A hydrophilic nanocomposite functionalized by carrageenan for the

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A hydrophilic nanocomposite functionalized by carrageenan for the specific enrichment of glycopeptides Yingxin Chen, Qianying Sheng, Yayun Hong, and Minbo Lan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05578 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

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Analytical Chemistry

A hydrophilic nanocomposite functionalized by carrageenan for the specific enrichment of glycopeptides Yingxin Chen†, Qianying Sheng *,†, Yayun Hong †, Minbo Lan *,†,‡ †

Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, PR China. ‡

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China. ABSTRACT: A hydrophilic nanocomposite was synthesized by an easy route to improve the glycopeptides enrichment efficiency. This new composite, prepared with a method based on electrostatic interaction, was demonstrated to be efficient for immobilization of carrageenan on graphene oxide/polyethylenimine support (denoted as GO-PEI-Carr). Carrageenan, which has a large number of hydroxyl groups and fully negative charged, was a new modified phase of hydrophilic materials in glycoproteomics. The introducing of carrageenan provided the composite not only a perfect surface charge, but also a greater ability to enrich glycosylated peptides. Thirty-four glycopeptides from human serum immunoglobulin G (IgG) tryptic digests were obviously observed with greatly improved signal-to-noise (S/N) ratio. A good selectivity was still kept even when the molar ratio of IgG and BSA tryptic digest mixtures reached to 1:500. Meanwhile, 76 glycopeptides derived from 56 glycoproteins with 83 N-glycosylation sites were identified from human serum and 149 glycopeptides derived from 129 glycoproteins with 157 N-glycosylation sites were identified from mouse liver tissues, which showed the ability to enrich glycopeptides from complex biological samples. In addition, GO-PEICarr exhibited a unique repeatability and stability even after enrichment glycopeptides for 20 times. It also performed a higher sensitivity (1 fmol/μL IgG), a better enrichment capacity (up to ~300 mg/g), and an ideal enrichment recovery (90.8% and 109.5%) for glycopeptides enrichment, indicating a great potential for the application of glycoproteomic research.

Carrageenan is a kind of sulfated galactans containing D-galactose. And its derivatives linked to each other by dint of repeating (1→4) and (1→3) bonds1, 2. It’s a natural product which is a type of polysaccharides extracted from red seaweed (marine red algae) of the Rhodophycea family2. Carrageenan is widely used in the food industry as a coagulant or emulsifier3. Transparent, flexible, high ionic conductivity, ability to conduct electricity and environment friendly are some of the properties of carrageenan2, 4-6. Due to these advantages, more applications of carrageenan have been discovered which could be seen in many recent studies. For example, Suraya Abdullah3 employed kappa-carrageenan as active layers in LSPR sensor for Pb (II) ion detection. Yuri S. Khotimchenko1 created a new drug consisting of carrageenans for elimination of metals from the body or targeted delivery of these metal ions for healing purposes. Fatima Boukhlifi7 prepared a K+-cross-linked kappa-carrageenan bead which was evaluated to remove the cationic crystal violet (CV) dye from water. However, to the best of our knowledge, the present paper is the first report on the using of carrageenan as the glycopeptides enrichment material.

Glycosylation is one of the most important types of posttranslational modifications in cell signal transduction and function regulation8. Because the amounts of glycopeptides or glycoproteins are extremely low which only accounts for about 1% of the total protein9, 10. Therefore, specific enrichment methods must be developed to enable efficient mass spectrometry analysis. Methods for enriching glycoproteins or glycopeptides mainly include immobilized lectin11, 12, hydrazide chemical method13, boric acid method14, 15, hydrophilic interaction chromatography16-18 and so on. These methods have accumulated much experience in the selectivity glycopeptides enrichment. And the recently new materials based on these methods tried to explore a path to provide a new solution for the limited enrichment capacity and poor selectivity. Hydrophilic interaction chromatography (HILIC) is considered as a mixed-mode mechanism, mainly based on hydrophilic partitioning and other interactions such as electrostatic interactions, dipole interactions, adsorption, hydrogen bonding and so on19. With excellent hydrophilicity and similar poly-hydroxy structure, several polysaccharide modified materials have been

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widely employed in HILIC mode to enrich glycopeptides, such as glucose20, maltose21, 22, cellulose23, glucose-6-phosphate24, chitosan25, 26, , sepharose27, dextran28, 29. There are some advantages such as high glycosylation coverage and unbiased recognition ability for glycopeptides, as well as excellent compatibility with MS analysis. However, there are still some difficulties to be improved. For example, the differences between saccharides of glycopeptides are often slight which makes the development of synthetic receptors for saccharide discrimination very difficult. And materials with a greater repeatability was ignored by most studies. Significantly improved the reusability of the composite can be more environmentally friendly. It is conventionally required a better molecular design with some excellent materials for stronger polar forces and perfect surface properties. In general, increasing the interaction power and sites are beneficial to improve the ability to identify the glycopeptides. In this work, carrageenan (Carr) as a new modified phase in the enrichment of glycopeptides was combined to graphene oxide/polyethylenimine support denoted as GO-PEI-Carr. The large number of hydrophilic groups in carrageenan increased polar forces and interaction sites of nanocomposites. In addition, carrageenan is a special polysaccharide which is negatively charged and can be combined with cross-linkers easily by electrostatic interaction owing to the existence of sulfate groups, making it easy to regulate the surface charge. In the HILIC mode, electrostatic interaction is a significant factor in the enrichment of glycopeptides. Theoretically, under acid conditions, most peptides and glycopeptides are positively charged. Materials with a strong positive charge may increase the repulsion force with glycopeptides. And in contrast, materials with negatively charged are tend to adsorb extra nonglycopeptides. According to the experiments, the GO-PEI-Carr nanocomposites were proved to perform better than GO-PEI especially in sophisticated systems owing to modification of carrageenan. Moreover, although the preparation process was simple and environmentally friendly, and no toxic solvents were involved, GO-PEI-Carr still exhibited a unique glycopeptides enrichment selectivity, versatility, mild enrichment conditions and great repeatability.

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Synthesis of GO-PEI-Carr. The graphene oxide (GO) was prepared by using the Hummers method 30 with some modification. GO-PEI was prepared by the following method: 100 mg of graphene oxide (GO) was dispersed by 100 mL deionized water. Then, 2 g PEI were added and dispersed by ultrasound for 5 min. Stir at room temperature for 12 h. Washed 3 times with deionized water. The result solid was freeze-dried overnight. At last, GO-PEI-Carr was prepared as followed: 50 mg of GO-PEI was dispersed with 50 mL deionized water under 60 ºC. Then 0.5 g carrageenans were added and dispersed by ultrasound for 5 min. Stir at 60 ºC for 12h. Wash 3 times with deionized water. The result solid was freeze-dried overnight. Characterization of GO-PEI-Carr. Zeta potential of the composites in the ultrapure water was measured at room temperature with a Nano-ZS90 instrument (Malvern, UK). X-ray photoelectron spectroscopy (XPS) spectra were obtained from an ESCALAB-250Xi (Thermo Fisher Scientific, USA). Field emission scanning electron microscope (FE-SEM) images were taken with a SU-70 field emission scanning electron microscope (Hitachi, Japan). Elemental analysis of the materials was measured with Elemental analyzer (ELEMENTAR, Germany) and EDX detector (EDAX, 133eV/Falion 60S, American). Fourier transform infrared (FT-IR) spectra were performed on a Nicolet 6700 using KBr pellets (Thermo Fisher Scientific, USA). Sample preparation. Human serum immunoglobulin G (IgG, 1 mg) or bovine serum albumin (BSA, 1 mg) was dissolved in 100 µL NH4HCO3 buffer (50 mM, pH 8.2) containing 8 M urea. Then, DTT was added to reduce the disulfide bridge at 56 ºC for 45 min. Subsequently, 20 µL of IAA (200 mM) was added for alkylation, and the mixture was incubated in the dark for 30 min. Finally, the mixture was diluted to 1mL with NH4HCO3 buffer and digested by trypsin for 17 h at 37 ºC with a 1:25 (w/w) enzyme/substrate ratio. Human serum samples were collected from healthy people and obtained from the Sixth People’s Hospital affiliated with Shanghai Jiaotong University according to the standard clinical procedures. The mixture containing 2 µL human serum and 16 µL 50 mM NH4HCO3 was centrifuged at 12000 rpm for 5 min. The supernatant was collected and denatured at 100 ºC for 10 min. After that, 200 mM DTT was added and the mixture was incubated at 56 ºC for 45 min, followed by alkylation in the dark for 30 min with the help of IAA. Finally, trypsin (trypsin:protein = 1:25, w/w) was added and the solution was incubated at 37 ºC for 17 h. The obtained tryptic digests were lyophilized and kept at -80 ºC for further use. The Wistar mice were bought from Shanghai SLAC Laboratory Animal. The mouse liver tissues were homogenized in a lysis buffer (100 mM Tris-HCl, 8 M urea, 1300 mM DTT, 20 mM EDTA, 20 mM PMSF, 2% protease cocktail, 1% Triton X100, 200 mM C3H7Na2PO6, 100 mM Na4P2O7, 200 mM NaF, and 200 mM Na3VO4, pH 7.4). The homogenates were sonicated at 400 W for 180 s and then clarified by centrifugation for 40 min. Afterward, five volumes of precipitation solution (acetone/ethanol/acetic acid=50:49.9:0.1, v/v/v) was added to the mixture and then incubated 4 h to precipitate the protein. The protein precipitate washed by ice acetone and ice 75% alcohol respectively. Precipitate was collected by centrifugation and resuspended in 50 mM NH4HCO3 buffer containing 8 M urea. Protein concentration was determined by using BCA (Pierce, Rockford, 1 L). Extracted proteins were stored at -80 ºC until

EXPERIMENTAL SECTION

Reagent and materials. Graphene powders are provided by Sinopharm (Shanghai, China). Chemical reagent KMnO4, H3PO4, H2SO4, and HNO3 were obtained from Shanghai Lingfeng (Shanghai, China). Chemical reagent polyethylenimine (PEI), carrageenan (Carr) were purchased from Aladdin (Shanghai, China). Chemical reagent ammonium bicarbonate (NH4HCO3), urea, protease cocktail, Triton X-100, βglycerophosphate disodium salt hydrate (C 3H7Na2PO6), sodium pyrophosphate tetrabasic (Na4P2O7), sodium fluoride (NaF), ethylenediaminetetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), sodium orthovanadate (Na3VO4), dithiothreitol (DTT), iodoacetamide (IAA), trypsin, human serum immunoglobulin G (IgG), 2,5-dihydroxybenzoic acid (DHB), bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PNGase F and glycobuffer was purchased from New England Biolabs (Ipswich, MA, USA). Acetonitrile (ACN) and trifluoroacetic acid (TFA) were chromatographic grade. Water used in experiments was purified using a Milli-Q system (Millipore, Bedford, MA, USA).


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use. Extracted proteins from mouse liver were reduced in 200 mM DTT at 56 ºC for 45 min and alkylated with 200 mM IAA for 30 min in the dark. After the reaction, the mixture was diluted to 1mL with NH4HCO3 buffer. The proteins were digested with trypsin (enzyme/substrate ratio of 1:25, w/w) at 37 ºC with rotation 17 h. The digestions were quenched by the addition of formic acid (FA) and then centrifuged for 20 min. Finally, the samples were dried by vacuum centrifugation and stored at -80 ºC for further analysis. Enrichment of glycopeptides from IgG Tryptic Digests. 6 µL of IgG digest were diluted with 200 µL loading buffer (ACN/H2O/TFA=90:9:1, v/v/v), and then added 500 µg GOPEI-Carr nanocomposites and incubated at room temperature for 30 min. After incubation, nanocomposites were centrifuged for 2 min and rinsed with 100 µL washing buffer (ACN/H2O/TFA=90:9.9:0.1, v/v/v) for three times. Subsequently, 10 µL eluent buffer (ACN/H2O/TFA=30:67:3, v/v/v) were used to elute the glycopeptides with 20 min shake. The eluent was collected and analyzed by MALDI-TOF MS. Enrichment of glycopeptides from IgG/BSA mixture digests. The peptides mixtures (IgG and BSA at a molar ratio of 1:50, 1:100, 1:500) were first mixed with 500 ug of GO-PEICarr (in ACN/H2O/TFA=90:9:1, v/v/v) and shaken for 30 min followed by centrifugation for 2 min. After that, the supernatant was removed, and the precipitation was washed with 200 µL washing buffer (ACN/H2O/TFA=90:9.9:0.1, v/v/v) three times and centrifuged for 2 min. Then, the sorbent was eluted with 10 µL eluent buffer (ACN/H2O/TFA=0:97:3, v/v/v), and the eluent was collected for analysis by MALDI-TOF MS. Enrichment of glycopeptides from real biological samples. Real biological samples (2 µL human serum tryptic digest or 50 µL mouse liver tissues) was first mixed with 500 ug of GO-PEICarr composites in 500 µL of loading buffer (ACN/H2O/TFA=90:9:1, v/v/v) and was shaken 30 min followed by centrifugation for 2 min. After that, the supernatant was removed, and the precipitation was washed with 500 µL washing buffer (ACN/H2O/TFA=90:9.9:0.1, v/v/v) three times and centrifuged for 2 min. Then, the sorbent was eluted with 100 µL eluent buffer three times (ACN/H2O/TFA=0:97:3, v/v/v). After centrifugation for 2 min, the supernatant was collected and deglycosylated for analysis of N-linked glycosites. Deglycosylation of N-glycopeptides by PNGase F. The freeze-dried glycopeptides were redissolved in 17 µL H2O. And then 2 µL glycobuffer and 1 µL PNGase F were added. The mixture was rotated at 37 ºC for 17 h. Finally, the solution was lyophilized for nano-LC-MS/MS analysis. MS analysis. MALDI-TOF MS experiments were performed under reflect positive ion mode on a 4800 Plus MALDI-TOF MS (AB Sciex, USA) with a Nd:YAG laser emitting at 355 nm. 1.0 µL analyte was dropped on the plate, then dropped 1.0 µL matrix (DHB, 25 mg/mL, ACN/H2O/H3PO4 = 70:29:1, v/v/v) and analyzed by MALDI-TOF MS. The scan range was 23003300 m/z. Glycopeptides enriched from human serum or mouse liver tissues were resuspended with 10 µL loading samples (H2O/FA=99.9:0.1) and then separated by nano LC and analyzed by on-line mass spectrometry. The experiments were performed on a Thermo EASY-nanoLC 1000 Nano HPLC System (Thermo Fisher Scientific, Bremen, Germany) connected to a

quadrupole-Orbitrap mass spectrometer (Q-Exactive plus, Thermo Fisher Scientific, Bremen, Germany) equipped with an online nano-electrospray ion source. The specific process can be seen in our previous study31. 1 µL peptide sample was separated on the analytical column (reverse capillary analytical C18, 75µm x 15cm) with a linear gradient. The elution method was carried out as follows: 0−4% mobile phase B for 2 min; 4−35% mobile phase B for 90 min; 35− 45% mobile phase B for 10 min; 45−90% mobile phase B for 5 min; 90% mobile phase B for 5 min; and finally equilibration with mobile phase A for 15 min (A: water with 0.1% formic acid; B: ACN with 0.1% formic acid). The column was re-equilibrated at initial conditions for 10 min. The column flow rate was maintained at 300 nL/min and column temperature was maintained at 25 ºC. The Q-Exactive mass spectrometer was operated in data-dependent MS/MS acquisition mode. The electrospray voltage of 2.0 kV versus the inlet of the mass spectrometer was used. Survey full-scan MS spectra (m/z 400-2000) were acquired with a mass resolution of 70K. The 12 most intense parent ions with charge states≥2 from the full scan were fragmented by higherenergy collisional dissociation (HCD) with a normalized collision energy (NCE) of 27%. The MS/MS acquisitions were performed in Orbitrap with a resolution of 35 000 (m/z 200), and the automatic gain control (AGC) target was set to 1×10 5 with a max injection time of 120 ms. Dynamic exclusion was set as 30 s. System control and data collection were carried out by Xcalibur software. Database Search. The MS raw data files were exported using the Proteome Discoverer software (Thermo Fisher Scientific, version 1.4) with Sequest HT search engine was used for all database searches, and the database was the Human or mouse UniProtKB/SwissProt database. The data retrieval parameters were as follows: precursor mass tolerance, 10 ppm; fragment mass tolerance, 0.05 Da. Two missed cleavages were allowed by trypsin digestion. False discovery rates (FDR) were controlled lower than 1% by the percolator algorithm at both the peptide and protein level. Carbamidomethyl on cysteine was set as a fixed modification. Oxidation on methionine and deamidation on asparagine were set as variable modifications. Only glycopeptides with the N-glycosylation consensus sequence (N-!P-S/T/C) were considered to be reliable.

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RESULTS AND DISCUSSION

Synthesis and characterization of GO-PEI-Carr. The graphene oxide (GO) was prepared by using the Hummers method30 with some modification. Then PEI, a cationic polymer, was self-assembled on the surface of anionic GO nanosheets by electrostatic interactions, which could greatly improve the hydrophilicity of GO owing to the large number of polar groups on the polymer chains. Subsequently, carrageenans were combined with GO-PEI by electrostatic adsorption. The preparation process was simple and environmentally friendly. Any toxic solvents were not involved. The prepared GO-PEI-Carr was further used as a new kind of saccharide-based-HILIC material for glycopeptides enrichment. Zeta potentials were measured to characterize the materials. As shown in Table 1, GO has a strong negative charge (-27.40 mv) due to the existence of carboxyl groups. Zeta potential measurements show that PEI changed the zeta potential from negative to positive (from -27.40 mv to +48.90 mv) due to the



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large amounts of amino groups. Then, the value of zeta potential was decrease to +6.76 mv demonstrating that carrageenan which was an acidic polysaccharide and negatively charged combined to GO-PEI successfully. The sulfur content of GO-PEI, GO-PEI-Carr and carrageenan were detected by Elemental analyzer (Table 1). Compared with GO-PEI, the content of sulfur of GO-PEI-Carr is increased to 0.46% which showed the successful modification of carrageenan. The calculated loading of carrageenan based on elemental analysis was 83 mg per gram of GO-PEI supports. Table 1. The charges and elemental analysis of the nanoparticles Materials

Charge(mv)

GO

-27.40

Element(S) counts (%) 0

GO-PEI

+48.90

0

GO-PEI-Carr +6.76 0.46 Carrageenan —— 5.57 In order to further confirm the preparation of GO-PEI-Carr, XPS was used to analyze different elements on the surface of GO-PEI-Carr. As is shown in Figure 1a, GO-PEI-Carr showed several main character peaks located at 286, 533, 401 and 169 eV, which were assigned to C1s, O1s, N1s and S2p signals. S2p spectra fitting analysis (Figure 1b) showed the peak of C-SOxC which was the characteristic of carrageenan32. Additionally, the surface chemical composition of the nanocomposites was measured as well (Table S-1). It can be inferred that the carrageenan was successfully modified. SEM image of GO, GO-PEI, GO-PEI-Carr were measured. As shown in Figure 1c, SEM image of GO consists of crumpled thin sheets which also observed with wrinkles and folds on the surface of GO nanosheets. After coated with PEI, GO-PEI nanofillers have resulted more compact morphology (Figure 1d). After modified with carrageenan, the structure of compact morphology was maintained (Figure 1e). The EDX spectra of GO-PEI-Carr was given in Figure 1f. The atomic and weight percentage of C, O, N, S were identified respectively. The weight and atomic percentage of S was 1.75% and 0.72% which represented that carrageenan was modified to GO-PEI support successfully. Fourier-transform infrared spectroscopy (FT-IR) was further applied to characterize the modification of carrageenan on the GO-PEI substrate. As shown in Figure 1g, for GO, the peak around 1728 cm-1 corresponds to the C=O stretching vibration of the carboxylic group. After PEI was self-assembled on the GO surface, the C=O stretching vibration almost disappeared, and new peaks at 2928 cm-1 and 2855 cm-1 ascribing to C-H stretch appeared, confirming the successful self-assembly33. Finally, after introducing carrageenan, some new peaks appeared because carrageenan has additional oxygen atoms attached to the sulphate (O=S=O) functional group. The bands at 1223 cm1 and 844 cm-1 represent the O=S=O symmetric vibration and C4-O-S stretching vibration respectively3.

Figure 1. Characterization of materials. a: XPS image of GO-PEICarr. b: S2p spectra of GO-PEI-Carr. c: SEM image of GO. d: SEM image of GO-PEI. e: SEM image of GO-PEI-Carr. f: EDX spectra of GO-PEI-Carr. g: Fourier-transform infrared spectroscopy of GO, GO-PEI, GO-PEI-Carr.

Optimization of glycopeptide enrichment with GO-PEICarr. In this work, N-glycosylated peptides were enriched specifically. The glycosylated Asn is located within a consensus sequence of N-X-S/T/C (where X can be any amino acid except proline). The N-linked glycan chains all contain a common trimannosyl-chitobiose core with one or more antennae attached to each of the two outer mannose residues. The common precursor is Glc3Man9GlcNAc2-PP-Dol which is synthesized by an evolutionary highly conserved process in the ER 34. Compared to nonglycopeptides, glycopeptides have N-linked glycans with higher hydrophilicity. Herein, Human serum immunoglobulin G (IgG) was chosen to be the standard glycopeptide to establish the enrichment model. IgG is a typical N-glycosylated protein whose amino acid sequence are EEQYNSTYR and EEQFNSTFR. Their glycan composition and amino acid sequence can be seen in Supporting Information (Table S-2). Glycopeptides were enriched by centrifugation in HILIC mode, and the enrichment procedure can be seen in scheme 1. According to literature, the ratio between ACN and H2O was quite significant for the enrichment of glycopeptides, and it was also the key to find an optimized mobile phase system. Hence, different loading buffers (ACN/H2O/TFA=95:4:1, v/v/v, ACN/H2O/TFA=92:7:1, v/v/v, ACN/H2O/TFA=90:9:1, v/v/v, ACN/H2O/TFA=88:11:1, v/v/v, ACN/H2O/TFA=85:14:1, v/v/v) were inspected as loading mobile phase, and six highabundant glycopeptides were selected as markers. As displayed in Figure S-1, when using ACN/H2O/TFA=90:9:1 (v/v/v) as loading mobile phase, the signal intensities of 6 markers reached the maximum. The MALDI-TOF mass spectra of glycopeptides enriched by GO-PEI-Carr under different washing and eluent conditions were illustrated as well. In this investigation, the concentration of TFA was carefully optimized for the


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best performance to separate glycopeptides and nonglycopeptides. According to Figure S-1, washing buffers contain 0.1% TFA for nonglycopeptides separation and elute buffers contain 3% TFA for glycopeptides enrichment were the best. Before enrichment, the IgG peptides had a strong signal of nonglycopeptide which inhibited the detection of glycopeptides, and only 4 glycopeptides were detected by MALDI (Figure 2a). However, after treatment by GO-PEI-Carr, 34 glycopeptides were obviously observed with greatly improved signal-to-noise (S/N) ratio (Figure 2b). The nonglycopeptides were completely separated which signified that a least intervention to the detection of glycopeptides was effectively achieved. Scheme 1. Demonstration of glycopeptides enrichment from protein digestion using GO-PEI-Carr.

nanocomposites. These experiments showed that GO-PEI-Carr exhibited a unique repeatability and stability, which are much more than most published papers35-37.

Figure 2. Mass spectra of the tryptic digest of IgG. a: direct analysis, before enrichment. b: after enrichment by GO-PEI-Carr. Arabic numerals indicate glycopeptides. Each number relative to a glycopeptide. The peptide information is in Table S-2.

The enrichment capacity of the GO-PEI-Carr towards glycopeptides was also investigated by adding different amounts of nanocomposites to a fixed amount of IgG (6 µg). Then, the elutes were analyzed by MALDI with a certain laser intensity. The intensity of six high-abundant glycopeptides increased first and after that it had an approximate constant with the increase of nanocomposites. As shown in Figure S-3, the loading capacity can be calculated to ~300 mg/g for glycopeptides, which was higher than that of many published HILIC materials25, 38, 39. In addition, the recovery was measured by using the quantitative approach of stable isotope dimethyl labeling. The massto-charge ratio (m/z) of the two deglycosylated peptides was 1158.5 and 1190.5. After labeling by dimethyl groups and deuterated dimethyl groups, the mass-to-charge ratio (m/z) of the former peptide increased to 1186.6 and 1190.6. And the latter increased to 1218.5 and 1222.5. The spectrums of these peptides can be seen in Figure S-4. The recovery rate was 90.8% and 109.5% which was reached by calculating the ratio between the signal of heavy hydrogen and light hydrogen (Table 2). Table 2. Recovery of two deglycosylated peptides from human IgG tryptic digest after enriched by GO-PEI-Carr.

Investigation of enrichment performances. The detection limit is another key performance criterion to evaluate the enrichment performance of affinity materials. In this experiment, different concentrations of IgG tryptic digest (5 fmol/µL and 1fmol/µL) were used as samples (Figure 3a and 3b). The results suggested that the signals of 9 glycopeptides could still be well detected even when the concentration of IgG digest was as low as 1 fmol/µL. Material repeatability was investigated to detect whether the enrichment results repeated after multiple enrichment with the same composite. In this experiment, 500 ug nanocomposites were used to enrich glycopeptides circularly for 20 times. All the mass figures were analyzed by the Data Explorer (TM) Software with an optimized condition. Herein, the resolution of peak was set to 0.95 and the signal to noise ratio (S/N) was bigger than 3. The relationship between the number of glycopeptides and the reused times can be seen in Figure S-2. And the mass figures of reusing 17 and 20 times can be seen in Figure 2c and d. There were 30 glycopeptides when enriched by 17 times. After enriched 18 times, the number of glycopeptides were less than 30, due to the increasingly reduction of

Ratio (%) D/H1 D/H2 D/H3 Average recovery


EEQFN#STFR (m/z=1158.5) 90.5 93.2 88.6 90.8±2.4

EEQYN#STYR (m/z=1190.5) 106.9 112.8 108.7 109.5±3.3


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Figure 3. The study of detection limit. a: 5 fmol/µL. b:1 fmol/µL. The study of composite repeatability. c: enrich IgG 17 times. d: enrich IgG 20 times. Arabic numerals indicate glycopeptides. Each number relative to a glycopeptide. The peptide information is in Table S-2.

In order to prove that carrageenan did increase the selectivity. GO, GO-PEI and GO-PEI-Carr were used to enrich the IgG and BSA tryptic digest mixture at a molar ratio of 1:50. In the complex systems, we also optimized the enrichment conditions especially the concentration of ACN in eluent buffer (Figure S-5). As shown in Figure S-6, GO had some hydrophilic groups. But it failed to obtain an ideal selectivity. Only 15 glycopeptides could be detected. After modified by PEI, 20 glycopeptides were detected with a proved selectivity. However, there are 31 glycopeptides detected by GO-PEI-Carr with an excellent selectivity and greatly improved S/N ratio. Compared Figure S6b to Figure S-6c, we concluded that the introducing of carrageenan took an active role in glycopeptides enrichment. In order to further explore the difference between GO-PEI and GO-PEICarr, the two composites were used to enrich the IgG and BSA tryptic digest mixture at a molar ratio of 1:100. In this case, the enrichment selectivity of the two composites varied dramatically. As shown in Figure 4a and Figure 4b, while GO-PEI only enriched 5 glycopeptides, GO-PEI-Carr still enrich 26 glycopeptides with a good selectivity. Even when the molar ratio of IgG and BSA tryptic digest mixture reached to 1:500, 19 glycopeptides with a good selectivity also obtained by GO-PEI-Carr (Figure 4c). It may have several reasons that GO-PEI-Carr showed a better enrichment performance than GO-PEI. First, after combined with carrageenan, the number of functional groups on composites grew rapidly. Second, according to the zeta potential measurements, GO-PEI had a strong positive charge (+48.90 mv). So, the repulsive power between GO-PEI and glycopeptides was very intense. After modified by carrageenans, the value of zeta potential of GO-PEI-Carr was decreased to +6.76 mv. In this condition, the electrostatic repulsion was weak, and thus composite had a much better capacity to identify glycopeptides. The selectivity of nanocomposites was as good as some other HILIC material16, 25, 33.

Figure 4. IgG and BSA tryptic digest mixture at a molar ratio of 1:100. a: after enriched by GO-PEI. b: after enriched by GO-PEICarr. IgG and BSA tryptic digest mixture at a molar ratio of 1:500. c: after enriched by GO-PEI-Carr. Arabic numerals indicate glycopeptides. Each number relative to a glycopeptide. The peptide information is in Table S-2.

Glycopeptides enrichment from the real biological sample. Inspired by the above advantages of GO-PEI-Carr for


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Analytical Chemistry capacity of GO-PEI-Carr; Investigation of enrichment recovery of GO-PEI-Carr; Enrich glycopeptides from IgG and BSA tryptic digest mixture (at a molar ratio of 1:50); Effect of ACN concentration on glycopeptide enrichment with different systems; Detailed information of glycopeptides obtained from human serum extracts and mouse liver tissues extracts. (PDF)

glycopeptide enrichment, such as good selectivity, excellent repeatability and high sensitivity, several biological samples were used to further detect the enrichment performance. Human serum is the most popular sample which contains abundance information regarding diseases and physiological and pathological conditions of the whole body40. The analysis of glycoproteins in serum substantially more difficult than that of whole-cell or other tissue samples, because 22 highly abundant proteins constitute 99% of serum proteins, while the remaining 1% contains millions of proteins, the abundances of which span more than 10 orders of magnitudes. In this work, 76 glycopeptides derived from 56 glycoproteins with 83 N-glycosylation sites were detected by nano LC-MS from human serum (Table S-3). The mouse liver tissue is another real sample whose glycopeptides is site-specific N- and O-glycosylation. There are different sialic acid variants in both N- and O-linked glycans41. The saccharides of glycopeptides possess highly variable glycan structures. And the differences between saccharides are often slight. This makes the development of synthetic receptors for saccharide discrimination very difficult. In this work, 149 glycopeptides derived from 129 glycoproteins with 157 N-glycosylation sites were detected by nano LC-MS (Table S-4).

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Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected].

ORCID Minbo Lan: 0000-0002-4247-0006

Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (21804041), the Natural Science Foundation of Shanghai (16ZR1407600), and the Shanghai Sailing Program (16YF1402400).

CONCLUSIONS

To sum up, carrageenan functionalized graphene hydrophilic nanocomposite (GO-PEI-Carr) was synthesized as a new HILIC material via a facile surface modification strategy. The preparation method was easy, fast, effective, and safe. Combined good specific surface area of graphene, efficient compatibility response of PEI and excellent hydrophilicity of carrageenan, GO-PEI-Carr nanocomposite showed good application prospects in enrichment of glycopeptides. 34 glycopeptides were obviously observed with greatly improved signal-to-noise (S/N) ratio. When IgG and BSA tryptic digest mixture was at a molar ratio of 1:500, GO-PEI-Carr still enrich 19 glycopeptides with high selectivity. GO-PEI-Carr showed some potentials in glycopeptides enrichment and glycosylation analysis form complex real samples. There were 76 glycopeptides derived from 56 glycoproteins with 83 N-glycosylation sites were identified from only 2 μL of human serum and 149 glycopeptides derived from 129 glycoproteins with 157 N-glycosylation sites were identified from 50 μL mouse liver tissues. In addition, GO-PEICarr also exhibited an excellent repeatability (20 times) and a high sensitivity (1 fmol/μL IgG) for glycopeptides enrichment. The enrichment capacity for glycopeptides was up to ~300 mg/g. And the enrichment recovery was 90.8% and 109.5%. Owing to these good performances, we believe that carrageenan will be well suitable for future applications in the isolation and identification of low abundance glycopeptides biomarkers.

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AUTHOR INFORMATION

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REFERENCES

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ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:. The surface chemical composition of GO-PEI-Carr measured by XPS; Detailed information of N-glycopeptides from IgG tryptic digest; The effect of different loading buffers, washing buffers and eluent buffers; The investigation of the repeatability of GO-PEI-Carr; Evaluation of the loading



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Characterization of materials. a: XPS image of GO-PEI-Carr. b: S2p spectra of GO-PEI-Carr. c: SEM image of GO. d: SEM image of GO-PEI. e: SEM image of GO-PEI-Carr. f: EDX spectra of GO-PEI-Carr. g: Fouriertransform infrared spectroscopy of GO, GO-PEI, GO-PEI-Carr. 83x96mm (300 x 300 DPI)


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Mass spectra of the tryptic digest of IgG. a: direct analysis, before enrichment. b: after enrichment by GOPEI-Carr. Arabic numerals indicate glycopeptides. Each number relative to a glycopeptide. The peptide information is in Table S-2. 83x80mm (300 x 300 DPI)



The study of detection limit. a: 5 fmol/µL. b:1 fmol/µL. The study of composite repeatability. c: enrich IgG 17 times. d: enrich IgG 20 times. Arabic numerals indicate glycopeptides. Each number relative to a glycopeptide. The peptide information is in Table S-2. 152x72mm (300 x 300 DPI)


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IgG and BSA tryptic digest mixture at a molar ratio of 1:100. a: after enriched by GO-PEI. b: after enriched by GO-PEI-Carr. IgG and BSA tryptic digest mixture at a molar ratio of 1:500. c: after enriched by GO-PEICarr. Arabic numerals indicate glycopeptides. Each number relative to a glycopeptide. The peptide information is in Table S-2. 83x119mm (300 x 300 DPI)



Demonstration of glycopeptides enrichment from protein digestion using GO-PEI-Carr. 83x78mm (600 x 600 DPI)


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83x27mm (600 x 600 DPI)


A hydrophilic nanocomposite functionalized by carrageenan for the

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