Hydrophilic maltose-modified magnetic metal-organic framework for highly efficient enrichment of N-linked glycopeptides

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Hydrophilic maltose-modified magnetic metal-organic framework for highly efficient enrichment of N-linked glycopeptides Junyu Lu a,b, Jingyi Luan a, Yijun Li a,c, Xiwen He a, Langxing Chen a,∗, Yukui Zhang a,d a

College of Chemistry, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Nankai University, Tianjin 300071, China College of Chemistry and Biology Engineering, Hechi University, Yizhou 546300, China c National Demonstration Center for Experimental Chemistry Education (Nankai University), Tianjin 300071, China d Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, China b

a r t i c l e

i n f o

Article history: Received 12 October 2019 Revised 26 November 2019 Accepted 29 November 2019 Available online xxx Keywords: Maltose Metal-organic framework Magnetic Fe3 O4 Au nanoparticles Enrichment Glycopeptides

a b s t r a c t Biomedical sciences, and in particular disease biomarker research, demand highly selective and efficient glycoproteins/peptides enrichment platforms. In this work, a facile strategy to prepare hydrophilic maltose-functionalized magnetic metal-organic framework loaded with Au nanoparticles (denoted as magMOF@Au-maltose) for highly efficient enrichment of N-linked glycopeptides. In brief, carboxylfunctional Fe3 O4 nanospheres were firstly coated with a Zr-based MOF shell, the resulting MOF was then loaded with Au nanoparticles in situ and then modified with thiol-functional maltose via Au−S bonds to obtain magMOF@Au-maltose with core-shell structure. The physical property and adsorption of magMOF@Au-maltose to glycopeptides were investigated. The results showed magMOF@Au-maltose possessing the outstanding performance in glycopeptides enrichment with high selectivity (1:200, mass ratio of horseradish peroxidase to bovine serum albumin digest), a low limit of detection (10 fmol), a high recovery (over 83.3%), and a large binding capacity (83 μg•mg−1 ). The magMOF@Au-maltose nanocomposite can enrich 24 and 32 glycopeptides from tryptic HRP and human IgG digests, respectively. Moreover, the nanocomposite was applied to the selective enrichment of glycopeptides from the complex biological samples and a total of 123 unique N-glycosylation sites were identified from 113 glycopeptides in 1 μL of human serum, which were assigned to 46 different glycoproteins. These results showed the promising application of magMOF@Au-maltose in the detection and identification of low-abundance N-linked glycopeptides in complex biological samples. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Protein glycosylation, one of most common but complex posttranslational modifications plays a crucial role in many fundamental biological processes, including signal transduction, protein folding, immune system response, molecular recognition, cell proliferation, differentiation, migration, and host−pathogen interactions [13]. Alterations and aberrations in glycosylation have been proved to be associated with many human diseases especially cancers [4,5]. Thus, identifying the structures and glycosylation sites of glycoproteins is necessary to better understand disease mechanisms and enable early diagnosis [6]. Mass spectrometry (MS) technique has been proven to be a powerful tool for analyzing protein glycosylation. However, the low abundance of glycoproteins in the complex biological samples, and strong ion suppression of non-

∗

Corresponding author. E-mail address: [email protected] (L. Chen).

glycopeptides, and the heterogeneity of glycosylation sites make it difficult to identify glycosylation sites [7]. Therefore, it is indispensable to develop an effective and sensitive platform for glycopeptides enrichment from complex samples prior to MS analysis. Up to now, various strategies such as hydrazine chemistry [8,9], boronate affinity materials [10-12], lectin affinity chromatography [13], immobilized metal ion chromatography [14], and hydrophilic interaction liquid chromatography (HILIC) [15,16] have been made for glycopeptides enrichment. Among these strategies, the HILICbased approach, which features many advantages with MS analysis, such as mild enrichment conditions, high enrichment efficacy, high selectivity, broad glycan adaptability, and excellent MS compatibility, has been proven to be a promising method for glycopeptide enrichment [17]. A number of HILIC-based materials including maltose [18-20], zwitterionic polymers [21-23], cellulose [24], and chitosan [25] with an abundant amount of polar functional groups (amine and hydroxyl groups) have been developed for glycopeptides enrichment. The enrichment efficiency of HILIC-based materials mainly depends on hydrophilicity and the amounts of func-

https://doi.org/10.1016/j.chroma.2019.460754 0021-9673/© 2019 Elsevier B.V. All rights reserved.

Please cite this article as: J. Lu, J. Luan and Y. Li et al., Hydrophilic maltose-modified magnetic metal-organic framework for highly efficient enrichment of N-linked glycopeptides, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460754

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tional groups immobilized on the surface of materials. Therefore, it is a great demand to develop novel HILIC-based materials with the above properties for efficient enrichment of glycopeptides. Metal-organic frameworks (MOFs) are a novel class of porous crystalline materials built from the coordination of metal ions or clusters with a variety of organic ligands. Owing to their large specific surface area, tunable pore size, and facile modification, MOFs have attracted great attention in the area of sample preparation [26]. Since Yan and co-workers firstly reported that MOFs (MIL53, MIL-100 and MIL-101) could be applied to the enrichment of low-abundance peptides in 2011 [27], MOFs have been increasingly utilized in proteomics research [3,28]. However, the majority of MOFs was constructed by choosing organic ligands containing hydrophilic groups such as amino group, carboxyl group and sulfonic acid group and metal ions during MOFs synthesis [29,30], it has become a major limitation of MOFs’ application in sample pretreatment. Recently, post-modification of MOFs as a significant approach for preparation of functionalized networks after the synthesis of MOFs has gained a wide attention. In the past few years, some HILIC-based MOFs were obtained by postmodification with hydrophilic substances and applied for glycopeptides enrichment. For example, Bai et al. described the synthesis of a maltose-functionalized MOF (MIL-101(Cr)-maltose) via a simple two step post-synthetic modification of MIL-101(Cr)-NH2 [31]. They also synthesized a hydrophilic cysteine-functionalized MOF via the in situ loading of Au nanoparticles as linkers of l-cysteine immobilization [32]. As the direct use of functionalized MOFs as a sorbent for glycopeptides enrichment, centrifugation at high speed in the traditional is usually required, which is often time consuming and leads to sample loss during the extraction process [33]. To meet the requirement of fast separation and minimum sample loss, some magnetic MOFs containing hydrophilic groups were designed and synthesized recently [34,35]. Hence, the development of original magnetic MOF nanocomposite via a facile synthetic route for rapid separation and superior enrichment efficiency of glycopeptides is still a research hotspot until now. Herein, a novel HILIC maltose-functionalized magnetic metalorganic framework (denoted as magMOF@Au-maltose) was prepared by immobilization of thiol-functional maltose on Au nanoparticles via the robust Au−S bond. Firstly, a Zr-based MOF (UiO-66-NH2 ) was coated on the surface of carboxyl-functional Fe3 O4 nanospheres, the resulting MOF was then in situ loaded with Au nanoparticles (NPs) and grafting thiol-functionalized maltose via the robust Au−S bond. The large amount of loading Au NPs would improve the immobilization number of maltose and, in turn, the hydrophilicity of the synthesized nanocomposites, thus favor the highly efficient enrichment of glycopeptides. The prepared magMOF@Au-maltose with high hydrophilicity, strong magnetic responses, and excellent reusability exhibited highly sensitive and selective enrichment of glycopeptide from tryptic horseradish peroxidase (HRP) and human serum immunoglobulin G (human IgG) digests, as well as complex biological sample like human serum. 2. Experimental 2.1. Reagents and materials HRP, human IgG, bovine serum albumin (BSA), and peptide-Nglycosidase (PNGase F) were purchased from Sigma-Aldrich (USA). Trypsin was obtained from Sangon Biotech Co. Ltd. (China). Human serum was obtained from Tianjin First Central Hospital (Tianjin, China). Trifluoroacetic acid (TFA), HAuCl4 •3H2 O, cyanoborohydride, boron trifluoride diethyl etherate (BF3 •Et2 O), tetraethylene glycol (TEG), and Amberlite IR120 were obtained from J&K (China). 2,5-Dihydroxybenzoic acid (DHB) was obtained from TCI (Japan). Dithiothreitol (DTT) and iodoacetamide (IAA) were obtained from

Fig. 1. The strategy for (A) preparation of MagMOF@Au-maltose and (B) glycopeptides enrichment.

Solarbio. Zirconium tetrachloride (ZrCl4 ), 2-aminoterephthalic acid (H2 BDC–NH2 ), sodium borohydride (NaBH4 ), potassium thioglycolate (KSAc), and mercaptoacetic acid (MAA) were obtained from Alfa Aesar (Shanghai, China). D-(+)-maltose, ammonium bicarbonate (NH4 HCO3 ), anhydrous sodium acetate (NaAc), and sodium methanolate were purchased from Aladdin (Shanghai, China). Iron(III) chloride hexahydrate (FeCl3 •6H2 O), anhydrous sodium sulfate (Na2 SO4 ), ethylene glycol (EG), deuterated chloroform (CDCl3 ), dimethyl sulfoxide (DMSO), acetic anhydride (Ac2 O), tosyl chloride (TsCl), N,N-dimethylformamide (DMF), dichloromethane (CH2 Cl2 ), tetrahydrofuran (THF), ethyl acetate, n-hexane, formic acid, ethanol, ammonium solution (25 wt%), HPLC-grade acetonitrile (ACN), and methanol (MeOH) were obtained from Tianjin Chemical Reagent Company (Tianjin, China). Ultrapure water (18.25 M cm) was used for all experiments and purified by an Aquapro Ultrapure Water System (Chongqing, China). 2.2. Characterization Fourier transform infrared (FT-IR) spectra were recorded by a Bruker Tensor 27 FT-IR spectrometer using KBr pellets. Powder X-ray diffraction (XRD) patterns were collected on a Rigaku D/max/2500 X-ray diffractometer with a Cu Kα source. The morphological features of the as-prepared nanocomposites were determined by a JEOL JEM-2100 EX transmission electron microscope (TEM; Japan). X-ray photoelectron spectra (XPS) were recorded on a ESCALAB 210 electron spectrometer. Thermogravimetric analysis (TGA) was conducted on an Exstar TG/DTA 6300. Water contact angle analysis was performed on a OCA 20 instrument (Dataphysics, Germany). 1 H NMR spectra were obtained on a Bruker AV-400 spectrometer. MALDI-TOF MS analysis was carried out on a Bruker AutoflexIII LRF200-CID instrument (Bruker, Germany). Herein, 1 μL of elution was dropped on a MALDI plate, air-dried, added with 1 μL of DHB matrix solution (25 mg•mL−1 , ACN/H2 O/TFA, 70/29.9/0.1, v/v/v), and then dried for MS analysis. MALDI MS spectra were acquired in reflector positive-ion mode. Nano-LC-MS/MS experiments were performed on an EASY-nLC 10 0 0 system (Thermo Fisher Scientific, Waltham, MA, USA) connected to an Orbitrap Q-Exactive mass spectrometer. 2.3. Synthesis of magMOF@Au-maltose nanocomposite As a hydrophilic substance, thiol-functionalized maltose (maltose−SH) was synthesized according to the procedure described in previous literature [36] (see the Supplementary Material). The magMOF@Au-maltose nanocomposite was fabricated as shown in Fig. 1A. Firstly, Fe3 O4 nanospheres were prepared via a solvothermal reaction [37] as previously reported. Then, 100 mg of Fe3 O4 nanospheres was dispersed in 40 mL of ethanol

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Fig. 2. TEM images of (A) magMOF and (B) magMOF@Au-maltose nanocomposite.

containing 50 mg of MAA, and the mixture was sonicated for 10 min and vibrated for 12 h at 25 °C. The MAA-modified Fe3 O4 nanospheres were collected, washed thrice with ethanol, and then dried in vacuo at 50 °C. Subsequently, the MAA-modified Fe3 O4 nanospheres (50 mg) were added to a solution of ZrCl4 (70 mg) in DMF (40 mL) and stirred for 1 h. A solution of H2 BDC–NH2 (54.4 mg) in DMF (10 mL) was then added to this mixture. After sonication for 15 min, the homogenous mixture was placed in a 100 mL Teflon-lined stainless-steel autoclave and kept at 120 °C for 8 h. The obtained product was washed with DMF, then redispersed in a solution containing ZrCl4 and H2 BDC–NH2 in the concentrations as described above. After three cycles, the resulting product was washed several times with DMF and ethanol, and then dried under vacuum at 50 °C. 50 mg of magMOF was added to a solution of HAuCl4 (15 mM) in ethanol/water (1:1, v/v; 30 mL) and agitated in an ice-bath for 1 h. Then, 5 mL of NaBH4 (90 mM) solution was added to the mixture, which was subsequently stirred for 30 min. The product (magMOF@Au) was washed thrice with ethanol and water, added to a solution of maltose−SH (150 mg) in ethanol/water (1:2, v/v; 20 mL), and then shaken for 16 h at 25 °C. Finally, the magMOF@Au-maltose nanocomposites were washed thrice with ethanol, and dried in vacuo for use. 2.4. Sample preparation 1 mg of standard proteins (i.e., HRP, IgG, or BSA) was dissolved in 0.5 mL of 25 mM NH4 HCO3 (pH 8.0) and denatured at 95 °C for 10 min. The proteins were then reduced by 10 mM DTT at 56 °C for 1 h and alkylated in 20 mM IAA at 37 °C in the dark for 45 min. The samples were incubated with trypsin (trypsin:protein, 1:40, w/w) at 37 °C for 16 h. Finally, 2 μL of formic acid was added to the mixed solution to terminate the reaction. The solution was diluted with 25 mM NH4 HCO3 for further analysis. The tryptic digests were stored at −20 °C before use. 1 μL of human serum was diluted with 199 μL of 25 mM NH4 HCO3 and denatured at 95 °C for 10 min to obtain the human serum digest. The mixture was reduced by using DTT at 56 °C for 45 min and alkylated by IAA at 37 °C for 1 h. After that, the mixture was digested with trypsin (trypsin:protein, 1:40, w/w) at 37 °C for 16 h. Finally, the tryptic digest was lyophilized and stored at −20 °C until use. 2.5. Glycopeptides enrichment by magMOF@Au-maltose 60 μg of magMOF@Au-maltose was dispersed in 50 μL of loading buffer (ACN/H2 O/TFA, 88/11.7/0.3, v/v/v). Then, 50 μL of sample solution containing the proper concentration of HRP or IgG digest was added to the mixture, and was shaken for 30 min at room temperature. The nanocomposites were rinsed thrice with

100 μL of loading buffer to remove non-glycopeptides. Finally, the extracted glycopeptides were eluted for 6 min by using 3 × 50 μL elution buffer (ACN/H2 O/TFA, 30/69.9/0.1, v/v/v). The elutes were lyophilized, re-dissolved in 2 μL of elution buffer, and analyzed by MALDI-TOF MS. Glycopeptide enrichment of human serum was also conducted. The above-mentioned lyophilized sample was redissolved in 100 μL of loading buffer (ACN/H2 O/TFA, 88/11.7/0.3, v/v/v) and mixed with 300 μg of magMOF@Au-maltose. Glycopeptide enrichment was carried out as described above. The obtained eluent was lyophilized, dissolved in 20 μL of 25 mM NH4 HCO3 solution (pH 8.0), incubated with 10 0 0 units PNGase F at 37 °C for 16 h, lyophilized once more, re-dissolved, and desalted for nano-LCMS/MS analysis. 2.6. Recovery evaluation of glycopeptide enrichment Two groups of IgG (20 μg) digest were individually labeled with light and heavy isotopes. Thereafter, the heavy isotope-tagged IgG digest was enriched by magMOF@Au-maltose as described above. The collected elute was lyophilized, spiked into the light isotope-tagged group, and then enriched as described above. The elute was incubated with PNGase F, and the resulting deglycosylated peptides were analyzed by MALDI-TOF MS. Recovery was defined as the ratio between the signal intensity of the heavy isotope- and light isotope-tagged deglycosylated peptides. 3. Results and discussion 3.1. Synthesis and characterization of the as-prepared magMOF@Au-maltose The magMOF@Au-maltose nanocomposite was synthesized via a facile synthetic route, as shown in Fig. 1A. Briefly, UiO-66-NH2 was coated onto the surface of carboxyl-functional Fe3 O4 nanospheres with core-shell structure. The resulting magMOF containing amino groups was coordinated with HAuCl4 through electrostatic adsorption. Au NPs were grown in situ on the surface of MOF by using NaBH4 as a reductive agent to get magMOF@Au. Finally, maltose– SH was bonded to the surface of magMOF@Au by Au−S bonds. The high density of Au NPs not only improved the hydrophilicity and biocompatibility of the nanocomposites but also favored the anchoring of an abundance of hydrophilic maltose, thereby remarkably promoting excellent performance for glycopeptides enrichment is anticipated. The TEM images of magMOF and magMOF@Au-maltose are presented in Fig. 2. The obtained magMOF and magMOF@Au-maltose exhibit a spherical shape with a typical core-shell structure. The dark Fe3 O4 core is coated by a MOF shell, and the thickness of

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Fig. 3. (A) FT-IR spectra of (a) maltose-SH, (b) magnetic Fe3 O4 , (c) magMOF, and (d) magMOF@Au-maltose. (B) XRD patterns of (a) magMOF and (b) magMOF@Au-maltose. (C) XPS spectra of magMOF@Au-maltose.

Fig. 4. Water contact angles of (A) magnetic Fe3 O4 , (B) magMOF@Au, and (C) magMOF@Au-maltose.

Fig. 5. MALDI-TOF spectra of 1 pmol•μL

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the uniform coating is about 30 nm (Fig. 2A). As can be seen from Fig. 2B, the Au NPs are distributed homogeneously on the magMOF substrate. FT-IR was employed to investigate the coating of MOF layer on the Fe3 O4 and two step post-modification of UiO-66-NH2. Fig. 3A showed the FT-IR spectra of maltose–SH, magnetic Fe3 O4 , magMOF, and magMOF@Au-maltose. Absorption peaks at 586 cm−1 are attributed to the Fe−O stretching vibrations of Fe3 O4 (curve b-d). A broad peak at 3436 cm−1 and a sharp peak at 1620 cm−1 are also observed, these peaks could be assigned to N–H and C = =O stretching vibrations in UiO-66-NH2 (curve c). After immobiliza-

HRP digest (A) before and (B) after enrichment by magMOF@Au-maltose. Glycopeptide peaks are labeled with ∗ .

Please cite this article as: J. Lu, J. Luan and Y. Li et al., Hydrophilic maltose-modified magnetic metal-organic framework for highly efficient enrichment of N-linked glycopeptides, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460754

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Fig. 6. MALDI-TOF spectra of 100 fmol•μL

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IgG digest (A) before and (B) after enrichment by magMOF@Au-maltose. Glycopeptide peaks are labeled with ∗ .

Fig. 7. MALDI-TOF spectra of 100 fmol•μL−1 HRP after enrichment by (A) magnetic Fe3 O4 , (B) magMOF, (C) magMOF@Au, and (D) magMOF@Au-maltose. Glycopeptide peaks are labeled with ∗ .

tion of maltose–SH on the surface of magMOF, compared with the spectrum of magMOF, the new adsorption peaks at 3420 and 2920 cm−1 appeared in the spectrum of magMOF@Au-maltose, which originate from –OH and C–H stretching vibrations in maltose–SH (curve a and curve d). These results proved that the hydrophilic maltose has been successfully immobilized on the surface of magMOF through Au NPs linker. The XRD patterns of magMOF and magMOF@Au-maltose are shown in Fig. 3B. The diffraction profiles obtained are consistent with those of UiO-66-NH2 [38] and Fe3 O4 (JCPDS Card 19– 0629). New characteristic peaks at 38.3° (labelled by 111), 44.4° (200), 64.5° (220), and 77.5° (311) are attributed to the Au NPs and matched well with the XRD spectrum of crystalline Au (JCPDS Card 01–1172). The evidence proves that Au NPs are anchored on

the surface of magMOF and provide numerous binding sites for maltose–SH immobilization. The thermal stability of magMOF and magMOF@Au-maltose was investigated by TG analysis (Figure S1, Supplementary Material). The weight loss of magMOF@Au-maltose proves that maltose–SH is bonded to the surface of magMOF@Au. In addition, X-ray photoelectron spectroscopy (XPS) was used to explore the surface composition of magMOF@Au-maltose. The peaks corresponding to C, O, N, S, Fe, Zr, and Au were found in the XPS survey spectra of magMOF@Au-maltose (Fig. 3C). Both peaks at 81.5 and 85.2 eV in Figure S2a (Supplementary Material) were attributed to Au 4f7/2 , and Au 4f5/2 spin-orbital splitting photoelectrons, respectively [38, 39]. The peak located at 161.5 eV was observed in the S 2p core-level and is mainly due to the formation

Please cite this article as: J. Lu, J. Luan and Y. Li et al., Hydrophilic maltose-modified magnetic metal-organic framework for highly efficient enrichment of N-linked glycopeptides, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460754

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Fig. 8. MALDI-TOF spectra of N-glycopeptides enriched from different concentrations of HRP digests using magMOF@Au-maltose: (A) 100 fmol•μL (C) 1 fmol•μL−1 , and (D) 0.1 fmol•μL −1 . Glycopeptide peaks are labeled with ∗ .

of the Au−S bond (Figure S2b, Supplementary Material). The highresolution spectra of C 1 s showed three obvious peaks at 284.6, 286.2, and 288.1 eV (Figure S2c, Supplementary Material), which could reflect the existence of C − C, C − O, and C = =O groups, respectively. In Figure S2d (Supplementary Material), deconvolution of the O 1 s spectrum of the nanocomposite revealed two peaks located at 528.2 and 530.1 eV, which are indexed to C − O and O − H groups, respectively. The results prove the successful preparation of magMOF@Au-maltose. Contact angle analysis was carried out to evaluate the hydrophilicity of the as-synthesized nanocomposites. As can be seen in Fig. 4, the water contact angles of magnetic Fe3 O4 , magMOF@Au, and magMOF@Au-maltose are 26.8°, 19.3°, and 15.9°, respectively. Water contact angles obviously decreased after loading of the nanocomposite with Au NPs and maltose−SH onto the surface of magMOF, thereby demonstrating that the self-assembly process of maltose−SH is successful and significantly improves the hydrophilicity of the nanocomposites, such features are beneficial for the enrichment of glycopeptides. 3.2. Glycopeptide enrichment from standard protein digests by magMOF@Au-maltose The procedure of magMOF@Au-maltose nanocomposite in glycopeptides enrichment from complex samples was illustrated in Fig. 1B, and the enrichment procedure mainly included loading, washing, elution, and MS determination. To investigate the enrichment performance of magMOF@Au-maltose toward glycopeptides, HRP digest was selected as test sample. As shown in Fig. 5A, the mass spectra of 1 pmol•μL −1 HRP was obtained by direct MS analysis, only eight glycopeptides with low intensity were ob-

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, (B) 20 fmol•μL

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,

served respectively, because the signal responses of the glycopeptides were seriously suppressed by the massive interference of non-glycopeptides. However, after enrichment with magMOF@Aumaltose, up to 24 glycopeptides were detected, and the signal intensity obviously increased, non-glycopeptides were efficiently eliminated from the digest (Fig. 5B). Detailed information of glycopeptide enrichment in the HRP digest by magMOF@Au-maltose is listed in Table S1 (Supplementary Material). Furthermore, human IgG digest was also selected as test sample to investigate the universality of magMOF@Au-maltose for glycopeptides enrichment. As shown in Fig. 6A, the direct analysis of human IgG at a concentration of 100 fmol•μL −1 , only four glycopeptides were detected with low intensity due to the signal of glycopeptides were suppressed by non-glycopeptides. After being enrichment by magMOF@Au-maltose, 32 glycopeptides were clearly detected and non-glycopeptides were efficiently eliminated from the digest (Fig. 6B). The detailed information is listed in Table S2 (Supplementary Material). To demonstrate enriched peptides were glycopeptides, the glycopeptides extracted from human IgG were deglycosylated by PNGase F and detected by MALDI-TOF MS. As shown in Figure S3 (Supplementary Material), only two deglycosylated peptides (m/z = 1158.49, 1190.52) were observed, all other signals of identified glycopeptides with m/z values greater than 1500 disappeared, thus confirming that the peaks in Fig. 6B are glycopeptides. The performance of magMOF@Au-maltose was assessed by capturing glycopeptides from tryptic digests of HRP and IgG. The 24 and 32 N-linked glycans were identified from HRP and IgG digests after enrichment by magMOF@Au-maltose, respectively. As shown in Table S3 (Supplementary Material), the numbers of identified glycopeptides by magMOF@Au-maltose were

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Fig. 9. MALDI-TOF MS spectra of a mixture of HRP and BSA tryptic digests (A) before and after enrichment by magMOF@Au-maltose at different mass ratios of (B) 1:1, (C) 1:10, and (D) 1:100. Glycopeptide peaks are labeled with ∗ .

comparable to or better than those previously reported by HILIC materials such as Fe3 O4 -GO@nSiO2 -PAMAM-Au-maltose [19], MIL101(Cr)-maltose [31] and mMOF@Au@GSH [35] and boronated material, such as SPIOs@SiO2 @MOF [40], magG@PF@APB [41], APBAMCNTs [42], BTiC-Tip [43] and Fe3 O4 −MPBA [44] except that the numbers of glycopeptides enrichment were slightly less than those of enrichment by BTiC-Tip [43] and Fe3 O4 −MPBA [44]. For comparison, four different materials, including magnetic Fe3 O4 , magMOF, magMOF@Au, and magMOF@Au-maltose were applied to capture glycopeptides from 100 fmol•μL −1 HRP digest under the same conditions. As shown in Fig. 7, glycopeptides enrichment by magMOF@Au-maltose showed the higher efficiency and selectivity than that of magnetic Fe3 O4 , magMOF, and magMOF@Au, which indicates that surface-grafted maltose–SH can provide strong hydrophilic interactions and play a significant role in glycopeptides enrichment. These findings verify that the superior enrichment ability of magMOF@Au-maltose to glycopeptides is due to its outstanding hydrophilicity which is consistent with the results of water contact angle analysis. The high enrichment efficiency and sensitivity were important factor for a method to meet the requirements of ultralowabundance glycoprotein analysis in real biosamples. Thus, the sensitivity of magMOF@Au-maltose for glycopeptide enrichment was further examined with different concentration (100, 20, 1, and 0.1 fmol•μL −1 ) of HRP tryptic digests. As shown in Fig. 8, the number and signal intensity of the glycopeptides detected were gradually reduced along with the concentration of the HRP digest decreased. At a lower concentration of 100 fmol•μL −1 HRP tryptic digest, 24 N-linked glycans were identified after enrichment by magMOF@Au-maltose. When the concentration of HRP digest was decreased to 0.1 fmol•μL −1 (10 fmol), the MS signal of one glycopeptide could still be detected with a high S/N ratio after enrich-

ment (Fig. 8D). In comparison to functional MOF materials, such as MIL-101(Cr)-NH2 (20 fmol) [29], UiO-66-COOH (50 fmol) [30] and Fe3 O4 @PDA@Zr-SO3 H (10 fmol) [34], the proposed method showed similar detection sensitivity for the enrichment of glycopeptides. The results indicated that magMOF@Au-maltose can be applied to glycopeptide enrichment with high enrichment efficiency and sensitivity. The selectivity of the magMOF@Au-maltose nanocomposite for glycopeptides enrichment was examined by using different mass ratios of HRP and BSA tryptic digests as mimic a complex sample. As shown in Fig. 9, when the mass ratio of HRP:BSA was 1:100, no glycopeptide signal could be detected prior to enrichment (Fig. 9A) because of the serious interference from non-glycopeptides of BSA. However, after being enriched by magMOF@Au-maltose, 17 and 15 glycopeptide signals were identified at mass ratios of 1:1 (Fig. 9B) and 1:10 (Fig. 9C), respectively. When the mass ratio was further increased to 1:100, 11 glycopeptides could still be detected with enhanced signal intensity, and the signal of non-glycopeptides obviously decreased (Fig. 9D). After enrichment, there are still 8 peaks of glycopeptides which were clearly detected even at a mass ratio of HRP:BSA up to 1:200, while some interference of nonglycopeptides could also be observed (Figure S4, Supplementary Material). These results revealed that highly selective enrichment of glycopeptides from complicated samples could be achieved by using the proposed magMOF@Au-maltose nanocomposite. 3.3. Recovery, reusability, and binding capacity of magMOF@Au-maltose The stable-isotope dimethyl labeling method was used to investigate recovery of magMOF@Au-maltose for glycopeptides enrichment from Human IgG digest. The obtained average recoveries

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for EEQFN#STFR and EEQYN#STYR were 83.3% and 86.0% (Figure S5, Table S4, Supplementary Material), respectively. The reusability of magMOF@Au-maltose was studied by using 100 fmol•μL −1 human IgG digest. The used magMOF@Au-maltose was thoroughly washed with water and loading buffer before another enrichment cycle to eliminate residues. As shown in Figure S6 (Supplementary Material), the number and intensity of glycopeptide peaks identified after five consecutive recycles (Figure S6B) is almost the same as the third recycle (Figure S6A). There were 28 glycopeptides still clearly being detected. The ability of the nanocomposite to enrich glycopeptides kept little variation after five recycles of enrichment and elution, which indicated that magMOF@Au-maltose possesses high reliability and great reusability for glycopeptides enrichment. Meanwhile, the binding capacity of the nanocomposite was determined through enrichment of 5 μg of human IgG digest by different amounts of magMOF@Au-maltose (20–100 μg), the results is illustrated in Figure S7 (Supplementary Material). The intensity of six selected glycopeptides reached maximum when 60 μg of magMOF@Au-maltose was applied, and the binding capacity of the nanocomposite was calculated to be 83 mg•g −1 , which was higher than HILIC materials such as Fe3 O4 -DA-Maltose (43 mg•g −1 ) [18], but less than of MIL-101(Cr)-maltose (150 mg•g −1 ) [31], mMOF@Au@GSH (140 mg•g −1 ) [35], and Fe3 O4 −MPBA (150 mg•g −1 ) [44]. The large binding capacity may be ascribed to the higher amount of maltose and strong multivalent hydrophilic interactions between magMOF@Au-maltose and glycopeptides.

ternative material in the isolation and identification of N-linked glycopeptide from the complex biological samples.

3.4. Glycopeptide enrichment from human serum

References

The prepared magMOF@Au-maltose was further applied to profile glycopeptides and glycoproteins in human serum to evaluate its practical application to low-abundance glycopeptides enrichment in real biosamples. Herein, 1 μL of human serum sample was first digested with trypsin without any pre-treatment and then incubated with magMOF@Au-maltose. The eluted glycopeptides were lyophilized, deglycosylated by PNGase F, and then analyzed by nano-LC-MS/MS. A total of 123 unique N-glycosylation sites were identified in 113 glycopeptides, which were mapped to 46 different glycoproteins. Detailed information of the glycopeptide sequences, protein group accessions, and m/z data are displayed in Table S5 (Supplementary Material). The numbers of detected glycopeptides/glycoproteins from 1 μL human serum, are inferior to that of Fe3 O4 −MPBA [44]. However, compared with the results obtained by magG/PDA/Au/L-Cys [45], MIL-101(Cr)-NH2 [46], magG@PF@APB [41], and Fe3 O4 @SiO2 @PSV [47] nanocomposites, magMOF@Au-maltose showed the comparable performance in the glycopeptides enrichment with the higher sensitivity and selectivity. The results demonstrated that magMOF@Au-maltose can specifically capture the glycopeptides from the complex biological samples.

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4. Conclusions In this work, a novel HILIC material magMOF@Au-maltose with a core-shell structure was successfully fabricated via a facile synthetic route. Owing to the higher amount of maltose and strong multivalent hydrophilic interactions between magMOF@Aumaltose and glycopeptides, magMOF@Au-maltose was applied for the enrichment and identification of glycopeptides from HRP and IgG digests as well as complex biological sample such as human serum with high selectivity (1:200), a low limit of detection (10 fmol), a high recovery rate (over 83.3%), and a large binding capacity (83 μg•mg −1 ). Moreover, the magMOF@Au-maltose revealed rapid magnetic responses and excellent reusability. All the above results indicate that magMOF@Au-maltose may be an efficient al-

Conflict of Interest The authors declare that they have no conflicts of interest. CRediT authorship contribution statement Junyu Lu: Conceptualization, Validation, Investigation, Writing - original draft. Jingyi Luan: Investigation. Yijun Li: Resources, Formal analysis. Xiwen He: Validation, Supervision. Langxing Chen: Conceptualization, Funding acquisition, Project administration, Writing - review & editing. Yukui Zhang: Supervision. Acknowledgements This work was supported by the National Key Research and Development Program of China (No.2018YFC1602401), the National Natural Science Foundation of China (No. 21475067) and the Fundamental Research Funds for the Central Universities. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2019.460754.

Please cite this article as: J. Lu, J. Luan and Y. Li et al., Hydrophilic maltose-modified magnetic metal-organic framework for highly efficient enrichment of N-linked glycopeptides, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460754

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Please cite this article as: J. Lu, J. Luan and Y. Li et al., Hydrophilic maltose-modified magnetic metal-organic framework for highly efficient enrichment of N-linked glycopeptides, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460754