3-Carboxybenzoboroxole Functionalized Polyethylenimine Modified

Subscriber access provided by FLORIDA STATE UNIV

Article

3-Carboxybenzoboroxole Functionalized Polyethylenimine Modified Magnetic Graphene Oxide Nanocomposites for Human Plasma Glycoproteins Enrichment Under Physiological Condition Qiong Wu, Bo Jiang, Yejing Weng, Jianxi Liu, Senwu Li, Yechen Hu, Kaiguang Yang, Zhen Liang, Lihua Zhang, and YuKui Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04451 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on January 31, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

3-Carboxybenzoboroxole Functionalized Polyethylenimine Modified Magnetic Graphene Oxide Nanocomposites for Human Plasma Glycoproteins Enrichment under Physiological Condition

Qiong Wu1, 2#, Bo Jiang1#, Yejing Weng1, 2, Jianxi Liu1,3, Senwu Li1, 2, Yechen Hu1, 2

, Kaiguang Yang1, Zhen Liang1, Lihua Zhang1*and Yukui Zhang1

1. CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian 116023, China. 2. University of Chinese Academy of Sciences, Beijing 100049, China. 3. College of Environment Science and Engineering, Fujian Normal University, Fuzhou 350007, China. # These two authors contributed equally to this work. *To whom correspondence should be addressed. E-mail: [email protected]; Phone & fax: +86-411-84379720.

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 26

ABSTRACT Boronate affinity materials have been successfully used for the selective recognition of glycoproteins. However, by such materials, the large-scale glycoproteins enrichment from human plasma under physiological conditions is rarely reported.

In

this

work,

3-carboxybenzoboroxole

(CBX)

functionalized

polyethyleneimine (PEI) modified magnetic graphene oxide nanocomposites were synthesized. Benefited from the low pKa value of CBX (~6.9) and PEI dendrimer-assisted multivalent binding, the Freundlich constant (KF) for the adsorption of horseradish peroxidase (HRP) was 3.0−7.3 times higher than that obtained by previous work, displaying the high enrichment capacity. Moreover, PEI could improve the hydrophilicity of nanocomposites and reduce non-glycoprotein adsorption. Therefore, such nanocomposites were successfully applied to the analysis of human plasma glycoproteome under physiological condition, and the identified glycoproteins number and recognition selectivity was increased when compared to the results

obtained

by

previous

boronic

acid-functionalized

particles

(Sil@Poly(APBA-co-MBAAm)) under common alkaline condition (137 vs 78 and 67.8% vs 57.8%, respectively). In additional, thrombin (F2), an important plasma glycoprotein, labile under alkaline condition, was specifically identified by our method, demonstrating the great promising of such nanocomposites in the deep-coverage proteome analysis.

2


Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Human plasma proteome, especially glycoproteome, provides much valuable information for disease diagnosis and therapy.1-2 Unfortunately, the inherent

low-abundance

of

glycoproteins

hinders

the

deep-coverage

glycoproteome analysis.3-4 Recently, boronate affinity materials have attracted increasing attention for the selective enrichment of glycoproteins, due to the advantages of pH-controlled capture/release, unbiased recognition and compatibility with mass spectrometry.5-8 However, since the pKa values of commonly used boronic acid ligands, such as 3-acrylamidophenylboronic acid and 4-vinylphenylboronic acid, are higher than 8.0. The enrichment of glycoproteins is usually performed under alkaline conditions, resulting in the relatively low affinity and the risk of labile glycoproteins degradation.9-12 Therefore, the synthesis of novel boronate affinity materials with ligands of low pKa is indispensable to achieve the deep-coverage glycoproteome analysis of human plasma under physiological condition. Benzoboroxole (BX), an improved Wulff-type boronic acid, consists of a benzene ring fused with an oxaborole heterocycle,13 and displays the advantages of low pKa value (7.3), excellent hydrophilicity and improved affinity to saccharides under neutral condition.14 BX and its derivatives have been used as the ligands for the selective

binding

of

saccharides,15-16

oligosaccharide,17

nucleosides18

and

glycoproteins.9-10 Among BX family, 3-carboxybenzoboroxole (CBX) possesses the even lower pKa value (6.9),19 and higher affinity to cis-diol compounds, contributed by the electron-withdrawing effect of carboxyl. Therefore, CBX is considered as a powerful ligand to prepare boronate affinity materials. For example, Liu group prepared the CBX-functionalized monolithic columns for the selective enrichment

and

separation

of

nucleosides/glycoproteins,19

glycoproteins/glycopeptides20 and glycoprotein-binding DNA aptamers21 with improved selectivity and affinity under neutral or acidic condition. However, such 3



materials have not been used in the large-scale glycoproteins enrichment, especially in human plasma proteome analysis. Besides, according to the previous work,22-25 the binding capacity of monolithic columns is usually lower than that of nanocomposites. Therefore, the development of CBX-functionalized nanomaterials might be of great significance to achieve the deep-coverage glycoproteome analysis of human plasma under physiological conditions, and to reflect the real physiological status of proteins. Besides functional ligands, the structure and property of matrices also influence the binding affinity of boronate affinity materials. Two-dimensional graphene oxide (GO) has recently attracted much attention in proteomic study, due to its excellent physical and chemical properties.26-28 Apart from the layered structure with large theoretical specific surface area,29-31 GO nanosheets bear hydroxyl and epoxy groups on the graphitic planes and carboxylic acid groups at the sheet edges,32-35 preferable to achieve the functionalization. Furthermore, polyethyleneimine (PEI) with branched structure is verified as an excellent scaffold to amplify the number of boronic acids ligands, to improve the binding affinity toward glycoproteins.22, 36 The abundant amine groups on PEI chain can also improve the hydrophilicity of functionalized materials, beneficial to reduce the adsorption of non-glycoproteins.37-38 Therefore, in this work, we synthesized CBX-functionalized PEI modified GO nanocomposites, by which the enrichment of glycoproteins in human plasma under physiological condition was achieved.

EXPERIMENTAL SECTION Materials and Chemicals Graphene oxide (GO) was purchased from Plannano Nanotech (Tianjin, China). 3-carboxybenzoboroxole (CBX, 95%) was ordered from J&K Chemical (Shanghai, China). Polyethylenimine (PEI, average Mn~10000), 2-Morpholino-ethanesulfonic acid (MES, ≥99%), dopamine hydrochloride (DA), horseradish peroxidase (HRP), bovine serum albumin (BSA, >96%), dithiothreitol (DTT,≥99%), iodoacetamide (IAA, 4


Page 4 of 26

Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


≥99%),

1-Ethyl-3-(3-dimethylaminopropyl)

carbodiimide

(EDC,

≥98%),

N-hydroxysuccinimide (NHS, 98%), urea (99%), formic acid (FA, 98%), trypsin, trifluoroacetic acid (TFA, 99%), Tris(hydroxymethyl)methyl aminomethane (Tris, ≥99.8% ) and ethylene glycol (≥99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sinapinic acid (SA) was from Bruker (Daltonios, Germany). Acetonitrile (ACN, ≥99.9%) was of HPLC grade purchased from Merck (Darmstadt, Germany). Peptide-N-glycosidase (PNGase F) was obtained from New England Biolabs (Ipswich, MA, USA). Deionized water was purified using a Milli-Q system (Millipore, Molsheim, France). All other reagents were of analytical grade purchased from China. Human plasma was supported by the second hospital of Dalian Medical university.

Preparation of Fe3O4/PDA/GO/PEI/CBX The preparation procedure of Fe3O4/PDA/GO/PEI/CBX nanocomposites was depicted in Scheme 1, mainly including magnetic GO preparation, PEI self-assembly and CBX covalent bonding. Magnetic nanoparticles (Fe3O4 NPs) were synthesized via a solvent thermal reaction. In a typical reaction, 1.08 g FeCl3.6H2O and 0.20 g sodium citrate were dispersed in 20 mL ethylene glycol with the aid of ultrasonic, followed by the addition of 1.20 g sodium acetate. Then, the mixtures were stirred vigorously by a magnetic stirring for 30 min to form a brown suspension. Finally, the solution was sealed in a PTFE-lined autoclave and reacted at 200 °C for 10 h. After cooling to room temperature, the obtained Fe3O4 NPs were washed with deionized water and ethanol three times, respectively. Fe3O4 NPs were dried under vacuum overnight. Dopamine was easy to auto-polymerize on Fe3O4 NPs surface to form polydopamine (PDA) under weak alkaline buffer. In a typical step, a reaction mixtures of Fe3O4 NPs (100 mg) in 10 mM Tris-HCl buffer (50 mL, pH 8.5) were added by dopamine hydrochloride (100 mg). After stirring for 2 h at 25 °C, the resultant PDA encapsulated Fe3O4 NPs (Fe3O4/PDA) were washed with deionized water and ethanol three times, respectively. Fe3O4/PDA NPs were dried under vacuum overnight. To prepare magnetic GO nanocomposites, GO homogenous suspension (100 mL, 5



Page 6 of 26

1 mg mL-1) was first prepared by sonication of GO nanosheets, and then Fe3O4/PDA NPs suspension (20 mL, 5 mg mL-1) was added to GO suspension, followed by mechanical stirring at room temperature for 8 h. The synthesized Fe3O4/PDA/GO were separated from reaction mixtures by using external magnetic force and washed with deionized water for several times. Fe3O4/PDA/GO were then used as support for PEI self-assembly. In a typical step, 10 mL PEI solution (100 mg mL-1) was mixed with 50 mL Fe3O4/PDA/GO (2 mg mL-1) homogenous solution under mechanical stirring at room temperature for 10 h. The obtained Fe3O4/PDA/GO/PEI were separated from reaction mixtures by using external magnetic force and washed with deionized water for several times and dried under vacuum overnight. To fabricate Fe3O4/PDA/GO/PEI/CBX, 640 mg EDC and 198 mg NHS were added to the reaction mixtures of CBX (150 mg) in 30 mL MES buffer (0.1 M, pH 5.6) to activate carboxyl group of CBX at 40°C for 45 min. The pH of the above mixtures was then adjusted to physiological condition by adding 60 mL 0.1 M Na2HPO4 solution. Subsequently, 15 mg of Fe3O4/PDA/GO/PEI was added into the mixtures solution under mechanical stirring at room temperature for 4 h. The obtained Fe3O4/PDA/GO/PEI/CBX were separated from reaction mixtures by using external magnetic force and washed with deionized water for several times and dried under vacuum overnight.

Characterization Fourier-transformed Perkin-Elmer

Spectrum

infrared GX

spectroscopy

spectrometer

(FT-IR)

was

(Perkin-Elmer,

performed

Waltham,

on

USA).

Transmission electron microscopy (TEM) images were obtained by JEOL JEM-2000EX instrument operated at 120 kV (JEOL, Tokyo, Japan). Atomic force microscopy (AFM) was performed with a Multimode 3D atomic force microscope (Bruker, USA). X-ray photoelectron spectroscopy (XPS) measurements were conducted with Thermo ESCALAB250Xi spectrometer with Al Kα radiation as the X-ray source (Thermo, Waltham, USA). The zeta potential was detected by Malven 6


Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Nano-ZS90 dynamic light scattering (Malven, Worcester, UK). The contact angles were measured by DSA100 (Krüss, Hamburg, German). The content of B element was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) using Hitachi P-4010 instrument operated at 40.68 MHz.

Glycoprotein Adsorption Isotherms The adsorption isotherm of the nanocomposites was investigated by HPLC quantity method. In our experiment, 200 µg Fe3O4/PDA/GO/PEI/CBX were incubated with different concentrations of HRP solution (from 0.06 mg mL-1 to 1.25 mg mL-1, 100 mM phosphate buffer, pH 7.4) at room temperature for 2 h. Then, the suspension was collected by using an external magnetic force and the glycoproteins concentration was measured by HPLC at regular intervals. The adsorption capacity Q (mg/g) was calculated according to the equation below:

Where C0 (mg mL-1) was the initial concentration, Ct (mg mL-1) was the supernatant concentration, v (mL) was the volume of protein solution, and m (mg) was the mass of the materials.

Freundlich Analysis The Freundlich analysis was carried out according to a previously reported method. The amount of glycoproteins bound to the Fe3O4/PDA/GO/PEI/CBX was plotted according to the Freundlich equation to estimate the binding ability of the nanocomposites. The Freundlich relationship can be established using the following equation:

Where qe (mg g-1) and Ce (mg mL-1) were the amount of glycoproteins bound to the nanocomposites at adsorption equilibrium and the free concentration of glycoproteins 7



Page 8 of 26

at adsorption equilibrium, respectively. KF and n were the Freundlich constants related to the adsorption capacity and intensity, respectively. The equilibrium constants for these models were determined using linear regression analysis.

Glycoprotein Enrichment and Sample Preparation Standard

proteins

enrichment:

In

typical,

200

µg

nanocomposites

(Fe3O4/PDA/GO, Fe3O4/PDA/GO/PEI and Fe3O4/PDA/GO/PEI/CBX, respectively) were firstly suspended in loading buffer (100 mM phosphate, pH 7.4, 200 µL) containing 10 µg HRP. The above mixtures were incubated with strong shaking at 25 °C for 2 h. Secondly, the supernatant was removed with the aid of the external magnetic force, and the nanocomposites were washed 3 times with 400 µL loading buffer. Finally, 10 µL elution buffer (ACN: H2O: TFA =50: 49: 1, v/v/v) was added to release the glycoproteins from nanocomposites at 25 °C for 1 h, and 1 µL elution was deposited on plate for MALDI-TOF/TOF MS analysis. Moreover, to investigate the enrichment specify, Fe3O4/PDA/GO/PEI/CBX were used to enrich HRP in the presence of 500-fold mass excess of BSA following the similar procedure. Human plasma glycoproteins enrichment: 20 µL sample was dissolved in 100 mM phosphate buffer (500 µL, pH 7.4) and incubated with Fe3O4/PDA/GO/PEI/CBX (800 µg) at 25 °C for 2 h. After incubation, the supernatant was removed and the nanocomposites were washed 3 times with 100 mM phosphate buffer (500 µL, pH 7.4) to remove non-glycoproteins. Subsequently, 100 µL elution buffer (ACN: H2O: TFA =50: 49: 1, v/v/v) was added to release the glycoproteins from the nanocomposites at 25 °C for 1 h. The eluted proteins were dried by a vacuum concentrator, then denatured in 100 µL 8 M urea and reduced with 5 mM DTT at 56 °C for 2 h. After cooling to the room temperature, cysteine residues were alkylated in 12 mM IAA at room temperature for 30 min in the dark. The digestion was performed by adding trypsin into proteins solution with a substrate-to-enzyme ratio of 40:1 (m/m) and incubated at 37 °C for 12 h. Protein digests were desalted by a C18 trap column and further dried. The deglycosylation of the peptides was performed by adding PNGase F 8


Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(50000 U mL-1) at the enzyme/substrate ratio of 1000 Units mg-1 in 60 µL, 20 mM NH4HCO3. After incubation at 37 °C for 6 h, the resulting peptides were stored at −80 °C for MS analysis.

MALDI-TOF/TOF Analysis All MALDI spectra were taken from a Bruker Ultraflex III MALDI-TOF/TOF MS instrument (Bruker, Daltonios, Germany). A total of 1 µL of elution was dropped onto a MALDI plate, to which 1 µL of SA solution (20 mg mL-1, 0.1% TFA in 60% CH3CN aqueous solution) was added. Spectra were obtained in positive ionization mode using linear detection. The laser intensity was kept constant for all samples. External calibration of MALDI-TOF/TOF MS spectra were performed with commercial proteins.

Nano-LC-MS/MS analysis The Nano-LC-MS/MS analysis was performed using a Q-Exactive MS (Thermo Fisher Scientific, USA) equipped with EASY-nLC 1000 (Thermo Fisher Scientific, USA).The experimental condition of Nano-LC-MS/MS were as follows: C18 trap column (150 µm i.d., 3 cm length); C18 separation column (75 µm i.d.,13 cm length); mobile phase, 2% (v/v) ACN containing 0.1% (v/v) FA as mobile phase A, 98% (v/v) ACN containing 0.1% (v/v) FA as mobile phase B; flow rate, 300 nL/min; separation gradient: 0–0.1 min, 2–6% B; 0.1–83.1 min, 6–22% B; 83.1–98.1 min, 22–35% B; 98.1–103.1 min, 35–80% B; 103.1–113.1 min, 80% B; 113.1–113.2 min, 80%–2% B; 113.2–118.2 min, 2% B; the spray voltage was 2.2 kV and the temperature of ion transfer capillary was set as 275 °C; the Q-Exactive MS was operated in positive ion data dependent mode, and the 15 most intense ions were subjected to HCD fragmentation with normalized collision energy at 28%; the MS1 scans were performed at the resolution of 70,000, ranging from m/z 300 to 1800 (automatic gain control , AGC value, 1E6; maximum injection time, 50 ms); the MS/MS scans were performed at the resolution of 17,500 (AGC, 1E5; maximum injection time, 60 ms); 9



Page 10 of 26

the data were acquired in centroid mode using a 20 s exclusion window. Charge states +2 to +6 of peptides were selected for fragmentation.

Database Searching The raw data were analyzed by Proteome Discoverer (version 1.4.1.14, Thermo Fisher Scientific) using a Mascot server (version 2.3.2) and searched against UniProtKB human complete proteome sequence database (release 2015_04, 42121 entries). The mass tolerances were set at 7 ppm for the parent ions and 20 ppm for the fragments. Peptides were searched using fully tryptic cleavage constraint, and up to 2 missed cleavage. Carbamidomethylation (C, +57.02 Da) was used as the fixed modification. Oxidation (M, +15.99 Da), acetylation (protein N-terminus, +42.01 Da) and the deamidation of asparagine residues (+0.98 Da) were set as variable modifications. For glycopeptides confirmation, the peptides should not only have the deamidation

modification

at

asparagine

residues,

but

also

contain

the

Asn-X-Ser/Thr/Cys sequence motif (where X is any amino acid other than Pro). The peptide and protein identifications were filtered by PD to keep the FDR ≤1%. FDRs were calculated by using the following equation: FDR= n (rev)/n (forw), where n (forw) and n (rev) are the number of peptides identified in proteins with forward (normal) and reversed sequence, respectively. KEGG pathway and GO term analyses were performed using the functional annotation program DAVID v6.7 with p-values < 0.05.

RESULTS AND DISCUSSION

Synthesis and Characterization ofFe3O4/PDA/GO/PEI/CBX Nanocomposites

As shown in Scheme 1, magnetic GO was firstly prepared by π-π stacking and hydrogen bonding interaction between GO and polydopamine (PDA) 10


Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


encapsulated

Fe3O4

nanoparticles

(Fe3O4/PDA).

Secondly,

PEI

was

self-assembled to the surface of GO by electrostatic interaction. Finally, CBX was

covalently

bound

to

PEI

by

amide

reaction,

to

obtain

Fe3O4/PDA/GO/PEI/CBX nanocomposites. FT-IR spectra were applied to characterize the reaction intermediates and nanocomposites. As shown in Figure 1, the peak at 578 cm−1 appeared in all spectra was attributed to the stretching vibration of Fe–O of Fe3O4 NPs, confirming the successful preparation of Fe3O4 NPs. The peaks at 1603 cm−1 and 1505 cm−1 were corresponding to the aromatic ring C=C stretching vibration, and the peak at 1271 cm−1 was assigned to the phenolic C–O stretching vibration of PDA, indicating the auto-polymerization of dopamine on Fe3O4 NPs. For magnetic GO (Fe3O4/PDA/GO), the appearance of C=O stretching vibration at 1724 cm−1 and C–O stretching vibration at 1055 cm−1 indicated the successful introduction of GO. The peaks at 2964 cm−1 and 2850 cm−1 were ascribed to the C–H stretching vibration of PEI, verifying the self-assembly of PEI. Two new characteristic peaks at 1638 cm-1 (amide band I) and 1544 cm-1 (amide band II), and the peak at 1369 cm−1 should be derived from the vibration of B–O bonds,39 indicating the successful covalently bonding of CBX. Furthermore, the results of zeta potential (Figure S1) and the surface chemical bonds investigated by XPS (Figure S2) also confirmed the fabrication of Fe3O4/PDA/GO/PEI/CBX nanocomposites. The morphologies of synthesized materials were also investigated by TEM. As shown in Figure 2, Fe3O4 NPs with the diameter of 170-180 nm were wrapped with 8-12 nm PDA shell; the surface of exfoliated GO contained silk-like wrinkles; Fe3O4/PDA were anchored onto GO surface without agglomeration. Interestingly, the edge of GO sheet spread out of the Fe3O4/PDA to a large extent without conglomeration, thus Fe3O4/PDA/GO with large surface area could provide abundant accessible sites for PEI immobilization.

Compared with Fe3O4/PDA/GO, the 11


morphologies

of


Fe3O4/PDA/GO/PEI

and

Page 12 of 26

Fe3O4/PDA/GO/PEI/CBX

remained

almost

unchanged, contributed by the mild reaction conditions for PEI self-assembly and CBX covalently bonding. Furthermore, by AFM analysis (Figures S3a and 3b), the average thickness of PEI was calculated to be 1.35 nm, demonstrating that multi-layers PEI were attached on both sides of GO, beneficial to afford multiple binding sites, and thus to improve the binding affinity of glycoproteins. Besides, as depicted in Figures S3c and 3d, the hydrophilicity of Fe3O4/PDA/GO/PEI was improved after

PEI

self-assembly,

preferable

to

reduce

the

adsorption

of

non-glycoproteins.

Measurement of Adsorption Isotherm

The adsorption isotherm of Fe3O4/PDA/GO/PEI/CBX was studied by changing glycoprotein (Horseradish Peroxidase, HRP) concentration from 0.06 mg mL-1 to 1.25 mg mL-1 with constant amount of Fe3O4/PDA/GO/PEI/CBX. As illustrated in Figure 3a, the adsorption isotherm curve reached equilibrium when HRP concentration increased to 1.00 mg mL-1. The saturation adsorption capacity

was

164.70±1.70

mg

g-1,

higher

than

that

of

benzoboroxole-functionalized microspheres (92.90 mg g-1).9 Data obtained from adsorption isotherm was further fitted to the Freundlich adsorption model (Figure 3b). The Freundlich constant, KF, reflecting the adsorption capacity,40 was calculated to be 195.00 (mg g−1) (mL mg−1)1/n. The KF of Fe3O4/PDA/GO/PEI/CBX was 3.0−7.3 times higher than that of other boronic acid-functionalized materials,41 demonstrating the high enrichment capacity of our synthesized nanocomposites. Another Freundlich constant, n=1.18, where n reflected the adsorption intensity.40 The value of n varied with the heterogeneity of the adsorbent and n>1 represented favorable adsorption mechanism with Freundlich adsorption model.

12


Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Standard Glycoproteins Enrichment

We firstly applied HRP to evaluate the enrichment ability of three different nanocomposites under physiological condition. As shown in Figure 4, HRP could only be enriched by Fe3O4/PDA/GO/PEI/CBX nanocomposites, demonstrating the selective enrichment ability was mainly attributed from CBX. Furthermore, Fe3O4/PDA/GO/PEI/CBX nanocomposites were further used to capture HRP from proteins mixtures. As shown in Figure 5, HRP could be selectively identified even in the presence of 500-fold mass excess of BSA (non-glycoproteins), much better than other reported boronate affinity materials under physiological condition.9, 24, 42 The results verified the high selectivity of Fe3O4/PDA/GO/PEI/CBX to glycoproteins.

Human Plasma Glycoproteomics Analysis

Since human plasma could provide valuable information for diagnosis and therapy, the development of novel boronate materials for the large-scale glycoproteomics analysis is of great significance. However, by the commonly used boronate affinity materials, the enrichment of glycoproteins is performed under alkaline condition, which might lead to the degradation of labile glycoproteins. Therefore, the analysis of glycoproteome under physiological conditions is of great significance to reflect the real status of proteins in human plasma. Herein, by Fe3O4/PDA/GO/PEI/CBX, the enrichment of glycoproteins in human plasma under physiological condition was performed, followed by nano-RPLC-MS/MS analysis. In three parallel runs, a total of 202 proteins were identified from 20 µL human plasma. The list of identified glycoproteins and their gene IDs captured were shown in Table S1. The identified glycoproteins number results

and recognition selectivity was increased when compared to the obtained

by

previous

boronic

acid-functionalized

particles

(Sil@Poly(APBA-co-MBAAm)) under common alkaline condition (137 vs 78 13



Page 14 of 26

and 67.8% vs 57.8%, respectively),43 which should be contributed by the low pKa value of CBX, PEI dendrimer-assisted multivalent binding and the good hydrophilicity of the matrix. To better understand the significance of identified glycoproteins, we used DAVID (Database for Annotation, Visualization and Integrated Discovery) database to perform gene onthology term (GO term) enrichment analyses (Figure 6a).44 The biological processes included platelet degranulation, complement activation, immune response, metabolic process and so forth. Molecular function annotation analysis revealed that the glycoproteins exerted various enzymatic activity and binding functions. Complement and coagulation cascades had the highest enrichment coefficient in KEGG pathway analysis (P=2.8E-58). More importantly, compared with the results obtained with enrichment under alkaline conditions,43 we could identify eight more glycoproteins (the red part in Figure 6b) associated with complement and coagulation cascades pathway, beneficial to elucidate complement and coagulation cascades pathway. Furthermore, thrombin (F2), labile under alkaline condition, was successfully identified. Moreover, we captured ten low abundance glycoproteins (

3-Carboxybenzoboroxole Functionalized Polyethylenimine Modified

Recommend Documents