Accepted Manuscript 4-mercaptophenylboronic acid functionalized graphene oxide composites: preparation, characterization and selective enrichment of glycopeptides Bo Jiang, Yanyan Qu, Lihua Zhang, Zhen Liang, Yukui Zhang PII:
S0003-2670(16)30088-5
DOI:
10.1016/j.aca.2016.01.018
Reference:
ACA 234358
To appear in:
Analytica Chimica Acta
Received Date: 13 November 2015 Revised Date:
8 January 2016
Accepted Date: 10 January 2016
Please cite this article as: B. Jiang, Y. Qu, L. Zhang, Z. Liang, Y. Zhang, 4-mercaptophenylboronic acid functionalized graphene oxide composites: preparation, characterization and selective enrichment of glycopeptides, Analytica Chimica Acta (2016), doi: 10.1016/j.aca.2016.01.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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GO/PEI/Au/4-MPB composites were synthesized, and exhibited high selectivity to
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capture glycopeptides.
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4-mercaptophenylboronic acid functionalized graphene oxide
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composites: preparation, characterization and selective enrichment
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of glycopeptides
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Bo Jianga, Yanyan Qua, Lihua Zhanga*, Zhen Lianga and Yukui Zhanga
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a
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for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of
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Science, Dalian 116023, China.
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E-mail: [email protected]. Fax: +86-411-84379720.
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National Chromatographic R. & A. Center, Key Laboratory of Separation Science
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Abstract
Selective enrichment and isolation of glycopeptides from complex biological
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samples was indispensable for mass spectrometry (MS)-based glycoproteomics,
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however, it remained a great challenge due to the low abundance of glycoproteins and
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the ion suppression of non-glycopeptides. In this work, 4-mercaptophenylboronic acid
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functionalized graphene oxide composites were synthesized via loading gold
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nanoparticles on polyethylenimine modified graphene oxide surface, followed by
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4-mercaptophenylboronic acid immobilization by the formation of Au-S bonding
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(denoted as GO/PEI/Au/4-MPB composites). The composites showed highly specific
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and efficient capture of glycopeptides due to their excellent hydrophilicity and
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abundant boronic acid groups. The composites could selectively capture the
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glycopeptides from the mixture of glycopeptides and nonglycopeptides, even when
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the amounts of non-glycopeptides were 100 times more than glycopeptides.
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Compared with commercial meta-amino phenylboronic acid agarose, the composites
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showed better selectivity when the sample was decreased to 10 ng. These results
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clearly verified that the GO/PEI/Au/4-MPB composites might be a promising material
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for glycoproteomics analysis.
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Keywords: graphene oxide; polyethylenimine; 4-mercaptophenylboronic acid; 1
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glycopeptides; enrichment
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1. Introduction Protein glycosylation, associated with secreted and membrane proteins, regulated
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many key biological functions, such as cell growth, signal transduction, immune
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response and so on [1]. Furthermore, many clinical biomarkers and therapeutic targets
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were related with glycoproteins [2]. Therefore, the discovery and identification of
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glycoproteins played an important role in disease diagnosis and treatment. It was
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demonstrated that MS technology had great potential for detailed information
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characterization of glycoproteins because of its high efficiency and high sensitivity
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[3]. However, owing to the inherent low abundance of glycoproteins, and the strong
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ion suppression of non-glycopeptides, it remained a great challenge for better
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understanding of protein glycosylation through MS strategy [4]. Therefore, selective
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enrichment glycopeptides from complex samples was essential prior to MS analysis
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and elucidation of glycosylation sites.
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To date, several methods were developed for selective enrich glycopeptides from
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biological samples. The most commonly used lectin affinity chromatography showed
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excellent affinity toward high-mannose and hybrid N-glycans. However, it displayed
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low affinity toward bian-tennary N-glycans and no affinity toward tri- and
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tetra-antennary complex-type glycans, which limited its applications [5-10].
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Hydrazide chemistry was proved to be effective for glycopeptides enrichment, but
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both the reaction time and the sample complexity were significantly increased due to
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the oxide step. Furthermore, the glycan structure information was lost during the
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tedious procedure [11-15]. Porous graphitized carbon was developed to isolate
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glycopeptides with short amino acid sequences, but its application to tryptic
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glycopeptides was limited [16]. For hydrophilic interaction chromatography (HILIC),
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it was generally believed that hydrogen-bonding interactions between the hydroxyl
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groups on the glycans and functional groups on the supports play crucial roles in
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glycopeptides retention. HILIC displayed the advantages of high coverage and good
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compatibility with MS [17-23]. However, the co-elution of non-glycopeptides was
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inevitable during elution process [24]. Boronic acid functionalized materials were
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introduced to unbiased enrichment of glycopeptides through forming diesters with all
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glycans that contain cis-diol groups [25, 26]. Up till now, several boronate affinity
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methods have been developed for glycoproteins/glycopeptides enrichment. Firstly,
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boronic acid ligands were immobilized on solid supports (such as magnetic
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nanoparticles,
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glycoproteins/glycopeptides enrichment [27-37]. The boronate affinity materials
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showed excellent selectivity, but needed to off-line operation which led to sample loss.
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Secondly, boronic acid functionalized monolith columns were developed for on-line
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capture of glycoproteins/glycopeptides [38-40]. The boronate affinity monolith
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reduced samples loss, but suffered low boronic acid immunization amount. Finally,
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boronic acid functionalized gold nanoparticles and Si wafer were successfully applied
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for the on-plate selective capture of glycopeptides [41, 42]. The above results clearly
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demonstrated that boronate affinity materials were perfect glycan-oriented probes for
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specific glycopeptides enrichment. For boronate affinity materials, it was still need to
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improve the immobilized amount of boronic acid groups and hydrophilicity of
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supports. Therefore, it was crucial to develop novel boronate affinity materials with
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high immobilization amount and superior hydrophilicity.
mesoporous
silica
and
gold
nanoparticles)
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polymers,
Since its discovery, graphene has received more and more attention due to its
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intriguing physical and chemical properties [43]. Graphene oxide (GO), a novel
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two-dimensional carbon nanomaterial prepared from natural graphite, has recently
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attracted significant interesting as a precursor of chemically converted graphene
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[44]. Apart from the layered structure with a large theoretical specific surface area
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(2630 m2/g), GO nanosheets bear hydroxyl and epoxy groups on the graphitic
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plane and carboxylic acid groups at the sheet edges. The presence of such groups
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not only provides the GO sheets excellent hydrophilicity, but also offers potential
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application as substrates for the preparation of novel composites [45-47]. Recently
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Au nanoparticles (Au NPs) were decorated on GO surface as novel composites.
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GO/Au composites were sucessfully used for biocatalysis, cell imaging, SERS and
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glycoproteomics [48-51]. GO/Au composites, with excellent hydrophilicity and
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abundant reaction sites, were considered as potential support for immobilizing
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boronic
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acid
groups.
The
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of
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composites
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enrichement with excellent hydrophilicity and large amount of boronic acid groups.
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Herein, GO/PEI/Au/4-MPB composites were synthesized via loading Au NPs on GO
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by in situ growth using polyethylenimine as reducing and immobilizing reagents,
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followed by 4-MPB immobilization through Au-S bond to construct novel boronic
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acid functionalized materials.
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2. Experimental
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2.1. Chemical and materials
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GO was purchased from Xianfeng Nanotech (Nanjing, China). Polyethylenimine,
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(ethylenediamine end-capped, PEI, Mn 600), 4-mercaptophenylboronic acid (4-MPB),
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TPCK-treated trypsin, bovine serum albumin (BSA), horseradish peroxidase (HRP),
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asialofetuin (ASF), dithiothreitol (DTT), iodoacetamide (IAA), formic acid (FA),
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boronic acid agarose, urea and trifluoroacetic acid (TFA) were obtained from Sigma
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(St. Louis, MO, USA). 2, 5-dihydroxybenzoic acid (DHB) was obtained from Bruker
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(Daltonios, Germany). Acetonitrile (ACN) was of HPLC grade from Merck
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(Darmstadt, Germany). All other reagents were of analytical grade purchased from
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China. Water was purified using a Milli-Q system (Darmstadt, Germany).
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2.2. Synthesis of GO/PEI/Au/4-MPB composites In a typical process, 0.3 mL PEI (100 mg/mL) was added into 1 mL GO
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suspension (0.1 mg/mL) under vigorous stirring for 1 h, and unreacted PEI was
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removed by centrifugation. The obtained PEI functionalized GO composites (GO/PEI)
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was dispersed in 1 mL H2O and 20 µL HAuCl4.3H2O (100 mg/mL) was added to the
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above suspension. The resulting suspensions were heated at 60 ºC for 1 h. After
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washing and centrifugation, the resulted composites (GO/PEI/Au) were vacuum-dried
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for further use. In the next step, the resultant GO/PEI/Au composites were mixed with
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15.4 mg 4-MPB at 1 mL ethanol, with strong stirring at room temperature for 24 h.
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After reaction, the mixture was washed with ethanol for three times, and then
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vacuum-dried to obtain GO/PEI/Au/4-MPB composites.
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2.3. Sample preparation The standard glycoprotein was dissolved in 1 mL of 50 mM NH4HCO3 (pH 8.0)
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and denatured at 90 °C for 15 min. Then, TPCK-treated trypsin was added to a final
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volume of 1:40 w/w of glycoprotein. The tryptic digestion proceeded at 37 ºC for 12 h.
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BSA was dissolved in 50 mM NH4HCO3 (pH 8.0) containing 8 M urea, and then
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reduced in 10 mM DTT for 1 h at 56 °C. When cooled to room temperature, cysteines
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were alkylated in the dark in 20 mM IAA for 30 min at 37 °C. After being diluted
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ten-fold with 50 mM NH4HCO3 (pH 8.0), the solution was subsequently treated with
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trypsin at 37 °C (enzyme/protein ratio of 1:40, w/w) for 12 h. All digestion was
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stopped with FA to a final concentration of 1%. Tryptic digests were desalt by C18
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column and stocked at -20 ºC for further use.
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2.4. Glycopeptides enrichment under hydrophilic mode
Firstly, 20 µg material was dispersed in 50 µL ACN/H2O/FA (80: 20: 0.1, v/v/v)
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containing 0.1 µg HRP digests. The capture procedure was carried out under gentle
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agitation at room temperature for 10 min. After reaction, the supernatant was
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discarding by centrifugation. Secondly, 20 µL ACN/H2O/FA (80: 20: 0.1, v/v/v. 3
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times) was used to remove the non-glycopeptides. Finally, glycopeptides were eluted
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with 20 µL ACN/H2O/FA (60: 40: 0.1, v/v/v). The collected peptides were analysis by
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MALDI-TOF MS.
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2.5. Enrichment of glycopeptides by GO/PEI/Au/4-MPB composites The tryptic glycoprotein mixture was incubated in 200 µL NH4HCO3 (50 mM,
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pH 9.0) containing 100 µg GO/PEI/Au/MPB composites with strong shaking for 2 h
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at room temperature. After washing with 200 µL NH4HCO3 for three times, the
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solution of ACN/H2O/TFA (50:49:1 (v/v/v), 20 µL) as the elution buffer was added to
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release the glycopeptides. The eluent fraction was lyophilized in a SpeedVac (Thermo
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Fisher, San Jose, CA, USA), and dissolved in 2 µL DHB (20 mg/mL, 0.1% TFA in 60%
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ACN aqueous solution) for MALDI-TOF MS analysis.
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2.6. MS analysis and date research All MALDI spectra were taken from a Bruker Ultraflex III MALDI-TOF/TOF
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MS instrument (Bruker, Daltonios, Germany). A total of 1 µL of DHB solution was
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added to MALDI plate. The laser intensity was kept constant for all samples. External
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calibration of MALDI-TOF/TOF MS spectra was performed with ten commercial
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peptides.
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2.7. Characterization
Transmission electron microscopy (TEM) image was obtained by JEOL JEM-2000
transmission
electron
microscope
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High-resolution TEM (HRTEM) images were collected on a Tecnai G2 F30 S-Twin
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microscope operated at 300 kV (Tecnai G2, FEI, Eindhoven, Netherlands). Zeta
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potential was detected by Malven Nano-zs90 dynamic light scattering (Malven,
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Worcester, UK). Fourier-transformed infrared spectroscopy (FT-IR) characterization
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has been performed on Perkin-Elmer Spectrum GX spectrometer (Perkin-Elmer,
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Waltham, USA). X-ray photoelectron spectroscopy (XPS) measurements were
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conducted with Thermo ESCALAB250Xi spectrometer with Al Kα radiation as the
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X-ray source (Thermo, Waltham, USA). Thermo Gravimetric Analysis (TGA) was
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conducted with Netzsch STA449F3 thermal analyzer (Netzsch, Bavaria, Germany)
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that was fitted to a nitrogen purge gas at 10 ºC/min heating rate.
Tokyo,
Japan).
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3. Results and discussion
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3.1. Preparation of GO/PEI/Au/4-MPB composites
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(JEOL,
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Fig. 1 depicted the synthesis route of GO/PEI/Au/4-MPB composites. Firstly,
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PEI was assembled on the surface of GO by electrostatic and hydrogen bonding
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interaction. The attached PEI significantly enhanced the hydrophilicity of GO due to
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its abundant amino groups [51]. Secondly, Au NPs were in suit introduced on the
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surface of GO/PEI nanosheets using PEI as reducing and stabilizing reagents. The
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obtained composites were denoted as GO/PEI/Au composites. Finally, GO/PEI/Au
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composites were used as hydrophilic support for immobilizing 4-MPB to prepare 7
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GO/PEI/Au/4-MPB composites. Zeta potential of GO changed from -67 to 43 mv after the self-assembly of PEI,
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which suggested that a large amount of PEI was immobilized on GO surface. PEI
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served as primers for the adsorption of anionic AuCl4- [52]. By combination of the
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multifunctional PEI, the attachment of Au NPs on the GO surface was achieved. Fig.
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2 showed the typical TEM images of GO before and after generation Au NPs. It was
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found that well-dispersed GO nanosheets were observed in Fig. 2A. However, as
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shown in Fig. 2B and C, after Au NPs immobilization, Au NPs homogeneously
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spread on the surface of GO/PEI with high density, which greatly increased the
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number of reaction sites of the composites. High-resolution TEM (HR-TEM), as
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shown in Fig. S1, revealed the highly crystalline nature of Au NPs with a lattice
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spacing of 0.24 nm, validating the generation of Au NPs [53]. These results clearly
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verified that Au NPs were successfully prepared and immobilized on GO/PEI surface.
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The immobilized PEI not only stabilized Au NPs, but also improved the
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hydrophilicity of GO/PEI/Au composites. Under HILIC mode, tryptic HRP were
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applied to evaluate hydrophilic capacity of GO, GO/PEI and GO/PEI/Au composites.
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Only 1 glycopeptides was detected in the eluting fraction of GO (Fig. S2A). However,
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the non-glycopeptides were almost removed and 5 glycopeptides with enhanced MS
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intensities were founded in the eluting fraction of GO GO/PEI/Au (Fig. S2B and C).
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The results demonstrated that PEI significantly enhanced the hydrophilicity of
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GO/PEI/Au composites.
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The successful preparation of GO/PEI/Au/4-MPB composites was characterize
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by FT-IR. Fig. 3A, for GO, the peak around 1728 cm-1 corresponded to the C=O
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stretching vibration of the carboxylic group. After PEI was self-assembled on the GO
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surface, the C=O stretching vibration almost disappears, and new peaks at 2933 and
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2838 cm-1 ascribing to C–H stretch appear, confirming the successful self-assembly of
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PEI. GO/PEI/Au composites showed the similar adsorption peaks as GO/PEI
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nanosheets. Compared with GO/PEI/Au composites, there appeared new B-O
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vibration (1338 cm-1) peak in the curve of GO/PEI/Au/4-MPB composites [31],
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which demonstrated the successful immobilization of 4-MPB. The survey XPS 8
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N, S, B and Au elements, indicating the successful preparation of GO/PEI/Au/4-MPB
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composites [33]. The immobilization amount of 4-MPB played an important role for
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enrichment efficiency of GO/PEI/Au/4-MPB composites. As shown in Fig. 4,
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GO/PEI/Au/4-MPB composites showed the similar mass loss curves to that of
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GO/PEI/Au composites. Slower mass loss (∼5%) over the whole temperature range
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between 600 and 800 ºC can also be seen. Taking into accounts of the data of
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GO/PEI/Au and GO/PEI/Au/4-MPB at 700 ºC, 4-MPB content in GO/PEI/Au/4-MPB
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was calculated as 14.3% wt (∼143 mg/g), which was higher than previous boronic
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acid functionalized material [25]. The high immobilization amount might be attribute
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to a large surface area of GO and a large number of Au NPs. The morphology of
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prepared GO/PEI/Au/4-MPB composites was characterized by TEM. As shown in Fig.
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S3, abundant dispersed Au NPs was found, and scarcely any Au NPs dissociated from
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GO/PEI/Au/4-MPB composites was observed. We could expect that the prepared
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GO/PEI/Au/4-MPB composites, with excellent hydrophilicity, large amount of
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boronic acid groups, had great potential for the selective enrichment of glycopeptides
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from complex samples.
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3.2. Enrichment Efficiency of GO/EPI/Au/4-MPB Composites
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Tryptic HRP was employed as samples to evaluate the performance of
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GO/PEI/Au/4-MPB composites. According to previous research, NH4HCO3 (50 mM,
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pH 9.0) and ACN/H2O/TFA (50/49/1, v/v/v) were used as loading and elution buffers,
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respectively [41]. As shown in Fig. 5A, before enrichment, almost no peaks matching
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with glycosylated peptides could be determined, which was attributed to the existence
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of abundant high-abundance non-glycosylated peptides. However, as shown in Fig.
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5B, after enrichment, non-glycopeptides were almost removed and 12 glycopeptides
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with enhanced MS intensities and S/N dominated the MS spectra (glycopeptides
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information was shown in Table. S1). Even the concentration of tryptic HRP was
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decreased to 0.05 ng/µL (10 ng), 7 glycopeptides were positively identified (Fig. 5C).
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For comparison, an equivalent amount of commercial boronic acid agarose was used
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signals representing glycopeptides could be detected, indicating that the
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GO/PEI/Au/4-MPB composites have better sensitivity than the commercial boronic
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acid agarose. The results evaluated that GO/PEI/Au/4-MPB composites show high
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selectivity to low concentration glycopeptides. The superior performance was
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attributed to high immobilized amount of boronic acid groups of GO/PEI/Au/4-MPB
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composites.
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The GO/PEI/Au/4-MPB composites were also applied to enrich glycopeptides
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from tryptic ASF. Fig. 6 displayed the MALDI-TOF MS spectra of 5 ng/µL tryptic
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ASF before and after enrichment by GO/PEI/Au/4-MPB composites. It was found that
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glycopeptides and the fragments dominant the mass spectra after enrichment, and the
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number of glycopeptides increased from 3 to 6 (glycopeptides information was shown
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in Table. S2). Inspired by the satisfying enrichment results, GO/PEI/Au composites
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were also applied to capture glycopeptides form tryptic ASF under the same
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conditions. It was found that the GO/PEI/Au composites showed no enrichment
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ability to glycopeptides, suggesting it was the boronic acid groups that displayed
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affinity to glycopeptides.
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GO/PEI/Au/4-MPB composites were further applied to isolate glycopeptides
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from the different mass ratios of tryptic BSA and ASF. Fig. 7 showed the
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MALDI-TOF MS spectra of identified glycopeptides from different mass ratios of the
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tryptic mixture of ASF and BSA. As shown in Fig. 7A, 5 glycopeptides were clearly
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detected from 0.5 ng/µL tryptic ASF. Five glycopeptides were still detected with
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non-glycopeptides scarcely identified in both 1:1 and 10:1 (Fig. 7B, C). Even the ratio
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was further increased to 100:1, the ion signal of glycopeptides was remain observable
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and 4 glycopeptides were identified (Fig. 7D). The superior enrichment capacity was
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ascribed to the excellent hydrophilicity of GO/PEI/Au/4-MPB composites, which
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greatly reduced the non-specific adsorption. The results demonstrated the reliability of
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GO/PEI/Au/4-MPB composites for separating glycopeptides from complex biological
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samples.
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4. Conclusion In summary, hydrophilic GO/PEI/Au/4-MPB composites were prepared and used
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to enrich glycopeptides from complex samples. GO/PEI/Au/4-MPB composites
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exhibited high selectivity and sensitivity in the capture of N-linked glycopeptides.
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Due to higher hydrophilicity of composites, no nonspecific binding was observed in
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the presence of prominent tryptic BSA, demonstrating specificity of binding.
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Comparison with commercial boronic acid resin, GO/PEI/Au/4-MPB composites
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showed better selectivity and sensitivity. Accordingly, with our newly developed
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method, various kinds of N-glycopeptides could be specifically selected and enriched
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for further analysis. We also expected it could be further applied to complex
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biological samples. The work not only offered an boronate affinity material, but also
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opened a new field for applications of GO.
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Acknowledgements
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This work was supported by National Nature Science Foundation (21190043, 21235005
and
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(2012CB910601 and 2013CB911201), and Creative Research Group Project by
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NSFC (21321064).
National
Basic
Research
Program
of
China
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Fig. 1. Synthetic schematics of GO/PEI/Au/4-MPB composites.
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Fig. 2. TEM images of (A) GO, (B) and (C) GO/PEI/Au composites.
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Fig. 3. (A) FI-IR spectra of GO, GO/PEI/, GO/PEI/Au and GO/PEI/Au/4-MPB
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composites, (B) XPS survey spectra of GO/PEI/Au/4-MPB composites.
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Fig. 4. TGA curves of GO, GO/PEI, GO/PEI/Au and GO/PEI/Au/4-MPB composites.
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Fig. 5. MALDI-TOF MS spectra of (A) 5 ng/µL HRP before enrichment, (B) 5 ng/µL
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HRP after enrichment by GO/PEI/Au/4-MPB composites, (C) 0.05 ng/µL HRP after
518
enrichment by GO/PEI/Au/4-MPB composites, (D) 0.05 ng/µL HRP after enrichment
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by commercial boronic resin. V= 200 µL.
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Fig. 6. MALDI-TOF MS spectra of (A) 5 ng/µL ASF before enrichment, (B) 5 ng/µL
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ASF after enrichment by GO/PEI/Au/4-MPB composites. V=200 µL.
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Fig. 7. MALDI-TOF MS spectra of 0.5 ng/µL ASF with different mass ratio of BSA
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(A)1:0, (B) 1:1, (C) 10:1 and (D) 100:1. BSA/ASF, V= 200 µL.
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Fig. 1. Synthetic schematics of GO/PEI/Au/4-MPB composites.
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Fig. 2. TEM images of (A) GO, (B) and (C) GO/PEI/Au composites.
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Fig. 3. (A) FI-IR spectra of GO, GO/PEI/, GO/PEI/Au and GO/PEI/Au/4-MPB composites, (B) XPS survey spectra of GO/PEI/Au/4-MPB composites.
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Fig. 4. TGA curves of GO, GO/PEI, GO/PEI/Au and GO/PEI/Au/4-MPB composites.
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Fig. 5. MALDI-TOF MS spectra of (A) 5 ng/μL HRP before enrichment, (B) 5 ng/μL HRP after enrichment by GO/PEI/Au/4-MPB composites, (C) 0.05 ng/μL HRP after enrichment by GO/PEI/Au/4-MPB composites, (D) 0.05 ng/μL HRP after enrichment by commercial boronic resin. V= 200 μL.
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Fig. 6. MALDI-TOF MS spectra of (A) 5 ng/μL ASF before enrichment, (B) 5 ng/μL ASF after enrichment by GO/PEI/Au/4-MPB composites. V=200 μL.
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Fig. 7. MALDI-TOF MS spectra of 0.5 ng/μL ASF with different mass ratio of BSA (A)1:0, (B) 1:1, (C) 10:1 and (D) 100:1. BSA/ASF, V= 200 μL.
S0003-2670(16)30088-5
DOI:
10.1016/j.aca.2016.01.018
Reference:
ACA 234358
To appear in:
Analytica Chimica Acta
Received Date: 13 November 2015 Revised Date:
8 January 2016
Accepted Date: 10 January 2016
Please cite this article as: B. Jiang, Y. Qu, L. Zhang, Z. Liang, Y. Zhang, 4-mercaptophenylboronic acid functionalized graphene oxide composites: preparation, characterization and selective enrichment of glycopeptides, Analytica Chimica Acta (2016), doi: 10.1016/j.aca.2016.01.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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GO/PEI/Au/4-MPB composites were synthesized, and exhibited high selectivity to
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capture glycopeptides.
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4-mercaptophenylboronic acid functionalized graphene oxide
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composites: preparation, characterization and selective enrichment
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of glycopeptides
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Bo Jianga, Yanyan Qua, Lihua Zhanga*, Zhen Lianga and Yukui Zhanga
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a
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for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of
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Science, Dalian 116023, China.
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E-mail: [email protected]. Fax: +86-411-84379720.
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National Chromatographic R. & A. Center, Key Laboratory of Separation Science
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Abstract
Selective enrichment and isolation of glycopeptides from complex biological
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samples was indispensable for mass spectrometry (MS)-based glycoproteomics,
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however, it remained a great challenge due to the low abundance of glycoproteins and
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the ion suppression of non-glycopeptides. In this work, 4-mercaptophenylboronic acid
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functionalized graphene oxide composites were synthesized via loading gold
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nanoparticles on polyethylenimine modified graphene oxide surface, followed by
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4-mercaptophenylboronic acid immobilization by the formation of Au-S bonding
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(denoted as GO/PEI/Au/4-MPB composites). The composites showed highly specific
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and efficient capture of glycopeptides due to their excellent hydrophilicity and
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abundant boronic acid groups. The composites could selectively capture the
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glycopeptides from the mixture of glycopeptides and nonglycopeptides, even when
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the amounts of non-glycopeptides were 100 times more than glycopeptides.
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Compared with commercial meta-amino phenylboronic acid agarose, the composites
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showed better selectivity when the sample was decreased to 10 ng. These results
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clearly verified that the GO/PEI/Au/4-MPB composites might be a promising material
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for glycoproteomics analysis.
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Keywords: graphene oxide; polyethylenimine; 4-mercaptophenylboronic acid; 1
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glycopeptides; enrichment
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1. Introduction Protein glycosylation, associated with secreted and membrane proteins, regulated
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many key biological functions, such as cell growth, signal transduction, immune
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response and so on [1]. Furthermore, many clinical biomarkers and therapeutic targets
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were related with glycoproteins [2]. Therefore, the discovery and identification of
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glycoproteins played an important role in disease diagnosis and treatment. It was
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demonstrated that MS technology had great potential for detailed information
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characterization of glycoproteins because of its high efficiency and high sensitivity
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[3]. However, owing to the inherent low abundance of glycoproteins, and the strong
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ion suppression of non-glycopeptides, it remained a great challenge for better
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understanding of protein glycosylation through MS strategy [4]. Therefore, selective
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enrichment glycopeptides from complex samples was essential prior to MS analysis
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and elucidation of glycosylation sites.
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To date, several methods were developed for selective enrich glycopeptides from
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biological samples. The most commonly used lectin affinity chromatography showed
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excellent affinity toward high-mannose and hybrid N-glycans. However, it displayed
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low affinity toward bian-tennary N-glycans and no affinity toward tri- and
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tetra-antennary complex-type glycans, which limited its applications [5-10].
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Hydrazide chemistry was proved to be effective for glycopeptides enrichment, but
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both the reaction time and the sample complexity were significantly increased due to
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the oxide step. Furthermore, the glycan structure information was lost during the
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tedious procedure [11-15]. Porous graphitized carbon was developed to isolate
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glycopeptides with short amino acid sequences, but its application to tryptic
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glycopeptides was limited [16]. For hydrophilic interaction chromatography (HILIC),
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it was generally believed that hydrogen-bonding interactions between the hydroxyl
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groups on the glycans and functional groups on the supports play crucial roles in
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glycopeptides retention. HILIC displayed the advantages of high coverage and good
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compatibility with MS [17-23]. However, the co-elution of non-glycopeptides was
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inevitable during elution process [24]. Boronic acid functionalized materials were
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introduced to unbiased enrichment of glycopeptides through forming diesters with all
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glycans that contain cis-diol groups [25, 26]. Up till now, several boronate affinity
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methods have been developed for glycoproteins/glycopeptides enrichment. Firstly,
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boronic acid ligands were immobilized on solid supports (such as magnetic
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nanoparticles,
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glycoproteins/glycopeptides enrichment [27-37]. The boronate affinity materials
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showed excellent selectivity, but needed to off-line operation which led to sample loss.
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Secondly, boronic acid functionalized monolith columns were developed for on-line
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capture of glycoproteins/glycopeptides [38-40]. The boronate affinity monolith
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reduced samples loss, but suffered low boronic acid immunization amount. Finally,
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boronic acid functionalized gold nanoparticles and Si wafer were successfully applied
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for the on-plate selective capture of glycopeptides [41, 42]. The above results clearly
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demonstrated that boronate affinity materials were perfect glycan-oriented probes for
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specific glycopeptides enrichment. For boronate affinity materials, it was still need to
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improve the immobilized amount of boronic acid groups and hydrophilicity of
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supports. Therefore, it was crucial to develop novel boronate affinity materials with
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high immobilization amount and superior hydrophilicity.
mesoporous
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and
gold
nanoparticles)
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Since its discovery, graphene has received more and more attention due to its
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intriguing physical and chemical properties [43]. Graphene oxide (GO), a novel
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two-dimensional carbon nanomaterial prepared from natural graphite, has recently
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attracted significant interesting as a precursor of chemically converted graphene
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[44]. Apart from the layered structure with a large theoretical specific surface area
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(2630 m2/g), GO nanosheets bear hydroxyl and epoxy groups on the graphitic
112
plane and carboxylic acid groups at the sheet edges. The presence of such groups
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not only provides the GO sheets excellent hydrophilicity, but also offers potential
114
application as substrates for the preparation of novel composites [45-47]. Recently
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Au nanoparticles (Au NPs) were decorated on GO surface as novel composites.
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GO/Au composites were sucessfully used for biocatalysis, cell imaging, SERS and
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glycoproteomics [48-51]. GO/Au composites, with excellent hydrophilicity and
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abundant reaction sites, were considered as potential support for immobilizing
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boronic
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modification 4
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composites
with
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enrichement with excellent hydrophilicity and large amount of boronic acid groups.
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Herein, GO/PEI/Au/4-MPB composites were synthesized via loading Au NPs on GO
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by in situ growth using polyethylenimine as reducing and immobilizing reagents,
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followed by 4-MPB immobilization through Au-S bond to construct novel boronic
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acid functionalized materials.
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2. Experimental
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2.1. Chemical and materials
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GO was purchased from Xianfeng Nanotech (Nanjing, China). Polyethylenimine,
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(ethylenediamine end-capped, PEI, Mn 600), 4-mercaptophenylboronic acid (4-MPB),
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TPCK-treated trypsin, bovine serum albumin (BSA), horseradish peroxidase (HRP),
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asialofetuin (ASF), dithiothreitol (DTT), iodoacetamide (IAA), formic acid (FA),
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boronic acid agarose, urea and trifluoroacetic acid (TFA) were obtained from Sigma
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(St. Louis, MO, USA). 2, 5-dihydroxybenzoic acid (DHB) was obtained from Bruker
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(Daltonios, Germany). Acetonitrile (ACN) was of HPLC grade from Merck
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(Darmstadt, Germany). All other reagents were of analytical grade purchased from
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China. Water was purified using a Milli-Q system (Darmstadt, Germany).
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2.2. Synthesis of GO/PEI/Au/4-MPB composites In a typical process, 0.3 mL PEI (100 mg/mL) was added into 1 mL GO
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suspension (0.1 mg/mL) under vigorous stirring for 1 h, and unreacted PEI was
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removed by centrifugation. The obtained PEI functionalized GO composites (GO/PEI)
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was dispersed in 1 mL H2O and 20 µL HAuCl4.3H2O (100 mg/mL) was added to the
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above suspension. The resulting suspensions were heated at 60 ºC for 1 h. After
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washing and centrifugation, the resulted composites (GO/PEI/Au) were vacuum-dried
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for further use. In the next step, the resultant GO/PEI/Au composites were mixed with
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15.4 mg 4-MPB at 1 mL ethanol, with strong stirring at room temperature for 24 h.
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After reaction, the mixture was washed with ethanol for three times, and then
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vacuum-dried to obtain GO/PEI/Au/4-MPB composites.
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2.3. Sample preparation The standard glycoprotein was dissolved in 1 mL of 50 mM NH4HCO3 (pH 8.0)
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and denatured at 90 °C for 15 min. Then, TPCK-treated trypsin was added to a final
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volume of 1:40 w/w of glycoprotein. The tryptic digestion proceeded at 37 ºC for 12 h.
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BSA was dissolved in 50 mM NH4HCO3 (pH 8.0) containing 8 M urea, and then
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reduced in 10 mM DTT for 1 h at 56 °C. When cooled to room temperature, cysteines
156
were alkylated in the dark in 20 mM IAA for 30 min at 37 °C. After being diluted
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ten-fold with 50 mM NH4HCO3 (pH 8.0), the solution was subsequently treated with
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trypsin at 37 °C (enzyme/protein ratio of 1:40, w/w) for 12 h. All digestion was
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stopped with FA to a final concentration of 1%. Tryptic digests were desalt by C18
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column and stocked at -20 ºC for further use.
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2.4. Glycopeptides enrichment under hydrophilic mode
Firstly, 20 µg material was dispersed in 50 µL ACN/H2O/FA (80: 20: 0.1, v/v/v)
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containing 0.1 µg HRP digests. The capture procedure was carried out under gentle
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agitation at room temperature for 10 min. After reaction, the supernatant was
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discarding by centrifugation. Secondly, 20 µL ACN/H2O/FA (80: 20: 0.1, v/v/v. 3
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times) was used to remove the non-glycopeptides. Finally, glycopeptides were eluted
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with 20 µL ACN/H2O/FA (60: 40: 0.1, v/v/v). The collected peptides were analysis by
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MALDI-TOF MS.
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2.5. Enrichment of glycopeptides by GO/PEI/Au/4-MPB composites The tryptic glycoprotein mixture was incubated in 200 µL NH4HCO3 (50 mM,
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pH 9.0) containing 100 µg GO/PEI/Au/MPB composites with strong shaking for 2 h
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at room temperature. After washing with 200 µL NH4HCO3 for three times, the
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solution of ACN/H2O/TFA (50:49:1 (v/v/v), 20 µL) as the elution buffer was added to
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release the glycopeptides. The eluent fraction was lyophilized in a SpeedVac (Thermo
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Fisher, San Jose, CA, USA), and dissolved in 2 µL DHB (20 mg/mL, 0.1% TFA in 60%
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ACN aqueous solution) for MALDI-TOF MS analysis.
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2.6. MS analysis and date research All MALDI spectra were taken from a Bruker Ultraflex III MALDI-TOF/TOF
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MS instrument (Bruker, Daltonios, Germany). A total of 1 µL of DHB solution was
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added to MALDI plate. The laser intensity was kept constant for all samples. External
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calibration of MALDI-TOF/TOF MS spectra was performed with ten commercial
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peptides.
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2.7. Characterization
Transmission electron microscopy (TEM) image was obtained by JEOL JEM-2000
transmission
electron
microscope
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High-resolution TEM (HRTEM) images were collected on a Tecnai G2 F30 S-Twin
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microscope operated at 300 kV (Tecnai G2, FEI, Eindhoven, Netherlands). Zeta
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potential was detected by Malven Nano-zs90 dynamic light scattering (Malven,
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Worcester, UK). Fourier-transformed infrared spectroscopy (FT-IR) characterization
194
has been performed on Perkin-Elmer Spectrum GX spectrometer (Perkin-Elmer,
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Waltham, USA). X-ray photoelectron spectroscopy (XPS) measurements were
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conducted with Thermo ESCALAB250Xi spectrometer with Al Kα radiation as the
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X-ray source (Thermo, Waltham, USA). Thermo Gravimetric Analysis (TGA) was
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conducted with Netzsch STA449F3 thermal analyzer (Netzsch, Bavaria, Germany)
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that was fitted to a nitrogen purge gas at 10 ºC/min heating rate.
Tokyo,
Japan).
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3. Results and discussion
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3.1. Preparation of GO/PEI/Au/4-MPB composites
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(JEOL,
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Fig. 1 depicted the synthesis route of GO/PEI/Au/4-MPB composites. Firstly,
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PEI was assembled on the surface of GO by electrostatic and hydrogen bonding
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interaction. The attached PEI significantly enhanced the hydrophilicity of GO due to
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its abundant amino groups [51]. Secondly, Au NPs were in suit introduced on the
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surface of GO/PEI nanosheets using PEI as reducing and stabilizing reagents. The
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obtained composites were denoted as GO/PEI/Au composites. Finally, GO/PEI/Au
209
composites were used as hydrophilic support for immobilizing 4-MPB to prepare 7
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GO/PEI/Au/4-MPB composites. Zeta potential of GO changed from -67 to 43 mv after the self-assembly of PEI,
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which suggested that a large amount of PEI was immobilized on GO surface. PEI
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served as primers for the adsorption of anionic AuCl4- [52]. By combination of the
214
multifunctional PEI, the attachment of Au NPs on the GO surface was achieved. Fig.
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2 showed the typical TEM images of GO before and after generation Au NPs. It was
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found that well-dispersed GO nanosheets were observed in Fig. 2A. However, as
217
shown in Fig. 2B and C, after Au NPs immobilization, Au NPs homogeneously
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spread on the surface of GO/PEI with high density, which greatly increased the
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number of reaction sites of the composites. High-resolution TEM (HR-TEM), as
220
shown in Fig. S1, revealed the highly crystalline nature of Au NPs with a lattice
221
spacing of 0.24 nm, validating the generation of Au NPs [53]. These results clearly
222
verified that Au NPs were successfully prepared and immobilized on GO/PEI surface.
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The immobilized PEI not only stabilized Au NPs, but also improved the
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hydrophilicity of GO/PEI/Au composites. Under HILIC mode, tryptic HRP were
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applied to evaluate hydrophilic capacity of GO, GO/PEI and GO/PEI/Au composites.
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Only 1 glycopeptides was detected in the eluting fraction of GO (Fig. S2A). However,
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the non-glycopeptides were almost removed and 5 glycopeptides with enhanced MS
228
intensities were founded in the eluting fraction of GO GO/PEI/Au (Fig. S2B and C).
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The results demonstrated that PEI significantly enhanced the hydrophilicity of
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GO/PEI/Au composites.
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by FT-IR. Fig. 3A, for GO, the peak around 1728 cm-1 corresponded to the C=O
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stretching vibration of the carboxylic group. After PEI was self-assembled on the GO
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surface, the C=O stretching vibration almost disappears, and new peaks at 2933 and
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2838 cm-1 ascribing to C–H stretch appear, confirming the successful self-assembly of
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PEI. GO/PEI/Au composites showed the similar adsorption peaks as GO/PEI
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nanosheets. Compared with GO/PEI/Au composites, there appeared new B-O
238
vibration (1338 cm-1) peak in the curve of GO/PEI/Au/4-MPB composites [31],
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which demonstrated the successful immobilization of 4-MPB. The survey XPS 8
ACCEPTED MANUSCRIPT spectra of GO/PEI/Au/4-MPB composites were showed in Fig. 3B. It depicted C, O,
241
N, S, B and Au elements, indicating the successful preparation of GO/PEI/Au/4-MPB
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composites [33]. The immobilization amount of 4-MPB played an important role for
243
enrichment efficiency of GO/PEI/Au/4-MPB composites. As shown in Fig. 4,
244
GO/PEI/Au/4-MPB composites showed the similar mass loss curves to that of
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GO/PEI/Au composites. Slower mass loss (∼5%) over the whole temperature range
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between 600 and 800 ºC can also be seen. Taking into accounts of the data of
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GO/PEI/Au and GO/PEI/Au/4-MPB at 700 ºC, 4-MPB content in GO/PEI/Au/4-MPB
248
was calculated as 14.3% wt (∼143 mg/g), which was higher than previous boronic
249
acid functionalized material [25]. The high immobilization amount might be attribute
250
to a large surface area of GO and a large number of Au NPs. The morphology of
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prepared GO/PEI/Au/4-MPB composites was characterized by TEM. As shown in Fig.
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S3, abundant dispersed Au NPs was found, and scarcely any Au NPs dissociated from
253
GO/PEI/Au/4-MPB composites was observed. We could expect that the prepared
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GO/PEI/Au/4-MPB composites, with excellent hydrophilicity, large amount of
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boronic acid groups, had great potential for the selective enrichment of glycopeptides
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from complex samples.
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3.2. Enrichment Efficiency of GO/EPI/Au/4-MPB Composites
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Tryptic HRP was employed as samples to evaluate the performance of
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GO/PEI/Au/4-MPB composites. According to previous research, NH4HCO3 (50 mM,
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pH 9.0) and ACN/H2O/TFA (50/49/1, v/v/v) were used as loading and elution buffers,
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respectively [41]. As shown in Fig. 5A, before enrichment, almost no peaks matching
263
with glycosylated peptides could be determined, which was attributed to the existence
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of abundant high-abundance non-glycosylated peptides. However, as shown in Fig.
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5B, after enrichment, non-glycopeptides were almost removed and 12 glycopeptides
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with enhanced MS intensities and S/N dominated the MS spectra (glycopeptides
267
information was shown in Table. S1). Even the concentration of tryptic HRP was
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decreased to 0.05 ng/µL (10 ng), 7 glycopeptides were positively identified (Fig. 5C).
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For comparison, an equivalent amount of commercial boronic acid agarose was used
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signals representing glycopeptides could be detected, indicating that the
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GO/PEI/Au/4-MPB composites have better sensitivity than the commercial boronic
273
acid agarose. The results evaluated that GO/PEI/Au/4-MPB composites show high
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selectivity to low concentration glycopeptides. The superior performance was
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attributed to high immobilized amount of boronic acid groups of GO/PEI/Au/4-MPB
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composites.
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The GO/PEI/Au/4-MPB composites were also applied to enrich glycopeptides
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from tryptic ASF. Fig. 6 displayed the MALDI-TOF MS spectra of 5 ng/µL tryptic
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ASF before and after enrichment by GO/PEI/Au/4-MPB composites. It was found that
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glycopeptides and the fragments dominant the mass spectra after enrichment, and the
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number of glycopeptides increased from 3 to 6 (glycopeptides information was shown
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in Table. S2). Inspired by the satisfying enrichment results, GO/PEI/Au composites
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were also applied to capture glycopeptides form tryptic ASF under the same
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conditions. It was found that the GO/PEI/Au composites showed no enrichment
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ability to glycopeptides, suggesting it was the boronic acid groups that displayed
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affinity to glycopeptides.
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GO/PEI/Au/4-MPB composites were further applied to isolate glycopeptides
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from the different mass ratios of tryptic BSA and ASF. Fig. 7 showed the
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MALDI-TOF MS spectra of identified glycopeptides from different mass ratios of the
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tryptic mixture of ASF and BSA. As shown in Fig. 7A, 5 glycopeptides were clearly
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detected from 0.5 ng/µL tryptic ASF. Five glycopeptides were still detected with
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non-glycopeptides scarcely identified in both 1:1 and 10:1 (Fig. 7B, C). Even the ratio
293
was further increased to 100:1, the ion signal of glycopeptides was remain observable
294
and 4 glycopeptides were identified (Fig. 7D). The superior enrichment capacity was
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ascribed to the excellent hydrophilicity of GO/PEI/Au/4-MPB composites, which
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greatly reduced the non-specific adsorption. The results demonstrated the reliability of
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GO/PEI/Au/4-MPB composites for separating glycopeptides from complex biological
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samples.
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4. Conclusion In summary, hydrophilic GO/PEI/Au/4-MPB composites were prepared and used
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to enrich glycopeptides from complex samples. GO/PEI/Au/4-MPB composites
303
exhibited high selectivity and sensitivity in the capture of N-linked glycopeptides.
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Due to higher hydrophilicity of composites, no nonspecific binding was observed in
305
the presence of prominent tryptic BSA, demonstrating specificity of binding.
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Comparison with commercial boronic acid resin, GO/PEI/Au/4-MPB composites
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showed better selectivity and sensitivity. Accordingly, with our newly developed
308
method, various kinds of N-glycopeptides could be specifically selected and enriched
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for further analysis. We also expected it could be further applied to complex
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biological samples. The work not only offered an boronate affinity material, but also
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opened a new field for applications of GO.
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Acknowledgements
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This work was supported by National Nature Science Foundation (21190043, 21235005
and
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(2012CB910601 and 2013CB911201), and Creative Research Group Project by
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NSFC (21321064).
National
Basic
Research
Program
of
China
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Fig. 1. Synthetic schematics of GO/PEI/Au/4-MPB composites.
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Fig. 2. TEM images of (A) GO, (B) and (C) GO/PEI/Au composites.
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Fig. 3. (A) FI-IR spectra of GO, GO/PEI/, GO/PEI/Au and GO/PEI/Au/4-MPB
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composites, (B) XPS survey spectra of GO/PEI/Au/4-MPB composites.
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Fig. 4. TGA curves of GO, GO/PEI, GO/PEI/Au and GO/PEI/Au/4-MPB composites.
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Fig. 5. MALDI-TOF MS spectra of (A) 5 ng/µL HRP before enrichment, (B) 5 ng/µL
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HRP after enrichment by GO/PEI/Au/4-MPB composites, (C) 0.05 ng/µL HRP after
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enrichment by GO/PEI/Au/4-MPB composites, (D) 0.05 ng/µL HRP after enrichment
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by commercial boronic resin. V= 200 µL.
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Fig. 6. MALDI-TOF MS spectra of (A) 5 ng/µL ASF before enrichment, (B) 5 ng/µL
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ASF after enrichment by GO/PEI/Au/4-MPB composites. V=200 µL.
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Fig. 7. MALDI-TOF MS spectra of 0.5 ng/µL ASF with different mass ratio of BSA
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(A)1:0, (B) 1:1, (C) 10:1 and (D) 100:1. BSA/ASF, V= 200 µL.
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Fig. 1. Synthetic schematics of GO/PEI/Au/4-MPB composites.
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Fig. 2. TEM images of (A) GO, (B) and (C) GO/PEI/Au composites.
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Fig. 3. (A) FI-IR spectra of GO, GO/PEI/, GO/PEI/Au and GO/PEI/Au/4-MPB composites, (B) XPS survey spectra of GO/PEI/Au/4-MPB composites.
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Fig. 4. TGA curves of GO, GO/PEI, GO/PEI/Au and GO/PEI/Au/4-MPB composites.
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Fig. 5. MALDI-TOF MS spectra of (A) 5 ng/μL HRP before enrichment, (B) 5 ng/μL HRP after enrichment by GO/PEI/Au/4-MPB composites, (C) 0.05 ng/μL HRP after enrichment by GO/PEI/Au/4-MPB composites, (D) 0.05 ng/μL HRP after enrichment by commercial boronic resin. V= 200 μL.
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Fig. 6. MALDI-TOF MS spectra of (A) 5 ng/μL ASF before enrichment, (B) 5 ng/μL ASF after enrichment by GO/PEI/Au/4-MPB composites. V=200 μL.
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Fig. 7. MALDI-TOF MS spectra of 0.5 ng/μL ASF with different mass ratio of BSA (A)1:0, (B) 1:1, (C) 10:1 and (D) 100:1. BSA/ASF, V= 200 μL.
