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Facile and Selective Enrichment of Intact Sialoglycopeptides Using Graphitic Carbon Nitride Mo Zhang, Yujie Liu, Dan Zhang, Tianjing Chen, and Zhili Li Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 07 Jun 2017 Downloaded from http://pubs.acs.org on June 8, 2017

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Analytical Chemistry

Facile and Selective Enrichment of Intact Sialoglycopeptides Using Graphitic Carbon Nitride Mo Zhang, Yujie Liu, Dan Zhang, Tianjing Chen, Zhili Li* Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College, 5 Dongdan San Tiao, Beijing 100005, P.R. China *Corresponding author: Zhili Li, Tel/Fax: +86 10 69156479, E-mail: [email protected]. ABSTRACT: Combining powerful selectivity, high stability, convenient operation, mild condition, and eco-friendliness, a novel graphitic carbon nitride (g-C3N4) based enrichment method of intact sialoglycopeptides (SGs) was developed. The intact SGs could be simply enriched and separated from protein tryptic digests by hydrogen bonding without damage of glycan structures due to the specific structure of g-C3N4. By optimizing the enrichment and elution conditions, 45 and 38 of SGs were detected from the tryptic digests of bovine fetuin and transferrin, respectively. Under the synergistic effect of hydrogen bonding and electrostatic adsorption, the SGs could be enriched simply in less than 2 hours with a detection limit of 50 fmol. The method is repeatable due to the high stability of g-C3N4 and the simple protocol of the method, indicating the potential application of g-C3N4 in efficient and selective enrichment of intact SGs.

As one of the most important post-translational modifications, glycosylation has a major impact on the structure and function of proteins.1,2 The variations of glycan are closely related to many physiological and pathological processes.3,4 Salic acid (SA), an important part of glycoprotein normally connected to the terminal of glycan structure, plays key role in organism, such as mutual recognitions and interactions between cells, membrane transportation, immune response, and cancer metastasis.5,6 The up- or down-regulated sialoglycans are strongly associated with many human diseases, for instance, tumors, diabetes, and inflammatory disorders.7,8 Therefore, the development of selective enrichment and rapid detection of sialoglycans is of great importance for screening and study of meaningful biomarkers. Although lots of efforts have been made to enrich glycoproteins or glycopeptides, such as lectin affinity,9,10 boronate or hydrazide chemical methods,11,12 and hydrophilic interaction chromatography,13-15 selective enrichment of sialoglycopeptides (SGs) remains a challenge due to the heterogeneity of protein glycosylation and the low abundance of SGs in complex biological sample.16,17 SA residue linked to the terminal of glycans is liable to be lost during the pretreatment and analytical progress, which may be another barrier for study of SGs.18,19 Lectin affinity has been used to separate SAcontaining glycoproteins. Nevertheless, the high cost and undesirable specificity restrict its application.10,20 Titanium dioxide (TiO2) was reported to selectively enrich SGs via multipoint binding.21 Natural materials have been used as efficient candidates for selective enrichment of SGs.22 The quality control of the enriched material and batch-to-batch reproducibility are inevitable difficulties. Smart polymer can be an efficient SA receptor based on the saccharide−saccharide interactions.23 The preparation of the novel smart polymer is complicated and may introduce some environmental pollutants. Hence, facile enrichment and analytical separation of intact SGs with satis-

factory reproducibility and eco-friendliness are urgent and necessary. Graphitic carbon nitride (g-C3N4), an organic semiconducting polymer consistent with carbon and nitrogen atoms, is a two dimension layered material similar to graphene.24 Due to its eco-friendliness, simple preparation, strong adsorption capacity, fast mass transfer, and high chemical stability, g-C3N4 has attracted extensive attention in photocatalysis and sustainable chemistry.25-27 As most of the graphitic materials, g-C3N4 possesses a highly conjugated system and can absorb different molecules through π interactions.28 The magnetic graphitic carbon nitride anion exchanger has been reported to keep excellent retention for phosphopeptides at pH as low as 1.8.29 However, the potential of this polymer has not been well explored in selective enrichment of SGs. A considerate degree of disorder exists in g-C3N4 prepared by bulk synthesis route, indicating abundant triazine ring and N-H in its structure. This special structure presaged the hydrogen bonding between gC3N4 and -COOH, -OH from SGs, which may be efficient selective adsorption, indicating its potential for selective enrichment and efficient separation of intact SGs. Herein, a g-C3N4 based facile and selective enrichment method of SGs was developed. Due to the specific structure of g-C3N4, the intact SGs could be simply and quickly enriched and separated by hydrogen bonding without dephosphorylation of peptides and analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). By optimizing the enrichment and elution conditions, 45 and 38 of SGs were detected from the tryptic digests of bovine fetuin and transferrin in less than 2 hours with a detection limit of 50 fmol, respectively. The repeatability of the method is ascribed to the simple protocol of the method and the stability of gC3N4, indicating its potential application in efficient and selective enrichment of SGs.

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EXPERIMENTAL SECTION Materials. Bovine fetuin, transferrin, bovine serum albumin, α-cyano-4-hydroxycinnamic acid (CHCA), and 2,5dihydroxybenzoic acid (DHB) were purchased from SigmaAldrich (Steinheim, Germany). PNGase F, α2-3,6,8Neuraminidase, and RNase B were purchased from New England Biolabs (Massachusetts, USA). Trypsin was acquired from Roche Diagnostics (Mannheim, Germany). Dicyandiamide was purchased from Sinopharm Chemical Reagent Corp.(Tianjin China). All the other reagents were of analytical reagent grade and used without further purification. Preparation of g-C3N4 Material. The g-C3N4 material was prepared by pyrolysis of dicyandiamide in air atmosphere. The typical preparation of g-C3N4 was as follows: 5 g of dicyandiamide was heated from 30°C to 550°C in a Muffle Furnace with a heating rate of 3°C/min, followed by calcination at 550°C for 4 hours. The yellow products were washed with 0.1% (v/v) trifluoroacetic acid (TFA) and deionized water to remove the residue absorbed on the surface of g-C3N4. Then the products were dried at 80 °C for 12 h. Characterization of g-C3N4. The morphology of g-C3N4 was examined with a Hitachi SU-8010 field emission gun scanning electron microscopy (SEM) and a Hitachi HT7700 transmission electron microscopy (TEM) operated at an accelerating voltage of 100 kV. Fourier transform infrared spectroscopy (FTIR) measurement was conducted in transmission mode using a Bruker VERTEX 700 spectrometer. The X-ray photoelectron spectroscopy (XPS) measurement was performed using a PHI 5300 ESCA system with beam voltage at 3.0 kV. The binding energy was calibrated with the signal of adventitious carbon at 284.8 eV. The Brunauer–Emmett– Teller (BET) surface area measurements were performed by a micromeritics surface area analyzer (ASAP 2010 V5.02H). The zeta potentials were examined with a Nano particle sizing & Zeta potential Analyzer (Delsa Nano C, Beckman Coulter Ltd, USA). Enzymatic Digestion. All of the proteins used in this paper were digested by trypsin. For pretreatment of enzymatic digestion, 20 µL of protein (1 µg/µL) was denatured in boiling water bath for 6 min. Bovine β-casein was digested directly with an enzyme to substrate ratio of 1:40 (w/w) in 100 mM Tris– HCl (pH 8.5) at 37°C for 16 h. For the other proteins in this paper, 2.2 µL of NH4HCO3 (250 mM) and 0.6 µL of dithiothreitol (1 M) were added into the denatured protein solution and the mixture was kept in water bath at 56°C for 1h, followed by alkylation with 5.6 µL of iodoacetamide (500 mM) at 37°C for 45 min. Then 12 µL of trypsin (50 ng/µL) was added and the solution was kept at 37°C for 12 h. The peptides were divided into two halves. One of them was not treated and the other one was dephosphorylated by 1.5 units of alkaline phosphatase at 37°C for 4h. The enzymatic digests were lyophilized and stored at -80°C. Enrichment of SGs by g-C3N4. The g-C3N4 was dissolved in enriched solution and dispersed by ultrasound for 5 min. The enriched solution was a mixture of ACN and ultrapure water with the content of ACN from 10% to 90% (v/v). The lyophilized enzymatic digests was redissolved with 60 µL of g-C3N4 dispersion and shaken for 1 h. Then the mixture was centrifugated at 15000 g for 5 min. The supernatant was re-

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moved and 50 µL of eluent was added to disperse the precipitated g-C3N4. To optimize the elution conditions, 0.1% TFA, 0.2% formic acid (FA), ultrapure water, 5 mM NH4HCO3, and 0.025% NH3·H2O was used as eluents, respectively. The dispersion was shaken for 40 min followed by centrifugation at 15000 g for 5 min. The supernatant was drawn off and lyophilized for analysis. Mass Spectrometric Analysis. All the mass data were recorded on Apex-Ultra 9.4 T hybrid quadrupole Fourier transform ion cyclotron resonance mass spectrometer (Qh-FTICR MS) equipped with a 355 nm Nd:YAG Smartbeam II laser (200 Hz) (Bruker Daltonics, Billerica, MA, USA) in positive ion mode. A calibration standard with eight peptides was used for external calibration. Mass spectra were acquired over the range from 1500 to 7000 Da in positive ion mode and recorded by accumulating 50 scans at 50 laser shots per scan. The lyophilized enzymatic digests was redissolved with 2 µL of ultrapure water. 0.3 µL of sample solution was spotted onto MALDI target plate and dried at room temperature, followed by the addition of 0.3 µL of DHB (20 µg/µL, 50% (v/v) acetonitrile (ACN), 0.1% (v/v) TFA). The instrument resolution was 490,000 at m/z 400. The glycan structures were predicted by GlycoMod tool (http://web.expasy.org/glycomod/) based on the experimentally determined mass of glycopeptides, within an allowable mass error of ±1 Da. Some of the SGs structures were identified by tandem mass spectra using UltrafleXtreme MALDI-TOF/TOF mass spectrometry (Bruker Daltonics, Billerica, MA, USA). Binding Capacity of g-C3N4 towards SGs. To investigate the binding capacity, the g-C3N4-enriched SGs, derived from bovine fetuin tryptic digests, were enriched once again. Seven equal amounts of g-C3N4 (10 µg for each portion) were incubated with 10 µL of SGs solution with different concentrations (14.8 pg/µL, 29.7 pg/µL, 59.4 pg/µL, 111.4 pg/µL, 148.6 pg/µL, 163.4 pg/µL, and 185.7 pg/µL) for 1 h, respectively. Then, the solution was centrifuged at 15000 g for 5 min and the raffinate was analyzed by MALDI MS. The SGs could be detected only when the loading amount of the SGs was excess for the binding capacity. The concentration of the SGs was computed based on the recovery rate of g-C3N4 (c= (m0×I)/v. c is the concentration of SGs; m0 is the original mass of tryptic digests; I is the recovery rate of g-C3N4 towards SGs; v is the volume of the solution). Thus the binding capacity of the gC3N4 enrichment method for SGs could be estimated according to the minimum signals of SGs in raffinate.12 RESULTS AND DISCUSSION Material Characterization. The structure of g-C3N4 prepared by thermal polymerization of dicyandiamide is shown in Figure 1a (inset). There are tri-s-triazine rings cross-linked by N atoms and enormous N-H in its structure.


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Figure 1. The XPS spectrum (a), structure (inset in a), and FTIR spectrum (b) of g-C3N4. The element composition of g-C3N4 was revealed by XPS. As shown in Figure 1a, C1s and N1s peaks are observed at 286 ev and 397 ev, indicating the main components of C and N elements.30 FTIR spectrum of g-C3N4 shows typical breathing mode of the triazine units at 806 cm-1 and aromatic C-N heterocycle stretches at 1200-1700 cm-1 (Figure 1b). The broad peak at 2900-3200 cm-1 is attributed to the N-H and O-H bands, derived from the residual unpolymerized amino groups and the absorbed H2O molecules.31,32 The highly conjugated system and abundant N-H in the structure suggest that g-C3N4 may be an ideal hydrogen bond donor or acceptor to selectively enrich SGs. The morphology of g-C3N4 was characterized by SEM and TEM (Supporting information, Figures S1 and S2). The gC3N4 exhibited bulk structure. Brunauer–Emmett–Teller (BET) measurement revealed a surface area of 10.6 m2/g and a pore volume of 0.03 m3/g. The nitrogen sorption measurement showed a type III isotherm with H3 hysteresis loop (Supporting information, Figure S3), indicative for a material with slit pore.33 Although the lower specific surface area than normal adsorbent may be an unfavorable factor of g-C3N4, the adsorption efficiency is sufficient for trace SGs. In addition, the surface adsorption of bulk material may facilitate the subsequent elusion. Enrichment Mechanism of g-C3N4. The g-C3N4 can enrich SGs efficiently by hydrogen bonding and separate them from other peptides by simple centrifugation (Figure 2).

of ACN in enrichment solutions and 0.025% NH3·H2O as eluent. Purple diamond, SGs; octagonal star, glycopeptides without SA residue; triangle, peptides. To optimize the enrichment and elution conditions, the polarity of enrichment solution and the pH of eluate were adjusted (Figure 3). With fixed pH of eluent at 11.1 (0.025% NH3·H2O), the number of the detected SGs was increased and the intensities of the SGs were enhanced gradually by changing ACN content in enrichment aqueous from 10% to 80% (v/v). When ACN content was increased to 90%, the intensities of non-glycopeptides were enhanced, which was disadvantageous for analysis of SGs. Thus, enrichment solution was optimized as 80% ACN aqueous. With the optimized enrichment condition and changed pH of eluent from 2.0 to 11.1, the enriched SGs were increased obviously. The optimal eluent was 0.025% NH3·H2O aqueous (Figure 4).

Figure 4. Mass spectra of the g-C3N4-enriched SGs from the tryptic digests of bovine fetuin (8 pmol) with different pH of eluents. The eluents: 0.1% TFA, 0.2% FA, H2O, NH4HCO3, and 0.025% NH3·H2O. Purple diamond, SGs; octagonal star, glycopeptides without SA residue; triangle, peptides; star symbol, electric noise.

Figure 2. The workflow of the enrichment of SGs using gC 3N 4.

Figure 3. Mass spectra of the g-C3N4-enriched SGs from the tryptic digests of bovine fetuin (8 pmol) with different content

Figure 5. The zeta potentials of g-C3N4 dispersed in enrichment solution with different ACN content (a) and eluents with different pH (b). The enrichment mechanism was further investigated by zeta potential analyses of g-C3N4 dispersion in different enrichment solutions and eluents. As shown in Figure 5a, the zeta potential fluctuated between -20 mV to -30 mV with ACN content increased from 10% to 60%. When the ACN content increased beyond 60%, the zeta potential showed a monotonic increasing trend. SA is known to be a negative charged terminal monosaccharide, which is more inclined to be absorbed on positive charged or less negative charged materials.19 Thus, 80% and 90% of ACN content as enrichment solution is more advantageous. However, the solubility of SGs is much poorer than non-glycopeptides in 90% ACN due to the highly polar of SA. 90% ACN in enrichment solution may lead to obvious



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interferences to the hydrogen bonding between SGs and gC3N4 by non-glycopeptides. Under the synergistic effect of hydrogen bonding and electrostatic adsorption, the enrichment condition was optimized as 80% ACN aqueous solution. As shown in Figure 5b, the zeta potential of g-C3N4 was decreased along with the increased pH of dispersion. On the one hand, the alkaline condition is conductive to the cleavage of hydrogen bond. On the other hand, the negative charged gC3N4 tends to repel SGs in alkaline environment. Therefore, the eluent was optimized as 0.025% NH3·H2O.

Figure 6. Mass spectra of peptides and glycopeptides derived from the tryptic digests of bovine fetuin (8 pmol, a), the gC3N4-enriched SGs (b), the raffinate (c), the g-C3N4-enriched peptides from α2-3,6,8-neuraminidase-treated enzymatic digests (d) and the g-C3N4-enriched peptides from PNGase Ftreated enzymatic digests (e). Purple diamond, SGs; octagonal star, glycopeptides without SA residue; triangle, peptides; star symbol, electric noise. The MS analysis of enzymatic digests, eluent, and raffinate are shown in Figure 6. The tryptic digest of bovine fetuin was consistent with considerable non-glycopeptides and minute traces of glycopeptides. Due to the interferences of massive non-glycopeptides, only 12 of SGs with low relative intensity could be detected before enrichment (Figure 6a). In the adsorption process, SGs were inclined to adsorb on g-C3N4 in 80% ACN and separated from raffinate by centrifugation. Then they desorbed in 0.025% NH3·H2O aqueous solution. Surprisingly, 45 of SGs were detected through the enrichment of g-C3N4 (Figure 6b, Supporting information, Figure S4). The number of detectable SGs was nearly increased by 3 times and the peak intensities of the SGs were enhanced remarkably. The non-glycopeptides were left in the raffinate and detected in Figure 6c. In order to further verify the adsorption ability of g-C3N4 for SGs, the α2-3,6,8-neuraminidase-treated enzymatic digests and PNGase F-treated enzymatic digests from the tryptic digests of bovine fetuin (8 pmol) were enriched by the same method. As shown in Figure 6d, none of SG could be detected and only a few glycopeptides peaks were observed in the region of 3000-5000 Da after treated by the neuraminidase. When all of the N-glycans were removed after PNGase F treatment, only rare peptides could be detected (Figure 6e). These results indicate that the nonspecific adsorption of glycopeptides without sialylation and non-glycopeptides was not obvious. The proposed glycan structures of SGs were predicted by database search (Supporting information, Table S1). Some of the SG structures were identified by tandem mass spectra (Supporting information, Figure S5). The glycosyla-

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tion site and the structure of the SGs are consistent with the results of previous study.22 The repeatability of the method was verified by intra- and inter-day reproducibility. The variation coefficients of the detected SGs number were less than 3.5% and 3.4%, respectively (Supporting information, Figures S6 and S7). The detection limit of the method was 50 fmol, conducted by enrichment with different amounts of fetuin tryptic digests (Supporting information, Figure S8). The recovery of the method was obtain by comparing the total intensity of the 8 abundant detected SGs derived from the tryptic digests of bovine fetuin before and after enrichment and was estimated as 89.2% (Supporting information, Figure S9). The binding capacity of the g-C3N4 enrichment method towards SGs was estimated as 148 mg/g according to the minimum signals of SGs in the raffinate, indicating the dramatically enrichment ability of g-C3N4 towards SGs (Supporting information, Figure S10). These results indicate that g-C3N4 could act as a competitive candidate for SGs enrichment. The enrichment of SGs by g-C3N4 was further investigated by enrichment of transferrin tryptic digests with less sialylated glycans than bovine fetuin. 38 of SGs could be detected after the enrichment from transferrin tryptic digests, which was obviously increased compared to that without enrichment, indicating that g-C3N4 can enrich SGs selectively and efficiently (Supporting information, Figures S11 and Table S2). Some of the SG structures were identified by tandem mass spectra (Supporting information, Figure S12). None of SG could be detected and only a few glycopeptides peaks were observed in the region of 3000-5000 Da after treated by the neuraminidase. Rare peptides could be detected after all of the N-glycans were removed by PNGase F treatment. The results indicated that g-C3N4 could enrich SGs efficiently from proteins with different content of sialylated glycans. Enrichment Efficiency of g-C3N4 for SGs. RNase B (a high mannose glycoprotein without SA residual) and albumin (a non-glycoprotein) were conducted, respectively, to further confirm the selective enrichment ability of g-C3N4 for SGs. After the enrichment process, no glycopeptide was detected from RNase B and only a few non-glycopeptides were detected from albumin, indicating the poor adsorption efficiency of non-glycopeptides and glycopeptides without sialylation (Supporting information, Figure S13a and b). Further, four standard proteins (bovine fetuin, transferrin, RNase B, and albumin, 8 pmol of each protein) were mixed to simulate a complicated sample. 54 of SGs were enriched by g-C3N4 from the tryptic digests of the mixed proteins (Supporting information, Figure S13c), which were all from the tryptic digests of bovine fetuin and transferrin. These results indicate that gC3N4 can selectively enrich SGs in a complicated sample, suppressing the interference of non-glycopeptides and glycopeptides without sialylation.


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Figure 7. Mass spectra of the g-C3N4-enriched SGs from the tryptic digests of the mixtures of fetuin and albumin with the ratio of 1:1 (a), 1:20 (b), and 1:50 (c). Purple diamond, SGs; octagonal star, glycopeptides without SA residue; triangle, peptides; star symbol, electric noise. The selective enrichment ability of g-C3N4 was further investigated by enrichment from fetuin tryptic digests with stoichiometric amounts of albumin. As shown in Figure 7, about 30 of SGs were detected after enrichment from the tryptic digests of the mixture of fetuin and albumin (1:50, W/W), indicating the remarkable selectivity of g-C3N4 towards SGs. The g-C3N4 showed enhanced selective enrichment ability compared with kapok fiber and TiO2, especially for enrichment of peptides from the tryptic digests of bovine fetuin without dephosphorylation (Supporting information, Figure S14a). When bovine fetuin was digested without dephosphorylation, the kapok fiber-enriched SGs mainly distribute in a narrow m/z range of 4000-6000,22 with massive intensity of larger SGs weakened or lost (Supporting information, Figure S14b). Under the same condition, only two SGs could be detected after enrichment by TiO2 (Supporting information, Figure S14c),19 which was much less than that of g-C3N4. The undesirable selectivity of TiO2 for SGs may be attributed to its high affinity of phosphopeptides.19 More interestingly, when the fetuin tryptic digests were dephosphorylated, about 45 of SGs could be enriched by either kapok fiber or g-C3N4, indicating their highly selective enrichment ability (Supporting information, Figure S15a and b). By contrast, only four SGs could be detected after TiO2 enrichment from dephosphorylated digests (Supporting information, Figure S15c). These results demonstrate the difficulty to detect SGs directly by MALDI MS with the TiO2 method due to its low selectivity, indicating that the TiO2 enrichment approach is more suitable to be combined with liquid chromatographymass spectrometry.19 When the matrix was changed from DHB to CHCA, the intensities of the TiO2-enriched SGs were detectable (Supporting information, Figures S16 and S17). The TiO2-enriched SGs from dephosphorylated digests were much higher than that from peptides without dephosphorylation. The enhancement trend was not pronounced for the gC3N4 method. Although more TiO2-enriched SGs could be detected with CHCA as matrix, the signal to noise ratio was disappointing compared with that of the g-C3N4 method. And CHCA is not a proper matrix for SG studies due to the significant degree of the desialylation under the ionization step.34 These results further indicate that g-C3N4 can be a more promising material than kapok fiber and TiO2 for enrichment of SGs selectively and efficiently.

Zhu et al have shown that g-C3N4 is capable of phosphopeptide enrichment with the loading buffer at pH as low as 1.8 (ACN/0.2% TFA (1/1, v/v)).29 In our study, the loading buffer is ACN/H2O (4:1, v/v), which is not a proper enrichment condition for phosphopeptides enrichment , indicating that g-C3N4 is a promising enrichment material for both of phosphopeptides and SGs. When the SGs are the target products, the interference of phosphopeptides could decrease by optimizing the method. In order to further investigate the selectivity of gC3N4 method towards SGs, the tryptic digests of fetuin with stoichiometric amounts of a phosphoprotein, β-casein, was enriched. The SGs could be selectively enriched from the mixture of the tryptic digests of fetuin and β-casein with the ratio of 1:1 and 1:5 (Supporting information, Figure S18a and b), respectively. However, when there was a tenfold increase in amount of β-casein, the signal of SGs was obviously interfered (Supporting information, Figure S18c). The results indicated that the enrichment method with g-C3N4 could eliminate the interference of phosphopeptides in appropriate condition within limits. When the amount of phosphopeptides was far more than SGs, the signals of SGs were dramatically suppressed. The SGs enrichment ability of g-C3N4 from real protein was further investigated by enrichment from tryptic digests of haptoglobin, which was separated from human serum as described previously.35 17 of SGs were detected after enrichment (Supporting information, Figure S19 and Table S3), indicating that the method is selectively and efficiently for real protein. CONCLUSIONS In conclusion, a g-C3N4 based facile, selective, and ecofriendly enrichment method of SGs was developed. The intact SGs could be simply enriched and separated from raffinate by hydrogen bonding without damage of glycans due to the specific structure of g-C3N4. By optimizing the enrichment and elution conditions, the g-C3N4 exhibited enhanced performance in enrichment of SGs compared with kapok fiber and commercial TiO2. The method is repeatable due to the stability of g-C3N4 and the simple protocol of the method, indicating its potential in efficient and selective enrichment of intact SGs. We expect that this new enrichment approach will be widely applicable for the efficient enrichment of trace amounts of biomolecules with negatively charged groups.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website, including experimental details, SEM images, TEM images, N2 adsorption-desorption isotherms and the mass spectra of SGs.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]

ORCID

Zhili Li: 0000-0002-1190-7949 Conflict of Interest Disclosure The work presented in this report is the subject of a pending patent filed by Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College.



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(34) Kurogochi, M.; Nishimura, S. I. Anal. Chem. 2004, 76, 60976101.

ACKNOWLEDGMENT This work was supported by the National Science Foundation of China (Grant no 21575164) to Z.L..

(35) Wang, Y.; Song, G.; Wang, Y.; Qiu, L.; Qin, X.; Liu, H.; Li, F.; Wang, X.; Li, F.; Guo, S.; Zhang, Y.; Li, Z. J. Proteome Res. 2014, 13, 710-719.

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