor Xinmiao Liang at Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.

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Title: Sample preparation for mass spectrometric analysis of human serum N-glycans by using hydrophilic interaction chromatography-based solid phase extraction High hydrophilicity of the Click TE-Cys material, which is conferred by its zwitterionic surface structure, facilitates the retention and thus the selective purification of serum N-glycans for mass spectrometric analysis. See Long Yu, Xinmiao Liang et al., Analyst, 2014, 139, 4538.

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Cite this: Analyst, 2014, 139, 4538

Sample preparation for mass spectrometric analysis of human serum N-glycans using hydrophilic interaction chromatography-based solid phase extraction† Liwei Cao,‡a Ye Zhang,‡b Linlin Chen,b Aijin Shen,a Xingwang Zhang,c Shifang Ren,c Jianxin Gu,c Long Yu*a and Xinmiao Liang*a Expression levels of N-linked glycans derived from human serum glycoproteins have been shown to change during the progression of many diseases. Generally, N-glycans released from human serum proteins coexist with endogenous serum peptides, salts, and other contaminants. Effective removal of these contaminants is essential to obtain the glycan profile of human serum proteins. Here, we developed a sample preparation method for mass spectrometry (MS) analysis of N-linked glycans derived from human serum glycoproteins based on a zwitterionic hydrophilic material named Click TE-Cys. The high hydrophilicity of Click TE-Cys, resulting from its unique surface structure and charge distribution, facilitated removal of co-existing salts and endogenous serum peptides. Furthermore, the present enrichment approach was handled in parallel, thus saving time. Using this method, a total of 47 unique

Received 12th April 2014 Accepted 6th July 2014

N-glycans released from human serum proteins were identified. The intrabatch and interbatch coefficients of variation for the 47 N-linked glycans were 8.57%
0.96% and 9.22%
1.03%,

DOI: 10.1039/c4an00660g

respectively. These results demonstrate that the present method is suitable for fast purification of

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N-linked glycans derived from human serum glycoproteins, and has potential for clinical application.

1. Introduction Blood serum can be considered to reect the status of an individual's health because most of the proteins in human serum are secreted or shed from different cells or tissues.1,2 N-glycosylated proteins shed from diseased tissues are an interesting subset of circulating proteins, which may constitute useful biomarkers.3 The attached glycans have been found to be associated with many cellular activities including cell recognition, cell–cell interactions, and protein localization.4–6 Furthermore, the progression of many diseases, such as cancer and immunodeciency diseases, has also been considered to be related to changes in N-linked glycans derived from human serum glycoproteins.7–10 Therefore, comprehensive characterization of the

a

Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China. E-mail: [email protected]; [email protected]; Fax: +86-0411-84379539; Tel: +86-041184379541

b

The Second Affiliated Hospitals of Dalian Medical University, Dalian, 116044, China

c

Department of Biochemistry and Molecular Biology, Key Laboratory of Glycoconjugate Research, Ministry of Public Health, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China † Electronic supplementary 10.1039/c4an00660g

information

(ESI)

‡ These authors contributed equally to this work.

4538 | Analyst, 2014, 139, 4538–4546

available.

See

DOI:

N-glycans released from human serum proteins is essential to understanding of these biological processes. Typically, a mass spectrometry (MS)-based workow for N-linked glycan analysis comprises sample preparation, glycan release, glycan enrichment, chemical derivatization, MS analysis (or LC-MS/MS), and data interpretation.11–13 N-linked glycans derived from complex protein samples are released by enzymes such as PNGase F.14,15 The resulting glycans co-exist with peptides, salts, and other contaminants, and should be puried prior to MS analysis.16–18 Several strategies have been developed for purication of N-linked glycans, including solidphase extraction with porous graphic carbon (PGC),19–21 hydrophilic interaction chromatography (HILIC),22–24 reversed-phase liquid chromatography (RPLC),25–27 glycoprotein immobilization for glycan extraction (GIG),28 and glycoblotting.29,30 In terms of glycan purication, the PGC column is a medium widely used for removal of salts and small molecules from the glycan fraction.31 However, N-linked glycans and co-existing peptides such as endogenous serum peptides32 are likely co-eluted in the same fraction during this process. These peptides having higher ionization efficiency will suppress glycan signals during MS analysis. Although RPLC materials, e.g. C18 material, can separate peptides from the glycan fraction, salts co-elute with the glycan fraction during this process. The obtained glycan fraction requires to be further desalted prior to MS analysis.

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˚ was obtained from Acchrom (Beijing, China). (5 mm, 100 A) Water used in this study was puried by the Milli-Q system (Millipore, Bedford, MA). Human normal serum (n ¼ 1, female, age 43) was provided by the Second Affiliated Hospitals of Dalian Medical University according to Institutional Review Broad (IRB) approval.

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Fig. 1

The structure of the Click TE-Cys material.

Novotny and colleagues employed C18 and PGC microspin columns to remove peptides and salts from the glycan fraction, respectively.33 However, multiple glycan purication steps increased sample loss and resulted in low yield from the glycan isolation. The HILIC-based method has the potential to effectively remove peptides and salts from the glycan fraction in a single purication step. Under HILIC conditions, a stable water-rich layer formed on the surface of the polar material facilitates retention of the hydrophilic glycans, while the co-existing peptides and salts with less hydrophilicity tend to be retained in the bulk solvent and are eluted earlier.34 Some hydrophilic materials such as cellulose22 and cotton23 have been applied to selectively enrich N-linked glycans. As a result of low hydrophilicity, these materials cannot completely remove the coexisting peptides from the glycan fraction. Therefore, the development of a highly hydrophilic material-based enrichment approach is highly desirable for glycan purication. In the present study, we developed a method for purication of N-linked glycans derived from human serum glycoproteins based on a zwitterionic hydrophilic material named Click TECys (Fig. 1). The capability of Click TE-Cys for glycan enrichment was assessed using N-glycans released from human serum immunoglobulin G (IgG), and a mixture of the IgG N-linked glycans and a tryptic digest of human serum albumin (HSA). Subsequently, the present method coupled with MALDI-QITTOF MS was used to prole the N-linked glycans derived from human serum glycoproteins, resulting in identication of 47 unique glycan structures. The present method exhibited an ability to simultaneously remove salts and endogenous serum peptides from human serum N-linked glycans, and has potential for clinical application.

2. 2.1

Experimental section Materials and reagents

IgG, HSA, bovine ribonuclease B (RNase B), dithiothreitol (DTT), iodoacetic amide (IAA), and ammonium bicarbonate (NH4HCO3) were ordered from Sigma (St. Louis, MO). Acetonitrile (ACN) and ZIC Glycocapture Resin (the ZIC-HILIC material refers to ZIC Glycocapture Resin in this study unless otherwise stated) was ordered from Merck (Darmstadt, Germany). GELoader tips were purchased from Eppendorf (Hamburg, Germany). PNGase F was bought from New England Biolabs (Ipswich, MA). Formic acid (FA) and triuoroacetic acid (TFA) were ordered from Acros (Geel, Belgium). The PGC material was purchased from Grace (Deereld, IL). The Click TE-Cys material

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2.2

N-linked glycan release

The N-linked glycans derived from standard protein or human serum proteins were released according to a previous procedure.9 Briey, 100 ml of sample solution (2 mg of standard protein or about 5 mg of human serum proteins) was mixed with 100 ml of digestion buffer consisting of 200 mM NH4HCO3 and 10 mM DTT. The mixture was heated to 100  C for 5 min to denature the proteins. The N-glycans attached to standard protein or human serum proteins were released by incubating with PNGase F for 24 h at 37  C. Then, 800 ml of chilled ethanol was added. The obtained solution was frozen to 80  C for 1 h and subsequently centrifuged for 20 min at 13 200 rpm. The supernatant was collected and lyophilized to dryness. 2.3

Protein digestion

HSA (0.5 mg) was denatured using 8 M urea in 50 mM NH4HCO3 for 3 h. The denatured protein was reduced by DTT (50 mM, 4 ml) for 2 h at 37  C, followed by addition of IAA (50 mM, 5 ml) for alkylation. The obtained solution was incubated in the dark for 30 min and then diluted tenfold with NH4HCO3 (50 mM) buffer. The resulting solution was mixed with trypsin at an enzyme/ substrate ratio of 1 : 25 (w/w) and incubated for 16 h at 37  C. 2.4

N-linked glycan purication using Click TE-Cys

For enrichment of the N-linked glycans released from standard protein, 3 ml of sample solution was mixed with 1 mg of Click TE-Cys beads, and the total volume was adjusted to 40 ml by adding ACN/H2O/FA (80/18/2) solution. The obtained solution was agitated for 1 min and incubated without agitation for 10 min. The beads were collected by centrifugation at 2000 rpm for 1 min. Then the beads were mixed with 200 ml of ACN/H2O/FA (80/18/2), and agitated for 4 min. The beads were collected by centrifugation at 2000 rpm for 1 min. The wash cycle and centrifugation were repeated four times. The beads were subsequently mixed with H2O (0.1% FA) to elute the glycan fraction, and agitated for 4 min. The supernatant was collected by centrifugation at 2000 rpm for 1 min, and lyophilized to dryness. For enrichment of the N-linked glycans released from human serum proteins, 20 ml (released from 7 ml of human serum) of sample solution was mixed with 1 mg of the Click TECys beads. The total volume was subsequently adjusted to 40 ml by adding ACN/H2O/FA (80/18/2) solution. The washing and elution steps were the same as for standard proteins. 2.5 N-linked glycan enrichment with PGC solid-phase extraction An ACN slurry containing the PGC material was pushed into the GELoader tip. The packed microcolumn was washed with ACN/

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H2O/TFA (80/20/0.05, 90 ml) and equilibrated with 90 ml of H2O. Subsequently, the N-linked glycans released from standard protein or human serum proteins were redissolved in H2O and loaded onto the microcolumn. Then the microcolumn was washed with 90 ml of H2O. The glycan fraction was eluted with ACN/H2O/TFA (40 : 60 : 0.05). The obtained glycan fraction was lyophilized to dryness and reconstituted in H2O prior to MS analysis. 2.6 N-linked glycan enrichment with ZIC-HILIC solid-phase extraction Slurry containing the ZIC-HILIC material was pushed into the GELoader tip. The packed microcolumn was washed with ACN/ H2O/FA (50/45/5, 90 ml) and equilibrated with ACN/H2O/FA (80/ 15/5, 90 ml). Then the N-linked glycans released from standard protein or human serum proteins were redissolved in ACN/H2O/ FA (80/15/5) and loaded onto the microcolumn. The obtained microcolumn was washed with ACN/H2O/FA (80/15/5, 100 ml) to remove the contaminants. The glycan fraction was eluted with H2O/FA (100/0.1). The obtained glycan fraction was lyophilized to dryness and reconstituted in H2O prior to MS analysis. 2.7

Mass spectrometric analysis

Mass spectra were recorded on an Axima MALDI-QIT-TOF mass spectrometer (Shimadzu Corp., Kyoto, Japan) equipped with a 337 nm nitrogen laser in reector positive ionization mode. One microliter of analyte was spotted onto a standard MALDI plate and dried at room temperature. Then 1 ml of 2,5-dihydroxybenzoic acid (10 mg ml1 in 0.1% TFA, 49.9% H2O, 50% ACN, and 10 mM NaCl) was applied to the dried spot and dried at room temperature. Subsequently, 0.2 ml of ethanol was added onto the dried spot to recrystallize the sample. The laser power was set at 116 or 120 to obtain satised signal-to-noise (S/N) ratios as well as minimize the “in-source decay”.35 The m/z range was set from 850 to 5000. Each mass spectra contained 500 or 1000 proles that were accumulated from different points of laser irradiation, while two laser shots generated a prole.

3.

Results

3.1 Characterization of human serum N-glycans using Click TE-Cys, followed by MALDI-QIT-TOF MS To allow high-throughput analysis of the N-linked glycans derived from human serum glycoproteins, a HILIC-based method was developed and followed by MS analysis. The experimental procedure included the following steps (Fig. 2). (1) Glycan release: N-linked glycans derived from human serum glycoproteins were released using PNGase F. Subsequently, the residual proteins were precipitated using chilled ethanol. (2) Glycan adsorption: the mixture of the Click TE-Cys material and the sample solution was incubated without agitation for 10 min so that the N-linked glycans were effectively adsorbed on the Click TE-Cys beads as a result of hydrophilic interactions. (3) Removal of salts and peptides: the co-existing salts and endogenous serum peptides were washed out ve times with 200 ml of ACN/H2O/FA (80/18/2). Two percent of formic acid was

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Fig. 2 Schematic diagram of characterization of the N-glycans derived from human serum glycoproteins using Click TE-Cys, followed by MALDI-QIT-TOF MS.

added to the washing buffer to help separate the peptides from the glycan fraction. When the concentration of formic acid decreased to 1% (or 0.1%), some peptides co-eluted with the glycan fraction (Fig. S-1†). The results obtained with 5% FA were analogous to those obtained with 2% FA. The solution should also be agitated for 4 min (3–5 min) to effectively remove the salts and endogenous serum peptides from the glycan fraction. (4) Glycan elution: the glycan fraction was eluted with H2O (0.1% FA). Agitation of the solution for 4 min (3–5 min) was needed to effectively elute the N-linked glycans. (5) Glycan analysis: the glycan fraction was collected and analyzed on a MALDI-QIT-TOF mass spectrometer. The DHB matrix was recrystallized with ethanol to generate a uniform layer of thin, ne crystals. Of note, the enrichment approach (the two to four steps) could be handled in parallel, and the entire enrichment procedure of 24 samples took 4 h. 3.2 Assessment of the capability of the Click TE-Cys material for glycan enrichment The capability of Click TE-Cys for removal of salts from the glycan fraction was assessed using N-glycans released from IgG. Fig. 3A shows direct analysis of the IgG N-linked glycans by MALDI-QIT-TOF MS without any enrichment treatment. In total, eight N-linked glycan peaks with weak peak intensities and low signal-to-noise (S/N) ratios were detected as a result of the ion suppression effect of the co-existing salts. Aer enrichment with Click TE-Cys, the salts were efficiently removed from the glycan fraction, resulting in identication of 16 IgG N-linked glycans with improved peak intensities (Fig. 3B). The compositions of the identied N-linked glycans were searched through GlycoMod Tool (http://web.expasy.org/glycomod/) with

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MALDI mass spectra of the N-glycans released from IgG: (A) by direct analysis; (B) after enrichment with Click TE-Cys. N-linked glycans are labeled with their structures. -: N-acetylglucosamine; C: mannose or galactose; :: fucose; A: sialic acid.

Fig. 3

oligosaccharide molecular weight, and some were further veried by MS/MS analyses (Fig. S-2, ESI†). The capability of Click TE-Cys for removal of peptides from the glycan fraction was assessed using a mixture of the IgG N-linked glycans and the tryptic digest of HSA in a 1 : 1 weight ratio. Before enrichment, the peptide signals, e.g. 1140.165 (1+), 1373.175 (1+), and 1911.414 (1+), dominated the mass spectra, and only 1 N-linked glycan with weak peak intensity and low S/N ratio was identied (Fig. 4A). Purication of the mixture with Click TE-Cys led to effective removal of the co-existing peptides (Fig. 4B). Overall, 16 IgG N-linked glycans were identied, as shown in Fig. 4B. A mixture of the N-glycans released from RNase B and the tryptic digest of HSA (1 : 1, w/w) was selected to assess the capability of Click TE-Cys for high-mannose glycan enrichment. All ve previously reported high-mannose type N-linked glycans of RNase B were observed aer enrichment with Click TE-Cys (Fig. S-3B, ESI†).36,37 For comparison, the PGC and ZIC-HILIC materials were also used to enrich the N-linked

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glycans from the mixture of the IgG N-linked glycans and the tryptic digest of HSA (1 : 1, w/w). The peptide signals dominated the mass spectra, and the N-linked glycan signals could rarely be detected aer enrichment (Fig. 4C and D). These results indicate that Click TE-Cys can be used to isolate N-linked glycans from peptides and salts.

3.3

Proling N-glycans released from human serum proteins

The N-glycans released from human serum proteins co-exist with endogenous serum peptides,32 salts, and other contaminants. These contaminants severely suppressed the N-linked glycan signals during MS analysis (Fig. S-4†). Effective removal of these contaminants was observed with Click TE-Cys, resulting in identication of 47 unique N-linked glycans (Table 1). The proposed structures of major peaks are shown in Fig. 5A, according to previously reported studies.16,38,39 Among these, two fucosylated high-mannose type N-linked glycans were

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Fig. 4 MALDI mass spectra of a mixture of the IgG N-linked glycans and the tryptic digest of HSA (1 : 1, w/w): (A) by direct analysis; (B) after enrichment with Click TE-Cys; (C) after enrichment with the PGC material; (D) after enrichment with the ZIC-HILIC material. N-linked glycans are labeled with their structures. -: N-acetylglucosamine; C: mannose or galactose; :: fucose; A: sialic acid.

identied in low-abundance, 1727.243 (1+) and 1889.268 (1+).40 A fucosylated hybrid N-linked glycan was identied (observed at an m/z value of 1606.265), which was less evident in human serum.40 These results clearly support high detection sensitivity of Click TE-Cys for glycan enrichment. Meanwhile, PGC solidphase extraction was also used to enrich N-glycans released from human serum proteins, and 38 N-linked glycans were identied aer enrichment (Fig. 5B and Table 1). In particular, several low-abundance N-linked glycans, e.g. 1606.265 (1+), 1727.243 (1+), and 1889.268 (1+), could not be detected aer enrichment with PGC solid phase extraction. When the N-glycans released from human serum proteins were puried using the ZIC-HILIC material, the endogenous serum peptide signals, e.g. 1897.652 (1+), 2081.615 (1+), and 2171.721 (1+), dominated the mass spectra. Only 22 structures were identied aer enrichment (Fig. 5C and Table 1). We compared the N-linked glycans identied here to those identied via highly ordered mesoporous carbon materialbased enrichment, followed by MALDI-TOF MS.41 The Click TECys material exhibited remarkably higher glycan enrichment capability (47 to 25 N-linked glycans), and 96% of the glycans identied using the highly ordered mesoporous carbon material also could have been identied using Click TE-Cys. The number of N-linked glycans identied previously by Ruhaak et al.22 was comparable with that identied in the present study (47 to 47 N-linked glycans). However, only 31 N-linked glycans were simultaneously identied in both studies, accounting for 66% of the N-linked glycans identied here. Of note, negative

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linear mode MALDI-TOF-MS with DHB matrix was chosen in the study of Ruhaak et al.,22 whereas reector positive ionization mode MALDI-QIT-TOF MS with DHB matrix was applied in this study. Consequently, one possible explanation for this nding is that, owing to the characteristics of the matrix-assisted laser desorption of quadrupole ion trap, some N-linked glycans are fragmented during MS analysis in the present study leading to loss of their sialic acids. We also determined the intrabatch and interbatch repeatability of the present workow through analysis of a sample of enriched human serum N-linked glycans in triplicate and three batches of enriched human serum N-linked glycan samples once, respectively. The data were normalized by expressing the intensity of each N-linked glycan as a percent of the total intensity for all serum N-linked glycans identied in this study (Table 1). The intrabatch and interbatch coefficients of variation for the 47 N-linked glycans were 8.57%
0.96% and 9.22%
1.03%, respectively (Tables S-2, and S-3, ESI†).

4. Discussion A method for purication of N-linked glycans derived from human serum proteins was developed based on a cysteinebonded zwitterionic hydrophilic material named Click TE-Cys (Fig. 1), which was synthesized via the “Thiol-ene” click reaction between the polar amine cysteine and vinyl silica.42 Under HILIC conditions, the amino and carboxylic groups of the immobilized cysteine carry positive and negative charges,

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Table 1

Analyst Human serum protein-derived N-linked glycans detected in this study

Number

Observed m/z

Dmassa (Dalton)

Compositionb

Glycan type

Enrichment with Click TE-Cys

Enrichment with PGC

Enrichment with ZIC-HILIC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

933.140 1095.163 1136.188 1257.187 1282.209 1298.202 1339.220 1419.207 1444.232 1460.230 1485.258 1501.248 1542.264 1581.231 1606.265 1622.244 1647.274 1663.277 1688.271 1704.290 1727.243 1743.247 1777.228 1809.303 1814.265 1825.284 1850.324 1866.321 1889.268 1905.258 1976.296 2012.346 2017.276 2028.334 2122.322 2110.283 2174.316 2240.320 2289.311 2325.356 2341.349 2393.358 2435.344 2487.383 2638.389 2654.360 2800.468

0.176 0.206 0.208 0.235 0.245 0.246 0.255 0.268 0.274 0.271 0.275 0.280 0.290 0.296 0.294 0.310 0.312 0.304 0.341 0.317 0.342 0.333 0.421 0.336 0.340 0.349 0.341 0.339 0.370 0.375 0.362 0.372 0.409 0.379 0.394 0.483 0.455 0.509 0.424 0.439 0.441 0.487 0.449 0.465 0.484 0.508 0.458

H3N2 H4N2 H3N3 H5N2 H3N3F1 H4N3 H3N4 H6N2 H4N3F1 H5N3 H3N4F1 H4N4 H3N5 H7N2 H5N3F1 H6N3 H4N4F1 H5N4 H3N5F1 H4N5 H7N2F1 H8N2 H3N4F3 H5N4F1 H4N4S1 H6N4 H4N5F1 H5N5 H8N2F1 H9N2 H5N4S1 H5N5F1 H4N5S1 H6N5 H5N4F1S1 H4N7 H6N5F1 H3N7F2 H5N4S2 H5N5F1S1 H6N5S1 H7N6 H5N4F1S2 H6N5F1S1 H5N5F1S2 H6N5S2 H6N5F1S2

Complex High mannose Complex High mannose Complex Complex Complex High mannose Complex Hybrid Complex Complex Complex High mannose Hybrid Hybrid Complex Complex Complex Complex High mannose High mannose Complex Complex Complex Hybrid Complex Complex High mannose High mannose Complex Complex Complex Complex Complex Complex Complex Complex Complex Complex Complex Complex Complex Complex Complex Complex Complex

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes No Yes No Yes Yes Yes Yes Yes No Yes Yes Yes No Yes Yes No Yes Yes Yes Yes Yes Yes No Yes No Yes No

Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes No Yes Yes No Yes Yes Yes No No No No Yes Yes No No No No No Yes Yes No No Yes No No No Yes No No No No No No No No

a Dmass ¼ observed m/z  calculated m/z. b N-linked glycan compositions are given in terms of hexose (H), N-acetylhexosamine (N), fucose (F) and sialic acid (S).

respectively, resulting in the zwitterionic property of Click TE-Cys.34 The distribution of the positively and negatively charged groups is parallel to the surface of the silica gel, which is different from other zwitterionic materials such as the ZIC-HILIC material. This unique surface structure and charge distribution give Click TE-Cys high hydrophilicity. Consequently, the hydrophilic glycans are well retained on the surface of Click TE-Cys, whereas the less hydrophilic co-existing endogenous serum peptides tend to be retained in the bulk

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solvent and are eluted earlier. On the other hand, the zeta potentials of Click TE-Cys are close to zero and switch slightly from positive to negative between pH 3 and 7.42 The washing buffer used in this study contained 2% FA (pH 2.0). Consequently, the net surface charge of Click TE-Cys was positive under this condition. Meanwhile, the co-existing peptides also carried a positive charge under this condition.43,44 Therefore, the electrostatic interactions between the Click TE-Cys material and the co-existing peptides were electrostatic repulsion,

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Fig. 5 MALDI mass spectra of the N-glycans released from human serum proteins: (A) after enrichment with Click TE-Cys; (B) after enrichment with the PGC material; (C) after enrichment with the ZIC-HILIC material. N-linked glycans are labeled with their structures. -: N-acetylglucosamine; C: mannose or galactose; :: fucose; A: sialic acid.

facilitating removal of the peptides from the glycan fraction. HILIC materials also exhibited better performance than C18 materials for desalting glycopeptides.45 In this study, the coexisting salts were washed out ve times with ACN/H2O/FA (80/18/2, 200 ml). Another zwitterionic hydrophilic material, ZIC-HILIC, was applied to enrich the N-linked glycans released from human serum proteins. The co-existing endogenous serum peptides could not be effectively removed from the glycan fraction aer enrichment with the ZIC-HILIC material. Of note, the ZIC-HILIC material exhibited lower hydrophilicity

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than the Click TE-Cys material on polar compound separation,42 which decreases retention of the hydrophilic glycans on the surface of the ZIC-HILIC material. The surface charge of the ZIC-HILIC material was slightly negative over a wide pH range,46 whereas the co-existing peptides carried a positive charge under the present study conditions (80% ACN, 5% FA). Electrostatic attraction interactions between the ZIC-HILIC material and the peptides increase retention of the peptides on the surface of the ZIC-HILIC material. Therefore, the retention difference between the N-glycans released from human serum proteins and the co-

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existing peptides decreased, resulting in co-elution of the peptides and the glycan fraction. The PGC material is a medium widely used to purify N-glycans released from human serum proteins from salts and small molecules. However, the N-linked glycans and the co-existing endogenous serum peptides are likely co-eluted in the same fraction aer enrichment with the PGC material.31 Therefore, the performance of the Click TE-Cys material for glycan enrichment is superior to that of PGC and ZIC-HILIC materials. There is an urgent need for high-throughput methods for Nlinked glycan proling to allow thorough testing of glycan biomarker candidates within population studies.22 Here, we described a method for fast sample preparation of N-glycans released from human serum proteins. Once the N-linked glycans are available by PNGase F treatment of serum proteins, it takes less than 1.5 h to identify the N-linked glycan prole using Click TE-Cys beads, followed by MALDI-QIT-TOF MS. Furthermore, the present enrichment approach can be handled in parallel, and the entire enrichment procedure of 24 samples takes 4 h.

Conflict of interest The authors declare no competing nancial interests.

Acknowledgements This work was supported by the National High Technology Research and Development Program of China (863 Program, 2012AA020203) and the National Natural Science Foundation of China (Grant no. 21135005 and no. 21205116).

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