Cysteine-Functionalized MetalâOrganic Framework - ACS Publications

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Cysteine-functionalized metal-organic framework: facile synthesis and high efficient enrichment of N-linked glycopeptides in cell lysate Wen Ma, Linnan Xu, Xianjiang Li, Sensen Shen, Mei Wu, Yu Bai, and Huwei Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 24 May 2017 Downloaded from http://pubs.acs.org on May 26, 2017

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ACS Applied Materials & Interfaces

Cysteine-Functionalized Metal-Organic Framework: Facile Synthesis and High Efficient Enrichment of N-Linked Glycopeptides in Cell Lysate Wen Ma,a Linnan Xu,a Xianjiang Li,b Sensen Shen,a Mei Wu,a Yu Bai*a and Huwei Liua a

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry

and Molecular Engineering of Ministry of Education, College of Chemistry, Peking University, Beijing, 100871, P. R. China. b

Division of Metrology in Chemistry and Analytical Science, National Institute of Metrology,

Beijing 100029, P. R. China. ABSTRACT: Cysteine-functionalized metal-organic framework (MOF) was synthesized via a common and facile two-step method of in situ loading of Au nanoparticles on amino-derived MOF followed by L-Cysteine immobilization. Owing to the large specific surface area and ultrahigh hydrophilicity of this nanocomposite, excellent performance was observed in the enrichment of N-linked glycopeptides in both model glycoprotein and HeLa cell lysate. By using this nanocomposite, 16 and 31 glycopeptides were efficiently extracted from digest of horseradish peroxidase (HRP) and human serum immunoglobulin G (IgG), respectively. The short incubation time (5 min), large binding capacity (150 mg/g, IgG digest to material), good selectivity (1:50, molar ratio of IgG and BSA digest), high recovery (over 80%) and low detection limit (1 fmol) ensure the effectiveness and robustness of MIL-101(NH2)@Au-Cys in

ACS Paragon Plus Environment

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complex HeLa cell lysate. As a result, 1123 N-glycosylation sites corresponding to 1069 Nglycopeptides and 614 N-glycoproteins were identified from the lysate. Compared with those of previously reported hydrophilic methods, it was the best result as far as our knowledge goes. This work paves a new way for fast functionalization of MOF, and also providing a novel thought in material design in sample preparation, especially in glycoproteome and related analysis. KEYWORDS: metal-organic framework, fast functionalization, glycopeptide enrichment, cell lysate, mass spectrometry 1. INTRODUCTION Protein glycosylation mediates a range of fundamental biological processes such as protein folding, receptor activation, immune response, and intracellular transport.1 Dynamic changes in glycosylation have been found to accompany Alzheimer’s disease states and various cancer progressions.2 To date, mass spectrometry (MS)-based technology is still the most common and powerful tools for the bottom-up proteomics research, however, it is largely hampered by intrinsic drawbacks in protein glycosylation analysis. Complex glycosylation heterogeneity, the low abundance and severe signal interference by non-target compounds make the enrichment procedure becoming a prerequisite prior to MS detection. Therefore, efficient sample pretreatment techniques before MS analysis have been in urgent demand for in-depth glycoproteome profiling. Hitherto, different strategies such as hydrophilic interaction liquid chromatography (HILIC)

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hydrazide chemistry,4 lectin chromatography,5 and boronic acid affinity6 have been largely carried out to fulfil the requirements in glycopeptide recognition and extraction. HILIC-based


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methods, in particular, display various merits including broad glycan specificity, remarkable reproducibility, and excellent compatibility with MS analysis. Until now, a number of HILIC nanomaterials have been explored for highly sensitive extraction of glycopeptides.7 Saccharides8 and zwitterionic9 (ZIC) groups are two kinds of commonly used functional moieties when designing HILIC-based nanocomposites. ZIC-HILIC are well-known as superhydrophilic materials via hydrophilic interaction and electrostatic force, and ZIC-materials show ultralow biofouling.10 However, to obtain more hydrophilic nanomaterials, laboursome synthetic procedures were usually required for various moieties incorporation.9,11 Such tedious synthesis steps result in the relatively low specific surface area of materials which greatly limits the extraction efficiency, thus leading to a low enrichment selectivity and poor performance in highly complex biosample application. Therefore, ultrahydrophilic nanocomposites with common and facile synthesis procedures should be of highly desirable for obtaining superior performance in target enrichment, especially in N-linked glycopeptide enrichment in complex samples. The engineering of HILIC-based nanocomposites in glycopeptide enrichment is still a hot topic. Different enrichment efficacy were observed in various substrates such as mesoporous silica,12 magnetic nanoparticles,13 sepharose14 and metal-organic frameworks (MOFs).15 Among them, MOFs stand out due to their ultrahigh surface specific area,16 tunable nanostructure cavities, high acid resistant stability and tailorable chemistry. Therefore, they have been successfully emerged in separation science as stationary phases of gas- and liquidchromatography,17 solid-phase extraction material,18 and enantioselective separation materials.19 Additionally, MOFs have been found to be an effective stabilizing matrix for metal nanoparticles (e.g. gold nanoparticles (Au NPs)) encapsulation to prevent aggregation and fusion due to their


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confined microporous structures. Such MOF-metal composites were widely applied in hydrogen storage,20 heterogeneous catalysis,21 and surface enhanced Raman spectroscopy (SERS) analysis.22 However, conventional synthetic methods including solid grinding, chemical vapor deposition, and template synthesis23 usually suffer from tedious procedures and lack of generality. Herein, low cost and hydrophilic reagent cysteine was used for fast functionalization of MOF to form MIL-101(NH2)@Au-Cys via a one-pot absorption/reduction method followed by cysteine immobilization. This work is the primary attempt of the synthesis of cysteine modified MOF-metal composite through simply two-step route followed by the innovative application in the glycoproteome profiling. Compared with other HILIC-based nanocomposites, our MIL-101(NH2)@Au-Cys share three superior characters. First, the nanomaterial synthesis is designed on a simple and rapid two-step procedures starting from MIL-101(NH2). Compare to most other nanocomposites, for instance, magnetic nanoparticle which usually require a silanization process and complex modification, this design was easy to accomplish and the whole post-functionalization procedure was time-saving.15 Second, due to the strong adsorption ability of MOF and high hydrophilicity of zwitterionic cysteine, excellent enrichment performance can be achieved both in standard glycoprotein and whole cell lysate. More importantly, the synthesis of MIL-101(NH2)@Au set up an example for encapsulating Au NPs on other amino-derived MOF and this general method has great potential in the downstream functionalization of the framework through Au-S interaction. Therefore, we expect it opens a new perspective in the design of functionalized MOF-based nanocomposites and contribute to the target separation from the complicate matrix, especially in the in-depth glycoproteome research in biosamples. 2. EXPERIMENTAL SECTION


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2.1. Materials and Chemicals. Standard glycoproteins including horseradish peroxidase (HRP), human serum immunoglobulin G (human IgG), and trypsin, acetonitrile (ACN) were from Sigma-Aldrich. 2,5-dihydroxybenzoic acid (DHB) used for MS matrix, dithiothreitol (DTT) and iodoacetamide (IAA) used for reduction and alkylation were obtained from J&K Scientific Ltd. Chloroauric acid (HAuCl4•4H2O) were from Beijing Chemical Works. L-Cysteine, chromic nitrate hydrate was from Sinopharm Chemical Reagent Co., Ltd. Formic acid was from DIKMA Technologies inc. Sodium borohydride (NaBH4) was purchased from Guangdong Guanghua Chemical Factory Co., Ltd. 2-aminoterephthalic acid was from Alfa Aesar China Chemical Co., Ltd. Peptide-N-glycosidase (PNGase F) was from New England Biolabs (Ipswich, MA, USA). Sequencing grade modified trypsin and endopeptidase Lys-C used for HeLa cell were from Promega Biotech Co., Ltd. Dulbecco’s modified Eagle’s medium (DMEM) was from Hyclone and fetal bovin serum (FBS) was from Life Technologies Co., Ltd. Tablets of complete mini EDTA-free cocktail was from Roche Diagnostics Ltd. Water used for digestion and enrichment analysis was from Wahaha Group Co., Ltd. All reagents were of analytical grade, and ACN was of HPLC grade. 2.2. Characterization. Fourier-transformed infrared spectroscope (FT-IR) characterization was measured by Bruker Tensor 27 FT-IR. The resolution of spectrum was 4 cm-1 and average spectrum was obtained from 32-time measurements. TEM images were recorded on a JEOL JEM-2100 at 200 kV. X-ray photoelectron spectroscopy (XPS) spectra were obtained from an Imaging Photoelectron Spectrometer (Axis Ultra, Kratos Analytical Ltd.). Powder X-ray diffraction (PXRD) measurement was performed using a Rigaku D/Max 2000 diffractometer with monochromatic Cu Kα radiation (λ = 1.5406 Å). All patterns were obtained at an accelerating potential of 40 kV and a tube current of 100 mA with a scanning rate of 4°/min. N2


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adsorption-desorption experiment was conducted in ASAP 2020M apparatus. MIL-101(NH2), MIL-101(NH2)@Au, MIL-101(NH2)@Au-Cys were degassed at 110 ℃

in case of

decomposition. Relative pressure was recorded from 0 to 1, and the BET surface was calculated between 0.05 and 0.2. It revealed that the surface area were 2533.6 m2 g-1, 889.8 m2 g-1 and 240.4 m2 g-1 for MIL-101(NH2), MIL-101(NH2)@Au, and MIL-101(NH2)@Au-Cys, respectively. Zeta potential measurement was conducted on Brookhaven ZetaPlus instrument at room temperature. 2.3. Synthesis of MIL-101(NH2)@Au-Cys. The synthesis of amino-derived MOF MIL101(NH2) was according to a literature.24 In brief, 1500 mg chromic nitrate hydrate and 690 mg 2-aminoterephthalic acid were dispersed in 21 mL water, the mixture was heated for 24 h under 130 ℃ in a 30 mL autoclave. The green product (200 mg) was then dispersed in 20 mL, 0.025 M HAuCl4•4H2O solution containing water and ethanol solvent (1:1, v/v) and stirred continuously in ice bath for 1 h, then NaBH4 solution (10 mL, 0.05 M) was added. The reaction was conducted in ice bath for another 0.5 h to get black product MIL-101(NH2)@Au. MIL-101(NH2)@Au-Cys was obtained through the reaction between MIL-101(NH2)@Au and cysteine solution at room temperature for 24 h. For comparison, cysteine-modified gold nanoparticles (Au-Cys NPs) were synthesized through trisodium citrate method,25 followed by cysteine immobilization. 2.4. Tryptic digest of model glycoproteins and HeLa cell lysis. 2 mg HRP or IgG was dissolved in 1 mL solution containing 50 mM NH4HCO3 and 8 M urea. After that, proteins were reduced by 20 µL DTT (1M) at 56 ℃ for 1 h and alkylated by 7.4 mg IAA at 30 ℃ for 45 min. Then, the mixture was ten-fold diluted by 50 mM NH4HCO3, trypsin was added (enzyme-protein ratio (w/w), 1: 20) to digest glycoproteins into peptides. The solution was gently incubated at


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37 ℃ for 18 h, and the tryptic digests were stored at -20 ℃. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium. 10% fetal bovine serum (FBS) was added and the cells were incubated at 37 °C in an atmosphere of 5% CO2. The cells were harvested at about 80% density, washed by ice-cold PBS for three times. After adding cell lysis buffer (1 tablet of complete mini EDTA-free cocktail dissolved in 10 mL solution containing 8 M urea and 50 mM NH4HCO3), cells were homogenized using a homogenizer and in a sonic ice bath for 10 min. Then the mixture was centrifuged at 4 ℃ for 15 min and supernatants were taken for further BCA analysis to test the concentration of proteins. For protein mixture digest, certain amount of DTT (final concentration 5 mM) was added and the mixture was incubated at 56 ℃ for 30 min, and alkylated by IAA (final concentration 500 mM) at 30℃ in the dark for 1 h. The alkylation was stopped by the addtion of 4.5 µL DTT. Then, the solution was first digested by Lys-C enzyme at a 1:100 enzyme-to-protein ratio (w/w) and incubated for 4 h at 37 °C. The solution was diluted five-fold with 50 mM NH4HCO3, then trypsin was added (enzyme-protein ratio (w/w), 1: 50) to digest proteins into peptides and the digestion was conducted at 37 ℃ for 16 h. The peptides was desalted by Sep-Pak tC18 SPE column and the tryptic digests were stored at -20 ℃.

2.5. Glycopeptide enrichment in standard glycoprotein and cell lysate sample. 20 µg MIL101(NH2)@Au-Cys was suspended in 200 µL loading buffer containing ACN/H2O/FA (80:15:5, v/v/v). Then 10 µL tryptic digest HRP (2 µg) or 15 µL human IgG (3 µg) was added and the solution was incubated for 5 min. After washed by loading buffer for 3 times to purify glycopeptides, the captured glycopeptides were eluted with buffer containing ACN/H2O/FA (30:69.9:0.1, v/v/v). The extracted peptides were analyzed by MALDI-TOF MS. Glycopeptide


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enrichment from HeLa cell lysate was conducted as following, 500 µg MIL-101(NH2)@Au-Cys was added to 150 µg peptide mixture. The enrichment was gently carried out for 30 min, then washed by loading buffer for 4 times to remove other contaminants. Then, glycopeptides were eluted with 2×100 µL elution buffer and lyophilized. The collected peptides were deglycosylated for further MS analysis. The lyophilized glycopeptides were redissolved in 10 mM NH4HCO3 solution, followed by addition of 1 µL PNGase F. The deglycosylation procedure was carried out at 37 ℃ for 16 h. The resulting solution was further analyzed by MALDI-TOF MS or LCMS/MS. 2.6. Recovery evaluation. 10 mg IgG digests were labeled by light and heavy isotopes respectively according to the dimethyl labeling method.26 The heavy-tagged peptides were enriched by MIL-101(NH2)@Au-Cys and the elute was added into the light-tagged IgG digest. The mixture was further extracted by MIL-101(NH2)@Au-Cys and the elute was analyzed by MALDI-TOF MS. Six glycopeptides were selected as indicators and recovery was calculated by the intensity ratio of heavy and light tagged glycopeptides. 2.7. Mass Spectrometry Analysis. The MALDI-TOF MS spectra were obtained by a Bruker Daltonics Ultraflex TOF mass spectrometer in reflection mode. A mixture of 25 mg/mL DHB in ACN/H2O/H3PO4 (70:29:1, v/v/v) was prepared as the matrix. For eluted glycopeptides, 0.5 µL elute was mixed with 0.5 µL matrix on the steel plate for MS analysis. All LC-MS/MS were performed on a Thermo Q-Exactive mass spectrometer (Thermo, San Jose, CA) equipped with a nano-ESI ion source. The samples were vacuum-centrifuged to dryness, reconstituted in 0.1% formic acid, loaded onto a pre-column for 10 min and separated on a C18 capillary column. For a gradient separation, 3-9% B in 16 min, 9%-15% B in 50 min, 15%-33% B in 54 min, 33%-45% B in 20 min, 45%-95% B in 20 min, then held at 95% B for 10 min (A = 0.1% formic acid in


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water, B = 0.1% formic acid in 80% acetonitrile). Full MS scan was obtained from m/z=3501800 at a resolution of 70000. The first 15 ions with highest intensity were selected for subsequent CID (Collision Induced Dissociation) MS/MS. The dynamic exclusion was set as 30 s. All data files were submitted for Mascot (version 2.3.02) database search. The search parameters were set as follows: fixed modification of cysteine residues (+57.0215 Da), variable modification of methionine oxidation (+15.9949 Da) and deamidation (+0.9840 Da). The mass tolerances for precursor ions were set as 20 ppm and for fragment ions were set as 0.5 Da. Two missed cleavages were allowed at most. Peptide ion scoring cutoff value was set as 30 (individual ion scores ＞ 30 indicate identity or extensive homology) and p value was lower than 0.05. Only peptides with N-X-S/T (X≠Proline) conserved sequence were defined as highly reliable glycopeptides. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of MIL-101(NH2)@Au-Cys


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Scheme 1. (a) Synthesis Route of MIL-101(NH2)@Au-Cys and (b) the Enrichment Procedure of Glycopeptides The synthesis procedure of MIL-101(NH2)@Au-Cys is shown in Scheme 1a. The substrate MIL101(NH2) rapidly coordinated the Au(0) precursor HAuCl4 via a electrostatic interaction between NH3+ and [AuCl4]-,27 followed by the reduction of NaBH4 solution for 0.5 h. Then, numerous cysteine groups were further anchored on the obtained MIL-101(NH2)@Au by the simple reaction of Au and thiol-containing cysteine. The successful synthesis of MIL-101(NH2)@AuCys was first verified by TEM images. Fig. 1 showed the morphology of MIL-101(NH2), MIL101(NH2)@Au, and MIL-101(NH2)@Au-Cys, respectively, which indicated that the Au NPs were highly dispersed on MIL-101(NH2) substrate. The electrostatic interaction between amino ligands and AuCl4- formed the ion-pair (NH3)+AuCl4-, which driven the even distribution of Au NPs over the MOF channels. The high-magnification image clearly demonstrated a lattice spacing of Au NPs was 0.230 nm. As shown in FT-IR spectroscopy (Fig. S1), no obvious change was observed after encapsulation of Au NPs into MOF framework. Cysteine functionalization was clearly confirmed through the appearance of a wide shape characteristic peak of COOHgroup at 3033 cm-1 and fingerprint peaks of L-Cysteine below 1000 cm -1. After cysteine immobilization, zeta potential of nanocomposite shifted from 95.01±5.91 mV to -159.7±8.9 mV at neutral condition (pI of cysteine was about 5.1), demonstrating the strong repulsion between each particle and the successful functionalization of cysteine groups. Zeta potential of MIL101(NH2)@Au-Cys at different pH was illustrated in Table S1. The powder XRD pattern (Fig. S2) showed no significant loss of crystallinity in MIL-101(NH2)@Au in contrast to MIL101(NH2), which was in agreement with FT-IR results. Bragg's reflections from (111) planes of Au in MIL-101(NH2)@Au were clearly observed at 38.2°, indicating the successful


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incorporation of Au NPs into MOF framework. As illustrated in Fig. 2, besides H element, MIL101(NH2)@Au consisted of C, O, N, Cr, Au elements. After cysteine functionalization, two new peaks appeared, corresponding to S 2s, S 2p core-levels, respectively, indicating the successful synthesis of MIL-101(NH2)@Au-Cys. The specific surface area was also explored (Fig. S3), with 889.8 m2 g-1 for MIL-101(NH2)@Au and 240.4 m2 g-1 for MIL-101(NH2)@Au-Cys respectively, higher than those of graphene-based nanocomposites.28,29

Figure 1. TEM image of (a) MIL-101(NH2), (b) MIL-101(NH2)@Au and (c) MIL101(NH2)@Au-Cys and (d) HRTEM of Au NPs


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Figure 2. (a) XPS survey spectra of MIL-101(NH2)@Au, (b) MIL-101(NH2)@Au-Cys, (c) Deconvoluted Au 4f spectra and (d) S 2p spectra of MIL-101(NH2)@Au-Cys

3.2. Enrichment of glycopeptides from model samples. Tryptic digest of HRP glycoprotein was first employed as model sample to evaluate the efficacy of MIL-101(NH2)@Au-Cys in glycopeptide enrichment. The loading conditions including buffer (Fig. S4) and incubation time (Fig. S5) were carefully optimized to get the best performance. Under the optimal conditions, the mixture was gently incubated in loading buffer containing ACN/H2O/FA (80/15/5, v/v/v) for only 5 min. The incubation time was obviously reduced when comparing with that of zwitterionic magnetic nanoparticles,9 this mainly attributed to the large surface area of our nanomaterial and high adsorption ability of MOF. As shown in Fig. S6, nearly no glycopeptides could be detected directly from the digest due to the co-existence of high amount of nonglycopeptides. Meanwhile, neither MIL-101(NH2) nor MIL-101(NH2)@Au could extract glycopeptides efficiently under the optimized conditions. However, after enrichment by


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MIL-101(NH2)@Au-Cys, glycopeptides with various glycosylation sites dominated the mass spectrum and 16 glycopeptides were detected (Table S2). For the comparison, cysteine-modified Au nanoparticles (Au-Cys NPs) was synthesized. The direct comparison about performance of Au-Cys NPs and MIL-101(NH2)@Au-Cys in IgG digest enrichment was performed. Moreover, IgG digests with different number of glycosylation sites and glycan composition were also employed to test the feasibility of the material and the results were shown in Fig. 3. As we expected, several glycopeptides could be enriched by MIL-101(NH2) or MIL-101(NH2)@Au with very low intensity while the non-specific adsorption was serious. Although several glycopeptides could be detected after enrichment by Au-Cys NPs, the number of extracted glycopeptides was far from enough and the intensity was low. In contrast, non-glycopeptides were almost completely removed after enrichment by MIL-101(NH2)@Au-Cys and 31 glycopeptides with no interference were found in total (Table S3). For unambiguous verification, all peptides in Fig. 3e were deglycosylated by PNGase F enzyme. As clearly shown in Fig. 3f, the identified glycopeptides were totally disappeared after deglycosylation and 4 deglycosylated peptides were found instead, illustrating all peptides detected in Fig. 3e were ascribed to glycopeptides. These 4 deglycosylated peptides corresponded to two completely digest peptides (EEQFN#STFR, EEQYN#STYR) and two incompletely digest (TKRPEEQFN#STFR, TKPREEQYN#STYR) according to the previous report.15 The above-results reliably demonstrated the excellent efficiency of MIL-101(NH2)@Au-Cys in glycopeptide enrichment in model samples.


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Figure 3. MALDI-TOF MS spectra of 1.0 pmol/µL digest of IgG glycoprotein (a) before enrichment, after enriched by (b) MIL-101(NH2), (c) MIL-101(NH2)@Au, (d) Au-Cys NPs, (e) MIL-101(NH2)@Au-Cys and (f) deglycosylated by PNGase F. 3.3. Binding capacity, selectivity, recovery and limits of detection In addition, the binding capacity was also inspected. Different amount of MIL101(NH2)@Au-Cys (10-40 µg) was added into a certain amount of IgG digest (3 µg) and further incubated for 5 min. Six glycopeptides with different glycan composition were selected as markers. As illustrated in Fig. 4, the peak intensity of six markers reached maximum after the addition of 20 µg MIL-101(NH2)@Au-Cys and remained almost the same regardless of the ever-increasing amount of material. It was estimated that 1 g nanocomposite could extract 150 mg IgG digest efficiently, which was higher than most other nanocomposites.9 This high adsorption capacity mainly owed to the large surface area and the high hydrophilicity of MIL-101(NH2)@Au-Cys. The unique synthetic route


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ensured high density of Au NPs encapsulation, thus facilitating grafting large amount of hydrophilic cysteine groups. Meanwhile, the enrichment selectivity was explored using MIL-101(NH2), MIL-101(NH2)@Au, MIL-101(NH2)@Au-Cys, respectively. Different molar ratios of BSA tryptic digest was added as non-glycopeptides to mimic peptide mixtures. Without enrichment, no glycopeptides were observed when the concentration of non-glycopeptides was 10 times higher than that of IgG digest due to serious interference (Fig. S7). MIL-101(NH2) and MIL-101(NH2)@Au also failed to extract glycopeptides selectively. However, the intensity of non-target peptides dramatically diminished after enriched by MIL-101(NH2)@Au-Cys and several glycopeptides could be detected. Even the ratio of IgG and BSA digest was 1:50, the nanomaterial still worked well in complex peptide mixture and 11 glycopeptides with little interference was observed. Meanwhile, enrichment recovery was also investigated with the combination of dimethyl labelling method. The obtained average recovery was over 80%, indicating the high recovery of enrichment procedure and reliability of MIL-101(NH2)@Au-Cys (Table S4). Moreover, the detection sensitivity was also investigated considering the ultralow concentration of glycopeptides in complex biosamples. IgG digest with various amount of 15 ng/µL, 3 ng/µL, 0.3 ng/µL which corresponding to 50 fmol, 10 fmol, 1 fmol were treated with MIL-101(NH2)@Au-Cys, respectively. As shown in Fig. 5, 10 glycopeptides were detected with high intensity and almost no interference after enrichment. The limit of detection was as low as 0.3 ng/µL and 3 glycopeptides were selectively obtained according to S/N ratio larger than 3. This high sensitivity at fmol level should be of great value for selective recognition of trace glycopeptides in biosamples.


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Figure 4. The MS intensity of six glycopeptides from 3 µg IgG tryptic digest after enriched by different amount of MIL-101(NH2)@Au-Cys nanocomposite.

Figure 5. MALDI-TOF MS spectra of (a) 15 ng/µL (50 fmol), (b) 3 ng/µL (10 fmol), (c) 0.3 ng/µL (1 fmol) IgG digest after enriched by MIL-101(NH2)@Au-Cys 3.4. Enrichment of glycopeptides from HeLa cell lysate. Encouraged by above merits, such as short incubation time, good selectivity, large binding capacity, and high sensitivity obtained in glycopeptide enrichment from model


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digest, MIL-101(NH2)@Au-Cys was finally applied to glycoproteome profiling of HeLa cell lysate. The adenocarcinoma HeLa cell line frequently serves as a model sample for cancer research, especially for cervical cancer.30 Herein, 30 µg tryptic digest of protein mixture extracted from HeLa cells was analysed as described above. N-glycosylation sites were identified by conserved sequence N-X-S/T (X≠P) and deamidated asparagine (N). In three independent isolation replicates, 848, 792, 809 N-glycopeptides were identified respectively, among which 577 glycopeptides (70.7%) were identified in all three isolations, demonstrating high reliability and reproducibility of enrichment procedures. The detailed number of identified glycopeptides and glycoproteins were illustrated in Fig. 6. In total, among the 1712 detected peptides and 686 proteins, 1123 N-glycosylation sites corresponding to 1069 N-glycopeptides and 614 N-glycoproteins were found (Table S5). To the best of our knowledge, the number of extracted glycopeptides from Hela lysate was so far the best result compared with those of previously reported hydrophilic methods.31,32 The percentage of extracted glycopeptide was up to 62.4%, which illustrated the high specificity of MIL-101(NH2)@Au-Cys in complex biosample in contrast to most other HILIC-based nanocomposites.13,28


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Figure 6.

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Overlapping of identified (a) glycopeptides and (b) glycoproteins in HeLa cell

proteins after enrichment by MIL-101(NH2)@Au-Cys in three isolation replicates CONCLUSIONS In summary, MIL-101(NH2)@Au-Cys was developed via a facile two-step synthesis from MIL-101(NH2) for glycopeptide enrichment. Owing to large specific surface area and ultrahigh hydrophilicity of this nanocomposites, excellent performance was observed in both model glycoprotein digest and HeLa cell lysate. It is noteworthy that fast functionalization from aminoderived MOF could be realized through the common and facile method for Au NPs encapsulation and subsequent modification through Au-S interaction. This work paves the way for general functionalization of MOF nanocomposites and provides a novel material design method for sample preparation in complicate matrix, especially for the glycoproteome analysis in biosample. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org FT-IR spectrum, XRD spectrum and N2 adsorption–desorption isotherm of MIL-101(NH2)@AuCys; optimal conditions of loading buffer and incubation time; MALDI-TOF MS spectra of HRP tryptic digest after enrichment; enrichment selectivity; zeta potential of MIL-101(NH2)@Au-Cys at different pH; enrichment recovery evaluation; detailed information of glycopeptides extracted from HRP, IgG, and HeLa cell lysate AUTHOR INFORMATION


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Corresponding Authors * E-mail: [email protected]. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (21575007, 21322505, 21527809) . REFERENCES (1)

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For Table of Contents Only


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Scheme 1. (a) Synthesis Route of MIL-101(NH2)@Au-Cys and (b) the Enrichment Procedure of Glycopeptides 98x64mm (300 x 300 DPI)


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Figure 1. TEM image of (a) MIL-101(NH2), (b) MIL-101(NH2)@Au and (c) MIL-101(NH2)@Au-Cys and HRTEM of Au NPs 87x89mm (300 x 300 DPI)



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Figure 2. (a) XPS survey spectra of MIL-101(NH2)@Au, (b) MIL-101(NH2)@Au-Cys, (c) Deconvoluted Au 4f spectra and (d) S 2p spectra of MIL-101(NH2)@Au-Cys 74x37mm (300 x 300 DPI)


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Figure 3. MALDI-TOF MS spectra of 1.0 pmol/µL digest of IgG glycoprotein (a) before enrichment, after enriched by (b) MIL-101(NH2), (c) MIL-101(NH2)@Au, (d) Au-Cys NPs, (e) MIL-101(NH2)@Au-Cys and (f) deglycosylated by PNGase F. 88x91mm (300 x 300 DPI)



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Figure 4. The MS intensity of six glycopeptides from 3 µg IgG tryptic digest after enriched by different amount of MIL-101(NH2)@Au-Cys nanocomposite. 59x41mm (300 x 300 DPI)


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Figure 5.

MALDI-TOF MS spectra of (a) 50 fmol (0.5 µL), (b) 10 fmol (0.5 µL), (c) 1 fmol (0.5 µL) IgG digest after enriched by MIL-101(NH2)@Au-Cys 67x54mm (300 x 300 DPI)



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Figure 6. Overlapping of identified (a) glycopeptides and (b) glycoproteins in HeLa cell proteins after enrichment by MIL-101(NH2)@Au-Cys in three isolation replicates 54x34mm (300 x 300 DPI)


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