Glucose-6-Phosphate-Functionalized Magnetic Microsphere as Novel

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Glucose-6-Phosphate Functionalized Magnetic Microsphere as Novel Hydrophilic Probe for Specific Capture of N-linked Glycopeptides Yilin Li, Jiawen Wang, Nianrong Sun, and Chun-hui Deng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03708 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on October 1, 2017

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Analytical Chemistry

3

Glucose-6-Phosphate Functionalized Magnetic Microsphere as Novel Hydrophilic Probe for Specific Capture of N-linked Glycopeptides

4

Yilin Li, Jiawen Wang, Nianrong Sun* and Chui-hui Deng*

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Department of Chemistry and Institutes of Biomedical Sciences, Collaborative

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Innovation Center of Genetics and Development, Fudan University, Shanghai, 200433,

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China

1 2

8

1

ACS Paragon Plus Environment


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ABSTRACT: Developing cost-effective approaches based on hydrophilic interaction

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liquid chromatography (HILIC) has been the main tendency for low-abundance

11

glycopeptides capture before LC-MS/MS analysis. Carbohydrates with outstanding

12

biocompatibility and hydrophilicity are ubiquitous in the kingdoms of animal and

13

plant and could be a wonderful choice as functional groups for glycopeptides

14

enrichment. In this work, glucose-6-phosphate, as one of the indispensable cogs in

15

pivotal metabolic wheels of life, was chosen as functionalized groups to be grafted

16

onto the surface of Fe3O4 microspheres via one-step surface fabrication strategy. The

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acquired hydrophilic Fe3O4@G6P microspheres showed superior enrichment

18

performance for glycopeptides with high sensitivity (0.5 fmol/µL) and high selectivity

19

(1:100) and good repeatability (10 times at least). Furthermore, the Fe3O4@G6P

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microspheres also exhibited enrichment ability for glycopeptides in different

21

bio-samples. A total of 243 glycopeptides assigned to 92 glycoproteins and 183

22

glycopeptides corresponding to 74 different glycoproteins was identified from merely

23

2 µL serum and saliva, respectively.

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2


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Glycosylation plays significant and specific roles in many biological events, such as

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host-pathogen interaction, signal transduction and tumor immunology.1-3 Elucidation

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of the relationship between glycosylation and its bio-function requires to obtain more

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detailed information about glycosylation sites and glycan structures and peptide

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sequences, etc.4 Mass spectrometry (MS)-based technique including matrix-assisted

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laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and

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electrospray ionization mass spectrometry (ESI-MS) has occupied a primary position

32

in glycosylation analysis attributed to its high throughput and sensitivity and so forth.5

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However, direct MS analysis of glycopeptides is still a challenge, since glycopeptides

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merely account for a very minor proportion in complex bio-samples and the

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co-existing interferences including non-glycopeptides and salts would suppress their

36

signals seriously during MS analysis.6,7 Therefore, highly specific enrichment

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strategies for glycopeptides are necessary before MS analysis.

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At present, developing cost-effective strategies based on hydrophilic interaction has

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become the main tendency for low-abundance glycopeptides capture before

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LC-MS/MS analysis.8-10 Owing to their advantages of higher glycosylation coverage

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and unbiased recognition ability for glycopeptides, as well as excellent compatibility

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with MS analysis, a great amount of hydrophilic materials was designed and

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synthesized for glycopeptides enrichment.11-14 Especially hydrophilic magnetic

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materials have been obtaining increasing popularity in glycoproteomics analysis not

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only because of the above advantages but also the following factors.15-18 Firstly,

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magnetic microspheres are easily obtained and they are nontoxic and bio-compatible. 3



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Secondly, magnetic microsphere could offer strong magnetic responsiveness to realize

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rapid separation of glycopeptide-conjugated microsphere from sample solution.

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Thirdly, the surface of magnetic microsphere is apt to be modified with numerous

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linkers through various routes and functionalized with different purposeful groups

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furtherly.

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Carbohydrates, a kind of organic compounds, which own maximum content in

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nature and are ubiquitous in the kingdoms of animal and plant, are the cheapest one in

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body’s major nutrients and possess excellent biocompatibility and hydrophilicity.19

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Recently, carbohydrate-based magnetic materials have been most widely applied for

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glycopeptides enrichment. For instance, Li et al. grafted PEI onto the surface of

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magnetic

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azido-functionalization and followed modification with maltose via “click chemistry”,

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which could provide large amounts of hydrophilic groups and exhibit high specificity

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and high capturing capacity for glycopeptides;20 Xiong et al. reported the synthesis of

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multilayer polysaccharide coated magnetic microsphere using layer-by-layer

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assembly of hyaluronan and chitosan, which presented high selectivity, low detection

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limit and large binding capacity for glycopeptides enrichment;21 Zheng et al. also

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designed and prepared glucose-functionalized hydrophilic magnetic mesoporous

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nanoparticles (Fe3O4@mSiO2-glucose MMNs) via click chemistry, which could offer

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higher efficiency in glycopeptides enrichment compared to the previous reported

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approaches.7 However, these reports all needed multistep modification to acquire

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carbohydrate-coated magnetic microspheres that made the synthetic processes tedious.

microsphere

by

one-pot

solvothermal

reaction,

further

4


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Therefore, it is highly desirable to explore a facile and effective protocol to prepare

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carbohydrate-based magnetic materials for glycopeptides enrichment.

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According to literature,

8

due to the fact that there are plenty of Fe3+/Fe2+ binding

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sites on the surface of Fe3O4 microspheres, it is a feasible and effective way in which

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the functional groups were grafted directly onto the surface via chelation between

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metal ions and organic ligands. For example, dopamine derivatives (such as

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azido-terminal dopamine and nitro-linked dopamine, etc.) could pass for anchors on

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the surface of Fe3O4 microspheres owing to their robust binding effect of catechol unit

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to Fe3O4.22-25 Similarly, as we all know, metal ion affinity chromatography (IMAC),

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which depends on the specific affinity of metal ions (Ti4+, Ga3+, Fe3+, etc.) for

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phosphate groups, has been one widely employed for phosphoproteomics

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research.26-28 And phosphorylated carbohydrates with excellent hydrophilicity are

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obbligato cogs for several pivotal metabolic wheels of life.29 Hence, in this work, for

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the first time, we designed a one-step surface modification protocol to prepare

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hydrophilic glucose-6-phosphate functionalized Fe3O4 microspheres (denoted as

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Fe3O4@G6P). In particular, we chose glucose-6-phosphate as anchor to be

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immobilized directly on the surface of Fe3O4 microspheres by taking advantage of the

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specific interaction between metal ions and phosphate groups.26 The glucose that

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existed on the anchor endowed the microspheres with high hydrophilic surface

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simultaneously. Serum and saliva are common clinical specimen and potential sample

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for disease diagnose, respectively. So, we applied Fe3O4@G6P microspheres to

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specifically extract N-linked glycopeptides from complicated bio-samples including 5



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human serum and human saliva, and Fe3O4@G6P microspheres exhibited highly

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specific capture ability for glycopeptides.

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EXPERIMENTAL SECTION

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Materials and chemicals. Glucose-6-phosphate (G6P) were purchased from

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Beijing bailingwei Technology Co. Ltd. Dithiothreitol (DTT), iodoacetamide (IAA),

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2,5-Dihydroxy-benzoic acid (DHB), horseradish peroxidase(HRP), bovine serum

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albumin (BSA), and immunoglobulin G(IgG) were purchased from Sigma Aldrich.

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PNGase F was purchased from Genetimes Technology. Human serum was offered by

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Shanghai Zhongshan Hospital from a healthy volunteer. Trifluoroacetic acid (TFA),

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acetonitrile (ACN) was purchased from Merck (Darmstadt, Germany). All deionized

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water in the experiment was prepared by Milli-Q system (Millipore, Bedford, MA).

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All the other reagents are of analytical grade and were purchased from Shanghai

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Chemical Reagent.

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Synthesis of Fe3O4@G6P miscrospheres. The Fe3O4 microspheres were

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prepared by a solvothermal reation. In brief, 1.35 g FeCl3·6H2O were dispersed in 75

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mL of ethylene glycol. Then, 3.6 g NaAc was added into the mixture and stirred for

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30 min. The solution was then transferred to a Teflon stainless steel autoclave at

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200 °C for 12 h. The obtained microspheres were collected with a magnet and washed

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sequentially with ethanol and water for three times. 50 mg Fe3O4 microspheres were

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dispersed in mixture solution containing 25 mL of acetonitrile, 25 mL of H2O and 50

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µL of TFA under the condition of sonication. Then, 50 mg glucose-6-phosphate was

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added into the above mixture. The mixed solution was stirred at 25 °C for 6 h. The 6


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obtained microspheres (denoted as Fe3O4@G6P) were washed with deionized water

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and ethanol for three times in order and dried in vacuum at 50 °C overnight.

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Digestion of standard proteins and bio-sample. 2 mg standard protein (HRP

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or IgG) and 500 µL of ammonium bicarbonate (50 mM, pH 8.3) were mixed by

117

shaking, and then protein solution was denatured at 100 °C for 10 min. Afterward,

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500 µL of Milli-Q water was added into the solution to form a final concentration of 2

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mg/mL. Also, trypsin (trypsin/protein: 1/40, w/w) was then added into the solution

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incubated at 37 °C overnight.

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Human serum (2 µL) was diluted in 16 µL NH4HCO3 (25mM, pH 7.9) buffer and

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denatured for 10 min. The mixture was reduced by DTT at 37 °C for 30 min and

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alkylated by IAA at 37 °C for 1 h in the dark. Then the obtained mixture was

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incubated with trypsin (trypsin/ protein is 1/40, w/w) at 37 °C for 16 h. Tryptic digests

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were lyophilized for further enrichment and analysis. The treatment method of saliva

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was the same as human serum.

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Protocol of enrichment process. 200 µg Fe3O4@G6P microspheres were

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dispersed in 100 µL loading buffer (ACN/H2O/TFA, 90/9/1, v/v/v) containing 1

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pmol/µL HRP digestion. The obtained solution was incubated in a vortex at 37 °C for

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30 min. Then, the microspheres were separated from solution with a magnet and

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washed with 200 µL washing buffer (ACN/H2O/H3PO4, 85/14.5/0.5, v/v/v) three

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times to remove non-glycopeptides. After that, 10 µL of 50% ACN were added into

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the microspheres and vibrated for 30 min at 37 °C. Finally, the elution was collected

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under magnetic separation and was analyzed directly by MALDI-TOF MS. 7



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For the glycopepetides enrichment from human serum, the lyophilized tryptic

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digests of human serum were redissolved in 200 µL loading buffer (ACN/H2O/TFA,

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90/9/1, v/v/v) and then 400 µg Fe3O4@G6P microspheres were added into the above

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solution. The mixture was vibrated for 30 min at 37 °C. Then, the microspheres were

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separated and washed three times with washing buffer. After that, the glycopeptides

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were eluted twice from the microspheres with eluting buffer (50% ACN). Finally, the

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collected elution was lyophilized and redissolved in 60 µL 25 mM NH4HCO3 solution,

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and then 1 µL PNGase F was added and incubated at 37 °C for 16 h to remove the

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glycans. The solution was lyophilized and redissolved in 5% ACN/0.1%TFA solution

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for further LC-MS/MS analysis.

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Characterization and MS analysis. The detailed characterization and instruments

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were shown in the Supporting Information.

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RESULTS AND DISCUSSION

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Preparation and characteristics of Fe3O4@G6P microspheres. The

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fabrication procedure of Fe3O4@G6P microspheres through one-step modification

150

presented in Scheme 1. Briefly, Fe3O4 microspheres was prepared via solvothermal

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reation, and then glucose-6-phosphate was grafted onto the surface of Fe3O4 to obtain

152

the Fe3O4@G6P microspheres with excellent hydrophilicity and biocompatibility.

153

8


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Scheme 1. Synthetic procedure of Fe3O4@G6P microspheres.

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Firstly, transmission electron microscopy (TEM) and scanning electron microscope

157

(SEM) were employed to characterize the morphology of Fe3O4@G6P microspheres.

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From the Figure 1, the average diameter of Fe3O4@G6P microspheres was estimated

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to be around 200 nm. Also, the morphology and size of Fe3O4@G6P microspheres

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was studied by atomic force microscopy (AFM) and dynamic light scattering (DLS),

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the topographies of 5 µm2 area and 3D representations were shown in Figure S1

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(supporting information). The particle height measured by AFM was around 189.5 nm

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and the size distribution of Fe3O4@G6P via DLS measurement was around 245.8 nm

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with 0.177 of particle dispersion index (PDI), which were closely with the particle

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diameter from TEM. Then the magnetic properties of Fe3O4 and Fe3O4@G6P

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microspheres were explored. As shown in Figure S2 (supporting information), the

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saturation magnetic (Ms) values of Fe3O4, Fe3O4@G6P microspheres were estimated

168

to be 88.85 and 80.21 emu·g−1, respectively. This indicated that Fe3O4@G6P

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microspheres possessed great magnetic responsiveness, which could be verified by

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separating Fe3O4@G6P microspheres from water with the external magnetic field 9



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(Figure S3).

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Figure 1. SEM images of (A) Fe3O4@G6P microspheres. TEM images of (B，C)

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Fe3O4@G6P microspheres.

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The successful modification of glucose-6-phosphate on the surface of Fe3O4 was

176

confirmed by fourier-transform infrared spectrophotometer (FTIR). As shown in

177

Figure 2, the obvious adsorption peak at 570 cm-1 was assigned to the vibration of

178

Fe-O-Fe, which suggested the successful synthesis of Fe3O4. The adsorption peaks at

179

1220 cm-1 and 1080 cm-1 could be ascribed to the stretching vibration of P=O and

180

P-O30, respectively, indicating successful immobilization of phosphate groups on the

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surface of Fe3O4. The adsorption peaks at 3420 cm-1 and 2950 cm-1 were assigned to

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the stretching vibration of O-H and CH2, respectively, which were increased slightly

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compared to those in FTIR spectrum of Fe3O4. The differences between the FTIR

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spectrum of Fe3O4 and Fe3O4@G6P microspheres proved the successful graft of

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glucose-6-phosphate on the surface of Fe3O4 microspheres.

10


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Figure 2. FTIR spectra of (A) Fe3O4 microspheres and (B) Fe3O4@G6P

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microspheres.

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Next, X-ray photoelectron spectroscopy (XPS) was employed to characterize the

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surface composition of two kinds of nanoparticles (Fe3O4 and Fe3O4@G6P). As

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shown in Figure 3A, the peaks of O 1s, C 1s, Fe 3p and Fe 2p can be evidently

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observed. Meanwhile, the signal of P 2p can be observed at 134.2-135 eV obviously

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in narrow-spectrum, suggesting the successful binding of glucose-6-phosphate on the

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surface of Fe3O4. As shown in Figure 3B, the O 1s spectrum can be split into five

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peaks, curve a, b, c, d, e can be assigned to P=O, O-H, Fe-O, P-O-Fe, C-O bond

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respectively,

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glucose-6-phosphate furtherly.31-35

which

confirmed

the

bonding

mode

between

Fe3O4

and

11



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Figure 3. XPS spectra of (A) Fe3O4@G6P microspheres (a); Fe3O4 nanoparticles (b)

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and (B) O 1s.

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Additionally, thermogravimetric analysis (TGA) was adopted to evaluate the

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ingredients proportion of Fe3O4@G6P microspheres and the result was shown in

203

Figure S5 (supporting information). The weight loss of Fe3O4 was about 5% at

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650 ℃, while an additional weight loss of approximately 2.6% could be observed

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after modification with the glucose-6-phosphate. Moreover, as shown in Figure S6

206

(supporting information), elemental analysis of the Fe3O4@G6P microspheres

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revealed the existence of Fe, C, O and P. All the above results should be attributed to

208

the triumphant modification of glucose-6-phosphate, for which the Fe3O4@G6P

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microspheres would possess remarkable hydrophilicity and make great contribution to

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the glycopeptides enrichment.

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The contact angles of Fe3O4 and Fe3O4@G6P microspheres were measured to

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evaluate the relative hydrophilicity of surface by using deionized water as liquid

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probe (Figure 4). As expected, the contact angle became smaller after immobilization

214

with G6P, which indicated a better hydrophilic surface due to the successful 12


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preparation of Fe3O4@G6P microspheres. The contact angle of Fe3O4 and

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Fe3O4@G6P microspheres were 56.88° and 15.57°, respectively, implying that the

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presence of G6P was helpful to improve the hydrophilicity of microspheres.

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Figure 4. Water contact angles of (A) Fe3O4 and (B) Fe3O4@G6P microspheres.

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Optimization of incubation conditions of Fe3O4@G6P microspheres. The

221

ability of Fe3O4@G6P microspheres for specific capture of glycopeptides was tested

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by enriching glycopeptides from mixture of strandard protein digestion. The

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enrichment workflow of Fe3O4@G6P microspheres for glycopeptides is illustrated in

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Scheme 2. There could be classified into four main steps: enriching, washing, eluting

225

and MS analysis. After incubation, glycopeptides were retained on the surface of

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Fe3O4@G6P microspheres with non-glycopeotides washed away at the same time.

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Then the eluent was analyzed by MS.

13



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Scheme 2. The enrichment workflow of Fe3O4@G6P microspheres for glycopeptides.

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At first, horseradish peroxidase (HRP) was chosen as standard glycoprotein to

231

verify

232

glycopeptides and explore the optimal experiment conditions. According to previous

233

reports,36,37 the concentration of acetonitrile (ACN) is quite important for

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glycopeptides enrichment based on hydrophilic interaction methods. Therefore, we

235

selected three loading buffer containing different ACN concentration to conduct

236

enrichment experiment. As shown in Figure S7A (supporting information), when

237

using ACN/H2O/TFA (80/19/1, v/v/v) as loading buffer, there were many

238

non-glycopeptides in the MS spectrum after enrichment with Fe3O4@G6P

239

microspheres, and the number and intensities of glycopeptides were relatively lower

240

compared to those with high ACN concentration. From Figure S7B&C (supporting

241

information), the peak intensities improved significantly and the nonspecific

242

adsorption reduced considerably with the increase of ACN concentration. 85% ACN/1%

243

TFA was chosen as the loading buffer in the following enrichment experiment in

244

consideration of 90% ACN/1% TFA and 85% ACN/1% TFA showed similar results

the

enrichment

performance

of

Fe3O4@G6P microspheres

towards

14


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245

for glycopeptides enrichment. Next, we investigated the influence of incubation time

246

to the enrichment performance of Fe3O4@G6P microspheres, the results were

247

displayed in Figure S8. The results indicated that 20 min as incubation time was

248

enough to realize enrichment of glycopeptides. (Figure S8, supporting information).

249

Application of Fe3O4@G6P microspheres for the enrichment of N-linked

250

glycopeptides. By taking advantage of the optimized enrichment condition,

251

Fe3O4@G6P microspheres were applied to enrich glycopeptides from 100 fmol/µL

252

HRP digestion. As seen in Figure 5A, only 4 glycopeptides with low intensity were

253

detected by direct MS analysis and there were a great many non-glycopeptides in the

254

MS spectra. While 17 glycopeptides with enhanced signal intensity were detected by

255

applying Fe3O4@G6P microspheres to treat the same HRP digest (Figure 5C, detailed

256

information was listed in Table S1, supporting information). For comparison, Fe3O4

257

microspheres were also employed to experience the same process. As observed in

258

Figure 5B, Fe3O4 microspheres exhibited poor capture ability for glycopeptides,

259

which meant the excellent enrichment performance of Fe3O4@G6P microspheres

260

should be attributed to the good hydrophilic property of G6P. Furtherly, the

261

enrichment sensitivity of the Fe3O4@G6P microspheres for glycopeptides was

262

investigated by treating attenuation of the HRP digest. As displayed in Figure 6, when

263

the concentrations of tryptic HRP was decreased to merely 0.5 fmol/µL, there were

264

still four glycopeptides could be observed after enrichment by Fe3O4@G6P

265

microspheres, indicating that Fe3O4@G6P microspheres had a high sensitivity for

266

glycopeptides enrichment compared to those reported hydrophilic works8,10,36 (Table 15



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S2).

268 269

Figure 5. MS spectra of 100 fmol/µL HRP digest (A) before enrichment; enriched by

270

(B) Fe3O4 microspheres and (C) Fe3O4@G6P microspheres. Glycopeptides are

271

marked with a solid red circle.

16


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Figure 6. MALDI mass spectra of N-linked glycopeptides enriched from different

274

concentration of HRP digests (A) 10 fmol/µL; (B) 1 fmol/µL; (C) 0.5 fmol/µL using

275

Fe3O4@G6P microspheres. Glycopeptides are marked with a solid red circle.

276

In addition, the repeatability of Fe3O4@G6P microspheres for glycopeptides

277

enrichment was evaluated by using HRP digests as sample. The reused microspheres

278

would be washed with eluting buffer and loading buffer in order, for ensuring no

279

residual glycopeptides on the surface of Fe3O4@G6P microspheres, before next

280

enrichment procedure. Fe3O4@G6P microspheres could be recycled to capture 17



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glycopeptides for successive ten times at least, as seen in Figure S9 (supporting

282

information), compared to that result for the first time, the number and peak

283

intensities of glycopeptides presented little difference, indicating Fe3O4@G6P

284

microspheres could be reused for glycopeptides enrichemnt.

285

286 287

Figure 7. Glycopeptides enrichment by Fe3O4@G6P microspheres from mixture

288

containing HRP digests and different amount of BSA digest with different ratios

289

(w/w): (A) 1:10; (B)1:50; (C)1:100, respectively. Insert: direct MS analysis.

290

Glycopeptides are marked with a solid red circle. 18


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291

Afterwards, to demonstrate the ability of selective enrichment of Fe3O4@G6P

292

microspheres, glycopeptides analysis was carried out by adding different quantity of

293

non-glycoprotein BSA digest into HRP digest as interference peptides. The obtained

294

MS spectra showed the signals of glycopeptides were severely suppressed by

295

non-glycopeptides before enrichment with Fe3O4@G6P microspheres (Figure 7A,

296

insert), while 12 apparent glycopeptides with clear blackground could be observed

297

after the enrichment by Fe3O4@G6P microspheres (Figure 7A). When further

298

increasing mass ratio to 1:50 or 1:100, the peaks of glycopeptides could still be

299

obviously identified after enrichment with Fe3O4@G6P microspheres (Figure 7B&C),

300

suggesting the selective enrichment ability of Fe3O4@G6P microspheres for

301

glycopeptides and the enrichment potency of Fe3O4@G6P microspheres for

302

glycopeptides in bio-samples. Besides, immunoglobulin G (Ig G) digest with a

303

different glycan type from HRP was employed to test the unbiased enrichment

304

capability of Fe3O4@G6P microspheres for glycopeptides. As shown in Figure S10A,

305

glycopeptides

306

non-glycopeptides before the enrichment, whereas 14 peaks of glycopeptides could be

307

observed after the enrichment by Fe3O4@G6P microspheres, indicating the excellent

308

enrichment performance of Fe3O4@G6P microspheres furtherly.

were

hardly

observed

due

to

the

strong

interference

of

309

Encouraged by all the above results, we applied the Fe3O4@G6P microspheres to

310

enrich glycopeptides from different bio-samples. Human serum and human saliva as

311

the promising clinical specimen play a key role in disease diagnose. Herein we

312

employed different bio-samples including healthy human serum and human saliva for 19



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313

glycopeptides analysis. In brief, the human serum and saliva were reduced and

314

alkylated, and then treated with trypsin to obtain the peptides mixture. The acquired

315

glycopeptides after enrichment with Fe3O4@G6P microspheres were deglycosylated

316

by PNGase F and analyzed by Nano-HPLC-MS/MS. The results of three parallel

317

enrichment experiments using human serum as samples were displayed in Figure S11,

318

overlapping 118 glycopeptides and 60 glycoproteins could be identified, indicating a

319

good reproducibility of Fe3O4@G6P microspheres for complex samples. A total of

320

243 N-glycopeptides corresponding to 92 N-glycoproteins were identified, which

321

presented a better or similar ability to deal with complicated sample compared to

322

previous hydrophilic strategies8,10,36 (Table S2). Additionally, the venn diagram of

323

human saliva enrichment experiment was presented in Figure S12, overlapping 131

324

glycopeptides and 64 glycoproteins were identified, a total of 183 N-glycopeptides

325

from 74 N-glycoproteins were identified, furtherly demonstrating the reproducibility

326

of Fe3O4@G6P microspheres for complex samples.

327

in Table S4 and Table S5 in supporting information.

The detail information is listed

328 329

CONCLUSIONS

330

In conclusion, we have developed a high-efficiency approach to capture

331

glycopeptides from different biological samples by using the outstanding

332

hydrophilicity and biocompatibility of glucose-coated Fe3O4 magnetic microspheres.

333

Fe3O4@G6P microspheres were successfully synthesized via such a facile synthetic

334

route and simple operation, and the strong mangnetic responsiveness of Fe3O4 could 20


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335

realize the fast separation of Fe3O4@G6P microspheres from solution. Besides,

336

Fe3O4@G6P microspheres exhibited high selectivity and sensitivity and good

337

repeatability in the aspect of enrichment for glycopeptides using tryptic HRP as

338

standard sample, the unbiased enrichment ability for glycopeptides using tryptic HRP

339

and tryptic Ig G as samples. Moreover, different biological samples (human serum

340

&saliva) were applied to confirm that Fe3O4@G6P microspheres possess great

341

potential in glycopeptides profiling based on MS strategies.

342

ASSOCIATED CONTENT

343

Supporting Information. This material includes detailed experimental methods,

344

the supporting information is available free of charge on http://pubs.acs.org.

345

AUTHOR INFORMATION

346

Corresponding Authors

347

*N. R. Sun, E-mail: [email protected]. ORCID: 0000-0003-1511-7116

348

*C. H. Deng, E-mail: [email protected]. ORCID: 0000-0002-8704-7543

349

Notes

350

The authors declare no competing financial interest.

351

ACKNOWLEDGMENT

352

This work was financially supported by the National Natural Science Foundation of

353

China (21425518, 21405022 and 21675034) and National Basic Research Priorities

354

Program of China (2013CB911201).

355

REFERENCES

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TOC

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25



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Figure 1. SEM images of (A) Fe3O4@G6P microspheres. TEM images of (B，C) Fe3O4@G6P microspheres.

538x180mm (75 x 68 DPI)


Page 26 of 28

Page 27 of 28

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FTIR spectra of (A) Fe3O4 microspheres and (B) Fe3O4@G6P microspheres.

226x184mm (72 x 72 DPI)



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XPS spectra of (A) Fe3O4@G6P microspheres (a); Fe3O4 nanoparticles (b) and (B) O 1s.

338x139mm (150 x 150 DPI)


Page 28 of 28

Glucose-6-Phosphate-Functionalized Magnetic Microsphere as Novel

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