In-Depth Analysis of Glycoprotein Sialylation in Serum Using a Dual-Functional Material with Superior Hydrophilicity and SwitchableSurface ChargeXuefang Dong,†,§ Hongqiang Qin,†,§ Jiawei Mao,†,‡ Dongping Yu,†,‡ Xiuling Li,† Aijin Shen,†

Jingyu Yan,† Long Yu,† Zhimou Guo,*,† Mingliang Ye,*,† Hanfa Zou,†,∥ and Xinmiao Liang*,†

†Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences,Dalian 116023, P. R. China‡University of Chinese Academy of Sciences, Beijing 100049, China

*S Supporting Information

ABSTRACT: Sialylation typically occurs at the terminal of glycans, and itsaberration often correlates with diseases including neurological diseases andcancer. However, the analysis of glycoprotein sialylation in complex biologicalsamples is still challenging due to their low abundance. Herein, a histidine-bonded silica (HBS) material with a hydrophilic interaction and switchablesurface charge was fabricated to enrich sialylated glycopeptides (SGPs) from thedigest of proteomics samples. High selectivity toward SGPs was obtained bycombining the superior hydrophilicity and switchable-charge characteristics.During the enrichment of sialylated glycopeptides from bovine fetuin digest, seven glycopeptides were detected even at the ratioof 1:5000 with the nonsialylated glycopeptides, demonstrating the high specificity of SGP enrichment by using HBS material.Then, HBS material was further utilized to selectively enrich SGPs from the protein digest of human serum, and 487 glycositeswere identified from only 2 μL of human serum; 92.0% of the glycopeptides contained at least one sialic acid, indicating goodperformance for SGP enrichment by using HBS material. Furthermore, the prepared HBS material also has great potentialapplications in the analysis of glycoprotein sialylation from other complex biological samples.

Glycosylation, as one of the most common post-transla-tional modifications (PTMs) of proteins, plays significant

roles in a wide range of biological processes, including proteinfolding, secretion, uptake, cell recognition, etc.1−4 Aberrantglycosylation of protein is associated with many diseases, suchas immunological diseases and malignant tumors.5,6 As one ofthe key forms of glycosylation, sialylation often occurs at theterminal of glycans, and its aberration also correlates withdiseases including neurological diseases and cancer.7 However,analysis of protein sialylation on the proteome level remainsvery challenging, due to the low abundance of sialylatedglycopeptides (SGPs), as well as high complexity of peptidemixture in the protein digest. Thus, enrichment of SGPs fromprotein digests is the important premise for analysis of SGPs.Strong cation-exchange chromatography (SCX) has been

employed to enrich SGPs by taking advantage of the negativecharge of sialic acids.6 However, low efficiency was obtained,due to the presence of nonglycopeptides with acidic aminoacids. Titanium dioxide (TiO2) chromatography has also beenused to selectively enrich SGPs by using the affinity interactionbetween TiO2 and sialic acids.8,9 Yet, the weak affinityinteraction induced the low specificity and efficiency of SGPenrichment. Hydrazine chemistry is a popular method to enrichglycopeptides/glycoproteins via the selective oxidization of theglycan structures followed by capture of the glycopeptides/glycoproteins by using hydrazine beads.10 Recently, selective

oxidization of sialic acids by using medium oxidized buffer hasbeen adopted to enrich SGPs.11−13 This approach couldenhance the specificity of SGP enrichment. Yet, the conditionsof selective oxidization were difficult to control, which coulddecrease the repeatability for analysis of SGPs in complexbiological samples, as well as loss of the information onsialylation during SGP enrichment. Boronate affinity materialshave become one of the important kinds of materials forglycoprotein/glycopeptide enrichment.14,15 The molecularlyimprinted nanoparticles prepared by using a sialic acid templatecould be used to selectively image cancer cells and tissuesagainst normal cells and tissues.16 However, this method isseldom used to enrich SGP for proteomics analysis, probablydue to the low specificity. Thus, enrichment of SGPs with highefficiency is the vital step for analysis of sialylation in biologicalsamples.Human serum is widely used in clinic diagnosis and

biomarker discovery research and is an excellent model forproteomics research.17,18 In particular, sialylated glycoproteomehas attracted great attention in clinical proteomic research.9,19,20

However, the complex compositions of serum, including highlyabundant proteins, lipids, and other components, interfere with

Received: November 8, 2016Accepted: March 5, 2017Published: March 5, 2017

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the identification of low abundance SGPs. Although manyattempts have been made for SGP enrichment, it is still difficultto analyze protein sialylation in human serum with highsensitivity, due to the high dynamic range of proteins andinterference of highly abundant nonglycoproteins.8,20,21 As aprerequisite, highly abundant nonglycoproteins should beremoved before enrichment of SGPs, which could inevitablyinduce loss of SGPs, especially for low abundance sialylatedproteins.Hydrophilic interaction chromatography (HILIC) has been

applied to the glycopeptide enrichment via facile proce-dures.22−25 However, its low selectivity and suitability limit itsutility in SGP enrichment from biological samples. The intrinsichydrophilicity and negative charge of SGPs inspired us toprepare smart materials with multiple-modals material, whichshould possess superior hydrophilicity and different chargestates at different pH values. Indeed, this type of smart materialwas prepared in this study. It is a dual-functional materialmodified with histidine, which exhibited both hydrophilicityand switchable surface charge. This material could improve theenrichment selectivity and tolerance of a complex matrix in thecomplex samples. The SGPs could be captured by thecombination of hydrophilic interaction and electrostaticattraction in acetonitrile (ACN)/H2O solution at acidiccondition, while most of the tryptic nonglycopeptides withpositive charges cannot be adsorbed due to electrostaticrepulsion. The nonglycopeptides with similar hydrophilicityto SGPs could be harshly washed off with a high content ofH2O while the SGPs are still adsorbed on the materials byelectrostatic attraction. Then, the retained SGPs can be releasedunder basic conditions with a high content of H2O, bycombination of the increase of electrostatic repulsion anddecrease of hydrophilic interaction. The high selectivity of thismethod was demonstrated by successful enrichment of SGPsfrom bovine fetuin digest at the ratio of 1:5000 with thenonsialylated glycopeptides. This smart material was furtherutilized to selectively enrich SGPs from the protein digest ofhuman serum, and 487 glycosites were identified from only 2μL of human serum; 92.0% of the glycopeptides contained atleast one sialic acid, indicating the excellent selectivity of thismethod.

■ EXPERIMENTAL SECTIONPreparation of the Histidine-Bonded Silica (HBS)

Materials. A 240 g portion of silica gel (approximately 570mmol silanol groups) was dried at 120 °C overnight. Aftercooling to room temperature, 1000 mL of NaAc/HAc solution(0.1 M, pH 4.0) was added under nitrogen atmosphere. Then,240 mL of (3-glycidyloxypropyl)triethoxysilane (860 mmol)was added into the solution with vigorous stirring. The solutionwas heated to 90 °C and stirred for 24 h. The silanized silicawas filtered; washed successively with water (1000 mL),methanol (1000 mL), water (500 mL), and methanol (1000mL); and then dried at 80 °C overnight to obtain the epoxysilica. A 10 g portion of histidine was dissolved in 100 mL ofwater, and then, 6.5 g of Na2CO3 (∼61 mmol) was added toadjust the solution pH to about 8.6. Then, 10 g of the epoxysilica was added into the histidine solution, and the reaction wasallowed to stir for 12 h at 65 °C. Finally, the reaction mixturewas filtered, and washed with water (200 mL) and methanol(200 mL) in succession. The white solid was added to 100 mLof methanol and refluxed for 1 h. Then, the mixture was filteredand washed with methanol (100 mL). The product was dried at

80 °C overnight to obtain the resulting histidine-bonded silicamaterial (designated as HBS).

Characterization of the Histidine-Bonded Silica (HBS)Material. The resulting epoxy silica and HBS material was firstcharacterized by elemental analysis. Elemental analysis wasperformed on a VarioEL III elemental analysis system(Elementar, Hanau, Germany), and the resulting surfacecoverage of histidine was calculated on the basis of the increaseof nitrogen content according to the literature.26 In addition,HBS material was also characterized by FTIR and 13C CP/MASNMR. Fourier-transform infrared (FTIR) spectroscopy wasperformed on a Bruker Optics HYPERION 3000. 13C cross-polarization magic angle spinning nuclear magnetic resonance(CP/MAS NMR) was performed on a Bruker AVANCE 500MHz NMR spectrometer (11.7 T).

Enrichment of Glycopeptides from Protein Digests.Enrichment of Glycopeptides from the Bovine Fetuin TrypticDigests. GELoader tips were packed with 1 mg of HBS sorbentsuspended in 20 μL of ACN. The tip was washed with 20 μL of0.1% FA (formic acid) solution and then equilibrated with 20μL of 75% ACN/0.1% FA. A 20 μL portion of tryptic peptides(equivalent to 5 μg of fetuin) in 75% ACN/0.1% FA wasloaded on the sorbent and then washed with 75% ACN/0.1%FA (20 μL), 70% ACN/0.1% FA (20 μL), and 70% ACN/0.05% HAc (20 μL) successively. The sorbent was eluted with40% ACN/5 mM NH4HCO3 (pH 8.2), and the eluent wascollected for ESI-Q/TOF-MS analysis.For the case of commercial ZIC Glycocapture Resin, the

enrichment protocol was similar to the optimized method inthe previous reports.27 The procedures were described in briefas below. GELoader tips were packed with about 1 mg of ZICGlycocapture Resin. The tip was washed with 40 μL of 0.5% FAsolution and then equilibrated with 40 μL of 80% ACN/0.5%FA. A 40 μL portion of tryptic peptides (equivalent to 10 μg offetuin) in 80% ACN/0.5% FA was loaded on the sorbent andthen washed with 80% ACN/0.5% FA (40 μL). The sorbentwas eluted with 0.5% FA aqueous solution to obtain theglycopeptides.

Enrichment of Glycopeptides from BSA/Fetuin MixtureDigests. The peptides mixtures (10 μg fetuin, fetuin/BSA at amolar ratio of 1:100, 1:500, 1:1000, 1:5000) were first mixedwith 2 mg of HBS (in 75% ACN/0.1% FA) and shaken for 1 hfollowed by centrifugation at 10 000 g for 2 min. After that, thesupernatant was removed, and the precipitation was washedwith 75% ACN/0.1% FA (500 μL × 4) and centrifuged at10 000 g for 2 min. The precipitate was transferred into the Tipcolumn and washed with 70% ACN/0.1% FA (40 μL) and 70%ACN/0.05% HAc (40 μL) successively. Then, the sorbent waseluted with 40 μL of 40% ACN/5 mM NH4HCO3 (pH 8.2),and the eluent was collected for analysis by ESI Q/TOF-MS ornanoLC-MS/MS.

Enrichment of Glycopeptides from Human Serum TrypticDigest. The human serum tryptic desalted digest (5 μL ofinitial human serum) was first mixed with 5 mg of HBS materialin 0.5 mL of 75% ACN/0.1% FA and shaken for 1 h followedby centrifugation at 10 000 g for 2 min. After that, thesupernatant was removed, and the precipitation was washedwith 75% ACN/0.1% FA (0.5 mL), 70% ACN/0.1% FA (0.5mL), and 70% ACN/0.05% HAc (0.5 mL) successively andcentrifuged at 10 000 g for 2 min. Then, the sorbent was elutedwith 0.5 mL of 40% ACN/5 mM NH4HCO3 (pH 8.2). Aftercentrifugation at 10 000 g for 2 min, the supernatant wascollected and deglycosylated for analysis of N-linked glycosites.

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The TiO2 enrichment was performed as previously reported.The human serum tryptic desalted digest (5 μL of initial humanserum) was first mixed with 2.0 mg of TiO2 in 0.4 mL of 85%ACN/5% TFA (trifluoroacetic acid) /1 M glycolic acid, andshaken for 30 min followed by centrifugation at 10 000 g for 2min. After that, the supernatant was removed, and theprecipitation was washed with 80% ACN/1% TFA (0.6 mL)and 20% ACN/0.1% TFA (0.6 mL), and centrifuged at 10 000g for 2 min. Then, the sorbent was eluted with 0.5 mL of 10%NH3·H2O. After centrifugation at 10 000 g for 2 min, thesupernatant was collected and deglycosylated for analysis of N-linked glycosites. The collected supernatant was dried andredissolved into 50 mM NH4HCO3 solution with 200 units ofPNGase F (pH = 8.0), and the mixture was incubated at 37 °Cfor 20 h. After being quenched by pure FA, the deglycosylatedpeptides were dried and analyzed by nanoLC-MS/MS.

■ RESULTS AND DISCUSSIONIn order to selectively enrich SGPs, a dual-functional materialwas prepared, which possesses good hydrophilicity andswitchable charge at different pH. Also, SGPs could becaptured by the combination of hydrophilic interaction andelectrostatic attraction in high acetonitrile content at acidiccondition, while most of the nonglycopeptides with positivecharges cannot be adsorbed due to electrostatic repulsion.Then, nonglycopeptides with hydrophilicity could be removedby high content of H2O under acidic conditions, while theSGPs were still adsorbed onto the materials by the electrostaticattraction. Finally, the retained SGPs could be released by ahigh content of H2O under basic conditions, which couldenhance the electrostatic repulsion and decrease the hydro-philic interaction (shown in Figure 1a). Thus, the preparationand characterization of the dual-functional material was thecritical step for the selective enrichment of SGPs.Preparation and Characterization of the Dual-Func-

tional Material. Two significant issues should be consideredin the preparation of the multiple-modal material. First, thefunctional ligand should possess hydrophilic and switchable-

charge properties. Amino acids are a class of natural smartzwitterions, performing unique electrostatic properties underdifferent pH conditions, which have been used to functionalizematerials’ surfaces.28−30 Histidine, with hydrophilicity and aproper isoelectric point (pI 7.6), is ideal for multiple-modalligand. Second, nonspecific adsorption on the silica supportshould be reduced. Enlightened by the antifouling biomaterialsand size-exclusion chromatography of protein, epoxy silica canbe employed in the ligand bonding in which the residual epoxylgroups could be converted to hydrophilic inert diols.31−33 Onthe basis of the above considerations, histidine-bonded silica(designated HBS) materials were prepared by amino-epoxychemistry (Figure 1a). In addition, histidine groups were thenattached to the silica surface via the ring opening reaction ofepoxy. The resulting HBS material was first characterized byFTIR and 13C CP/MAS NMR. As shown in Figure S1, thestrong absorption peak at 1628 cm−1 was attributed to the CO group in the carboxyl. The peaks around 1408, 1443, and1483 cm−1 were assigned to the CN bond and the CCbond of the imidazole ring. Absorptions from 1000 to 1255cm−1 were assigned to the Si−O bond of the silica support. 13CCP/MAS NMR was utilized to characterized the dual-functional material. As shown in Figure S2, the signal at 179ppm was assigned to the carbon atoms of the carbonyl group,and peaks of 137 and 113 ppm were assigned to the carbonatoms of the imidazole ring. The resonances between 8 and 71ppm belonged to the carbon atoms. Also, it could be found thathistidine was successfully modified onto the surface of thematerials. The surface coverage of histidine was also importantfor the specificity of SGP enrichment, and the resulting HBSmaterial was characterized by elemental analysis. The carboncontent of HBS was 7.76%, and the nitrogen content was about1.09%. The surface coverage of histidine was 0.9 μmol/m2

calculated according to the literature.26

The hydrophilicity of HBS was tested by the chromato-graphic separation of fructo-oligosaccharides. As shown inFigure 1b, the result demonstrated the typical HILICcharacteristics of HBS materials. The surface charge of the

Figure 1. (a) Synthesis of the HBS materials with superior hydrophilicity and switchable charge for the extraction of SGP. (b) The chromatographyof fructo-oligosaccharides separated in HILIC mode by using HBS materials. (c) Zeta-potential test of HBS materials in buffers with different pH.

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HBS was also investigated by zeta-potential test and chromato-graphic evaluation. As shown in Figure 1c, the curve of zeta-potential under different pH conditions was obtained, whichclearly demonstrated the switchable surface charge of HBSmaterials: positive charge at pH lower than 6, and negativecharge at the pH higher than 6. Also, the high surface coverageof the histidine ligand is the primary contributor to theswitchable surface charge. Furthermore, sialic acid wasemployed as the test probe to test the retention of SGPs inchromatography. As we expected, the retention of the sialic aciddecreased dramatically with the increase of pH in mobile phase(shown in Figure S3). This could be explained that the

negatively charged sialic acid was attracted by the positivecharge of HBS at low pH conditions and repulsed by negativecharge at high pH conditions. Thus, the above resultsdemonstrated that the HBS materials have great potential forthe selective enrichment of SGPs.

Enrichment of SGPs from Fetuin Digest. Fetuin was ahighly sialylated blood glycoprotein, and its level in serum hasbeen proven to be correlated with some diseases.34,35 Herein,the bovine fetuin was used to evaluate the performance of HBSin SGP enrichment. First, the tryptic digests were loaded ontoHBS materials in acidic buffer (75% ACN/0.1% FA at pH 2.6).Then, the sorbent was harshly washed using washing buffer

Figure 2. (a) Glycopeptide enrichment from bovine fetuin tryptic digests, mixtures of bovine fetuin, and (b) bovine serum albumin tryptic digestswith molar ratio of 1:500 and (c) 1:5000.

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(70% ACN/0.1% FA, pH 2.6, and 70% ACN/0.05% acetic acid,pH 3.3) to remove the nonglycosylated peptides and neutralglycopeptides. Finally, the enriched SGPs were eluted by using40% ACN containing 5 mM NH4HCO3 at pH 8.2, and theeluent was analyzed by nanoelectrospray ionization-quadrupoletime-of-flight mass spectrometry (nanoESI-Q/TOF MS). Asshown in Figure 2a, a total number of 37 glycopeptides frombovine fetuin were detected, and most of them contained sialicacid termination. To further investigate the tolerance of HBS toa complex matrix, SGPs were selectively enriched from mixturesof bovine fetuin and bovine serum albumin (BSA, anonglycoprotein) digests with different ratios. As shown inFigure 2b, there were still 22 SGPs detected when the molarratio of fetuin/BSA was dramatically increased to 1:500. Also, 7SGPs could still be identified even when the molar ratio offetuin/BSA increased to 1:5000 (shown in Figure 2c). Theabove results demonstrated the high specificity of HBS materialtoward SGPs. It should also be noted that some interferingpeaks with high intensity not corresponding to SGPs of fetuinwere also detected. To further identify these peaks, nanoliquidchromatography-tandem mass spectrometry (nanoLC-MS/MS) was employed to analyze the eluent obtained from thefetuin/BSA mixture (1:5000). Also, 348 SGPs corresponding tomore than 20 other glycoproteins of bovine were identified(Table S1), which might be the trace impurities in standardBSA protein. According to the nanoLC-MS/MS result, 33SGPs of bovine fetuin could be still detected. The SGPs fromthe impurities were labeled in Figure 2c. Commercial ZICResins have been widely used in the enrichment ofglycopeptides.36,37 Also, the ZIC Glycocapture Resins (MerckSeQuant, Umea,̊ Sweden) were employed to enrich glycopep-tides from the mixtures of fetuin and BSA. As shown in FigureS4, many of the SGPs were detected from bovine fetuin digest,while only a few glycopeptides could be found from the digestsof mixed proteins at the fetuin/BSA ratios of 1:100 and 1:500by using ZIC beads. The performance of glycopeptideenrichment by using ZIC beads was obviously not as good asthose achieved with HBS (Figure S5). Clearly, good perform-ance, including high enrichment selectivity toward SGPs andgood resistance to bulk nonglycopeptide interference, could beachieved by HBS material. The above results indicated greatpotential of the HBS material for the analysis of sialylatedglycoproteins in complex biological samples.Enrichment of SGPs from Human Serum Digest.

Recently, it was reported that the aberrant modification ofterminal sialylation glycosylation in serum was closely related tothe progress of diseases.38 Yet, it is still challenging fordetermination of the site-specific glycoforms in serum, due tothe high dynamic range of proteins and interference of highabundance nonglycoproteins. In order to improve thespecificity of glycopeptide enrichment, high abundance proteinsshould be removed before the extraction.4 However, theremoval of high abundance proteins inevitably induces the lossof proteins, resulting in low detection sensitivity of proteinglycosylation. By taking the superior performance of glycopep-tide enrichment, HBS materials were employed to specificallyextract SGPs from human serum digest directly.The amounts of beads applied to enrich SGPs from human

serum digests have a significant influence on enrichmentperformance (shown in Table 1). As shown in Figure S6, thenumber of glycosites identified was between 349 and 462 byusing 0.5−20 mg of HBS materials. Also, up to 462 glycositescould be identified from 5 μL of human serum using 5 mg of

HBS materials, while the number of identified glycositesunexpectedly decreased with the use of greater amounts ofmaterials (10 and 20 mg). It should be noted that the selectivityof SGP enrichment was up to 60%−70% using 0.5−5 mg ofmaterials, while it decreased dramatically by using 10−20 mg ofmaterials. The reason for the different selectivity was that morenonglycopeptides were adsorbed onto the materials using alarger amount of materials, while the number of glycopeptideswas only changed a little (shown in Table S2 and Figure S6).For detailed analysis, it was noted that most of thenonglycopeptides were acidic peptides with low pI (shown inFigure S7). This could be caused by the electrostaticinteractions between the ligand and peptides. During theSGP enrichment, glycopeptides and nonglycopeptides com-petitively adsorbed onto the materials. Because of the highaffinity of glycopeptides to the materials, the adsorbed acidicnonglycopeptides would be replaced by the glycopeptides dueto the limited adsorption capacity. However, it would adsorbmany acidic nonglycopeptides along with the glycopeptideswith the use of greater amounts of materials, due to the highadsorption capacity of materials. Considering the number ofglycosites and selectivity of glycopeptide enrichment, 5 mg ofmaterials was selected for the enrichment of SGPs from humanserum.Titanium dioxide has also been widely used in the

enrichment of SGPs. There were only about 230 N-linkedglycosites identified from 1.3 of μL human serum in a 120 mingradient run by using TiO2, and 44.4% (1932/4352) of thepeptides were identified as glycopeptides (details in shownTable S3). As a comparison, there were 352 N-linked glycositesidentified from the same amount of human serum by usingHBS material, and 52.5% (3270/6233) of the peptides wereidentified as glycopeptides (shown in Figure S8). Obviously,the above results indicated that better performance, includinghigh enrichment selectivity toward SGPs and good resistance tobulk nonglycopeptide interference, could be achieved by HBSmaterial.To further demonstrate the specificity of SGP enrichment,

the data set of enriched intact glycopeptides was also processedby using the automated glycoproteomic method from ourprevious work.39 The initial LC-MS analysis of glycopeptidesenriched from human serum digest led to collection of 59 099MS/MS spectra, and 67.1% (39 659/59 099) of the spectracontained the signature ions of sialic acid (274.09, 292.10 Da).This was consistent with the enrichment selectivity defined bythe percentage of identified glycopeptides among all identi-fications (shown in Table 1). As shown in Table S4, there wereabout 723 intact glycopeptides identified, and 92.0% (665/723)of them contained sialic acid termination. The above resultsfurther demonstrated the high specificity and efficiency of SGPenrichment by HBS materials. In total, there were as many as

Table 1. Numbers of Identified Glycosites and theEnrichment Selectivity for the Analysis of Human SerumTrypsin Digests Using Different Amounts of Materials

amount/mg glycosites selectivity of glycopeptides (%)

0.5 433 73.11 451 64.12 439 63.85 462 61.310 419 52.120 349 42.7

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487 N-linked glycosites identified from only 2 μL of serum inthree runs, and good repeatability was obtained by using thismethod (shown in Figure 3a). The details of the glycoproteins

and glycosites were shown in Table S5. We further comparedthe glycosites with those identified previously from human livertissue in our database, which is the largest glycosite databasefrom one human tissue.40 Also, 82.6% (421/487) of theglycosites were also found in the large human glycosylationdatabase, confirming the accuracy of the identified glycosites. Inaddition, 66 new glycosites were identified for the first time byHBS materials, indicating this capability for glycopeptideenrichment, which could be favorable with the in-depth analysisof sialylation in human serum (shown in Figure 3b).

■ CONCLUSIONSIn summary, a dual-functional material with switchable chargeand good hydrophilicity was fabricated, and it was employed asan absorbent for the enrichment of sialylated glycopeptidesfrom human serum digests. In a comparison with theconventional HILIC materials, the combination of thehydrophilic and electrostatic interactions favored SGP enrich-ment. It was demonstrated that this new material wascomplementary with the analysis of glycoprotein sialylation ina complex biological matrix such as human serum. There were487 glycosites identified from only 2 μL of human serum, and92.0% of them contained sialic acid termination indicating thehigh specificity and sensitivity of the enrichment by using thisnew material. Taken together, our material provides an effectivetool for in-depth analysis of sialylation glycosylation, and will bepromising in studies of glycoproteomics, glyco-biomarkercandidate discovery, and medical diagnostics.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.anal-chem.6b04394.

Reagents, materials, instruments, experimental details,characterization data, and figures (PDF)Glycosites and glycoforms of fetuin tryptic digest(XLSX)Glycosites identified from human serum tryptic digestusing different amounts of materials (XLS)Comparison of glycosites identified by using TiO2 andHBS materials (XLSX)Intact glycopeptides determined from human serumtryptic digest (XLSX)Glycosites identified from human serum tryptic digest(XLSX)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: guozhimou@dicp.ac.cn.*E-mail: mingliang@dicp.ac.cn.*E-mail: liangxm@dicp.ac.cn.ORCIDHongqiang Qin: 0000-0002-7508-0872Mingliang Ye: 0000-0002-5872-9326Xinmiao Liang: 0000-0001-5802-1961Author Contributions§X.D. and H.Q. contributed equally to this work. All authorshave given approval to the final version of the manuscript.NotesThe authors declare no competing financial interest.∥Professor Hanfa Zou passed away on April 25, 2016.

■ ACKNOWLEDGMENTSThis work was supported by the National Natural ScienceFoundation of China (21135005, 21405156, 81430072,21525524), and the Natural Science Foundation of LiaoningProvince (2015021015).

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Figure 3. (a) Overlap of identified N-linked glycosites from humanserum digests in three runs. (b) The Venn map of glycosites identifiedfrom human serum tryptic digest and the total identified glycosites ofhuman tissues.40

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Analytical Chemistry Article

DOI: 10.1021/acs.analchem.6b04394Anal. Chem. 2017, 89, 3966−3972

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