An Accessible Protocol for Solid-Phase Extraction of N-Linked

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An accessible protocol for solid-phase extraction of N-linked glycopeptides through reductive amination by amine-functionalized magnetic nanoparticles Ying Zhang, Min Kuang, Lijuan Zhang, Pengyuan Yang, and Haojie Lu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac400733y • Publication Date (Web): 09 May 2013 Downloaded from http://pubs.acs.org on May 11, 2013

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Analytical Chemistry

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An accessible protocol for solid-phase extraction of N-linked glycopeptides through reductive amination by amine-functionalized magnetic nanoparticles

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Ying Zhang, † , § Min Kuang, ‡, § Lijuan Zhang†, Pengyuan Yang†,‡ and Haojie Lu†,‡,*

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[email protected]( Haojie Lu)

*Corresponding author Email

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Fax: (+86)21-5423-7961 §

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†

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200032, P. R.China.

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‡

These authors contributed equally to this work.

Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai

Department of Chemistry, Fudan University, Shanghai 200433, P. R. China

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ACS Paragon Plus Environment

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ABSTRACT

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In light of the significance of glycosylation for wealthy biological events, it is important to

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pre-fractionate glycoproteins/glycopeptides from complex biological samples. Herein, we

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reported a novel protocol of solid phase extraction of glycopeptides through a reductive

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amination reaction by employing the easily accessible 3-aminopropyltriethoxysilane (APTES)-

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functionalized magnetic nanoparticles. The amino groups from APTES, which were assembled

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onto the surface of the nanoparticles through one-step silanization reaction, could conjugate with

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the aldehydes from oxidized glycopeptides, therefore, completed the extraction. To the best of

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our knowledge, this is the first example of applying the reductive amination reaction into the

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isolation of glycopeptides. Due to the elimination of the desalting step, the detection limit of

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glycopeptides was improved by 2 orders of magnitude, comparing to the traditional hydrazide

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chemistry-based solid phase extraction, while the extraction time was shortened to 4 hours,

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suggesting the high sensitivity, specificity, and efficiency for the extraction of N-linked

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glycopeptides by this method. In the meantime, high selectivity towards glycoproteins was also

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observed in the separation of Ribonuclease B from the mixtures contaminated with bovine serum

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albumin. What’s more, this technique required significant less sample volume, as demonstrated

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in the successful mapping of glycosylation of human colorectal cancer serum with the sample

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volume as little as 5 µL. Because of all these attractive features, we believe that the innovative

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protocol proposed here will shed new light on the research of glycosylation profiling.

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KEY WORDS

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glycopeptides/glycoprotein, selective enrichment, magnetic nanoparticles, solid-phase extraction,

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mass spectrometry


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As one of the most ubiquitous post-translation modifications, glycosylation is involved in a

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variety of physiological and pathological processes.1,2,3 Protein glycosylation is essential for a

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wealth of biological events including protein folding, intracellular sorting, secretion, uptake, as

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well as cell and host-microbial recognition.4,5,6 However, comprehensive and detailed analysis of

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glycosylations on a proteome-wide scale is still a daunting challenge because of the low

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abundance and low ionization efficiency of glycopeptides in mass spectrometry analysis.7 To

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gain thorough insights into glycosylation and elucidate the functional relationships among

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proteins, efficient separation of glycoproteins for their further analysis is absolutely

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necessary.8,9,10

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To date, numerous techniques for isolation and identification of glycopetides have been

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established, including those approaches based on lectin affinity chromatography,11,12 boronic

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acid

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chromatography adsorbents.17,18 Pros and cons are also reported for each method. For example,

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different lectins have diverse affinities toward various glycans attached to glycoproteins, thus

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combining different types of lectins is needed for the separation of complex samples.19,20

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Hydrophility affinity methods, which rely on the hydrophility of glycan moiety to extract the

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hydrophilic glycopeptides, always suffer from the low specificity for the reason of

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simultaneously capturing nonglycopeptides which contain several hydrophilic amino acids.21

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Solid phase extraction by hydrazide chemistry for the enrichment of N-linked glycopeptides is

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also a popular method which has drawn extensive attention.22, 23 Nevertheless, it always turns out

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to be sample-consuming and involves a cumbersome procedure. Besides, with regards to the

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most commonly used commercial hydrazide resins, they face the drawbacks of difficult to

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redisperse and separate from solution phase. Furthermore, hydrazide resins are difficult to be

functionalized

nanoparticles,13,14

hydrazide

beads,15,16


hydrophilic

interaction

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prepared in lab and commercially available hydrazide nanoparticles are relatively expensive.

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Therefore, currently available methods for the specific extraction of N-glycoproteins are far from

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maturity.

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Herein ， a brand-new protocol based on the conjunction of aldehydes from oxidized

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glycopeptides to the amino-groups on the surface of Fe3O4@SiO2@NH2 magnetic nanoparticles

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via reductive amination reaction was employed for the extraction of glycopeptides. Reductive

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amination, including Schiff base formation and its subsequent reduction to a secondary amine

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with reducing agent, has been widely used for the derivatization of glycans.24,25 The reaction

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involves the initial formation of the intermediate carbinol amine which dehydrates to form an

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imine. With the aid of acid, the imine is protonated rapidly to form an iminium ion. Subsequent

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reduction of this iminium ion could enable the formation of alkylated amine product.26 Thus the

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versatile nature of this reaction opens the possibility to develop a novel approach for N-linked

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glycoproteomic profiling. Firstly, amine-functionalized magnetic nanoparticles were synthesized

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through the combination of solvothermal reaction, sol-gel reaction as well as the amino-groups

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modification. Then the cis-diol groups of glycoproteins were converted into aldehydes through

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the oxidation of sodium periodate. After incubation with amine-functionalized magnetic

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nanoparticles, non-specifically adsorbed substances could be washed away whereas

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glycopeptides/glycoproteins remained immobilized on the surface of nanoparticles. Afterwards,

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with the aid of specific release of N-linked glycopeptides/glycoproteins by PNGase F, excellent

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isolation performance together with glycosylation-sites identification can be achieved.

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EXPERIMENTAL SECTION

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Materials and reagents


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Asialofetuin from fetal calf serum (ASF), Bovine serum albumin (BSA), Myoglobin from horse

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heart (MYO), Ribonuclease B from bovine pancreas (RNase B) as well as dithiothreitol (DTT),

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Sodium cyanoborohydride (NaBH3CN), sodium periodate (NaIO4), sodium acetonitrile (ACN),

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ammonium bicarbonate (NH4HCO3), urea, MALDI matrix (α-cyano-4-hydroxycinnamic acid,

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CHCA) were all obtained from Sigma (St. Louis, MO). Acetonitrile (ACN, 99.9%,

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chromatographic grade) and trifluoroacetic acid (TFA) were purchased from Merck (Darmstadt,

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Germany). The glycerol free peptide-N-glycosidase (PNGase F, 500 units/µL) and SDS-PAGE

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molecular weight standards (6.5-175 KDa) were from New England Biolabs (Ipswich, MA).

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Sep-Pak® C18 columns were from Waters (Milford, MA). Bradford reagent together with Affi-

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Gel Hz hydrazide Gel were from Bio-Rad (Hercules, CA). Human serum from colorectal cancer

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patients were provided by Fudan University Shanghai cancer center and stored at -80 ℃ before

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analysis. Other chemical reagents were of analytical grade and obtained from Shanghai Chemical

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Reagent Co., Ltd., which were used as received without further purification. Water used in

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experiments was ultra-pure water prepared using a Milli-Q50SP Reagent Water System

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(Millipore, Bedford, MA).

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Sample preparation

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Preparation of amine-functionalized magnetic nanoparticles

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The synthetic routine of amine-functionalized magnetic nanoparticles (Fe3O4@SiO2@NH2) is

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illustrated in scheme 1(A). After coating a thin layer of silica on the magnetic cores, grafting

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with 3-aminopropyl-triethoxysilane (APTES) on the surface of the magnetic nanoparticles was

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followed. First of all, Fe3O4 was fabricated by a modified solvothermal reaction. Briefly,

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FeCl3•6H2O (1.350 g) was dissolved in ethylene glycol (70 mL). Subsequently, with the addition


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of sodium acetate anhydrous (3.854 g) and sodium citrate (0.400 g), a homogeneous black

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solution was formed under vigorous stirring for 1 h at 170 ℃. Then the mixture was transferred

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to a Teflon-lined stainless-steel autoclave (100 mL capacity). The autoclave was heated to 200

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℃ and maintained for 16 h. Finally, after cooling down to room temperature, the black products

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were rinsed with ethanol for several times with the aid of sonication and an external magnet

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before being dried at 50 ℃.

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Afterwards, the preparation of Fe3O4@SiO2@NH2 core/shell microspheres was carried out

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by a the Stober process27 with minor changes. The synthesized magnetic nanoparticles (0.040 g)

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were homogeneously dispersed in a mixture containing ethanol (128 mL), deionized water (36

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mL), and 28% (wt) concentrated ammonia aqueous solution (NH3•H2O), followed by the

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addition of 0.20 g of tetraethyl orthosilicate (TEOS). Then the reaction was allowed to proceed at

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0 ℃ for 1 h under sonication to get the silica embedded magnetic nanoparticles. Finally, 0.020 g

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3-aminopropyl-triethoxysilane (APTES) was added to the above mixture, and the reaction was

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performed for another 20 min under sonication. The harvested Fe3O4@SiO2@NH2 microspheres

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were collected with a magnet and washed repeatedly with ethanol and water to effectively

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remove nonmagnetic byproducts and residual reagents. The final products were dried under

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vacuum at 50 ℃ for further use.

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Characterization of amine-functionalized magnetic nanoparticles

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Scanning electron microscope (SEM) images were adopted by SUPERSCAN SSX-550 scanning

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electron microscope (Shimadz, Japan). Transmission electron microscopy (TEM) images were

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obtained with a JEOL 2011 microscope operated at 200 kV( JOEL, Tokyo, Japan). Samples for

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TEM measurements were suspended in ethanol and supported on a carbon-coated copper grid.


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Fourier-transformed infrared spectroscopy (FTIR) characterization was performed at a Nicolet

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Nexus 470 FT-IR spectrometer with KBr pellets (Nicolet, Wiscosin, USA). Zetasizer Nano

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(Malvern, England) was used for the measurements of zeta potential of the magnetic

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nanoparticles. To evaluate the crystalline nature of the synthesized magnetite colloidal

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nanocrystal clusters, X-ray Powder Diffraction analysis was conducted on D8 ADVANCE with

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DAVINCI (Bruker, Germany).The saturation magnetization curves were obtained by a MPMS

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SQUID VSM at room temperature (Quantum Design, USA).

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Preparation of protein digests.

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Model proteins (ASF and MYO) were dissolved in 25 mM NH4HCO3 (pH=8.0) and denatured by

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incubating at 100 ℃ for 10 min. After cooling down to room temperature, trypsin was added to

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the solution at an enzyme-to-substrate ratio of 1:30 (w/w). The digestion procedure was allowed

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to proceed at 37 ℃ overnight, followed by the lyophilization of the digested sample.

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Human blood was coagulated at room temperature for 10-20 min and then centrifuged at the

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speed of 2000-3000 rpm at 4 ℃ to collect the supernatant. The serum collected was immediately

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aliquoted and stored at -80 ℃. Subsequently, 5 µL of the serum was 3-fold diluted by denaturing

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solution which containing 60 mM NH4HCO3 and 8 M urea. The mixture was treated with 10 mM

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dithiothreitol (DTT) at 57 ℃ for 30 min and alkylated with 20 mM iodoacetamide (IAA) at room

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temperature for 1 h in the dark. Prior to digestion, the solution was diluted with 50 mM

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NH4HCO3 till the final concentration of urea was less than 1.5 M. Trypsin was added according

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to enzyme-to-substrate-ratio of 1:30 (w/w) and hydrolyzed for 16 h under gentle shaking. The

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digests were desalted by C18 columns and the eluted peptides were lyophilized for further use.


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Enrichment of N-linked glycopeptides with Fe3O4@SiO2@NH2 nanoparticles.

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The successful oxidation of cis-diols to aldehyde groups is the prerequisite for the enrichment of

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glycopeptides. The lyophilized peptides were firstly suspended in sodium acetate buffer (100

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mM sodium acetate and 150 mM NaCl, pH=5.5) and then oxidized with 10 mM sodium

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periodate at room temperature in the dark with constant shaking. After 1 hour incubation, sodium

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sulfite was introduced to the mixture till a final concentration of 20 mM and subsequently

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incubated for another 10 min with shaking at room temperature. The oxidized samples were

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lyophilized and resuspended in the coupling solution containing 70% methanol and 30% acetic

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acid (v/v). Amine-functionalized nanoparticles were prewashed twice with the coupling solution

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before adding to the above mixture. After constant shaking for 2 h at certain temperature (37, 45,

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60, 70 ℃), sodium cyanoborohydride was added to the mixture to the final concentration of 1M,

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followed by another 2 h incubation. Impurities were removed by rinsing the nanoparticles twice

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with each of the following solutions sequentially: coupling solution, water, 80% ACN/20% H2O

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(v/v), 50 mM NH4HCO3. Subsequently, after addition of 200 µL of fresh 50 mM NH4HCO3 and

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1 µL of PNGase F (500 units per µL) to the nanoparticles, the release of peptide moieties was

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performed overnight with shaking at 37 ℃. The supernatant of this procedure was collected

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through magnet separation for MALDI-MS analysis.

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Mass spectrometry analysis.

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For standard glycopeptides, matrix-assisted laser desorption/ionization (MALDI) mass

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spectrometric (Applied Biosystems, Framingham, MA, USA) analysis was carried out on 5800

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Proteomics analyzer in positive ion mode. The UV laser was operated at a 400 Hz repetition rate

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with wavelength of 355 nm. Then the mixture was spotted on a MALDI target plate (AB


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SCIEX). The matrix employed was 10 mg/mL CHCA (α-cyano-4-hydroxycinnamic acid)

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dissolved in 50% ACN (v/v) containing 0.1% TFA). The automated acquirement of 5800

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MALDI-TOF mass spectra were accomplished through the average of 1000 laser shots. Before

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analyzing, MYO digests were used to calibrate the mass instrument with internal calibration

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mode.

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The deglycosylated peptides solutions purified from 5 µL human serum were lyophilized

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using a vacuum centrifuge and resuspended with 5% ACN containing 0.1% FA, then separated

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by nano-LC and analyzed by online electrospray tandem mass spectrometry. The Nano-LC

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MS/MS analysis was performed on an LC-20AD system (Shimadzu, Tokyo, Japan) connected to

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an LTQ orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) equipped with an

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online nanoelectrospray ion source (Michrom Bioresources, Auburn, CA). Samples were

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injected into a CAPTRAP column (0.5×2 mm, MICHROM Bioresources, Auburn, CA) in 4 min

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with a flow rate of 20 µL/min. Subsequently, a linear gradient of acetonitrile of from 5-45%

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(95% ACN in 1% FA) over 100 min at a flow rate of 500 nL/min was applied. The separated

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samples were introduced into the mass spectrometer via an ADVANCE 30 µm silica tip

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(MICHROM Bioresources, Auburn CA). The spray voltage was set at 1.6 kV and the capillary

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was heated to 180 ºC. The mass spectrometer was operated in data-dependent mode. For each

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cycle of duty, it consisted of one full-MS survey scan at the mass range 400~2000 Da with

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resolution power of 100,000. Then MS/MS scan was conducted for eight of the most abundant

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precursor ions by LTQ section with a dynamic exclusion duration of 90s. Only Peaks with the

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charge of 2+ and 3+ could be selected for MS/MS run. The AGC expectation during full-MS and

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MS/MS were 1,000,000 and 10,000, respectively. All tandem mass spectra were collected

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through the LTQ section using collision-induced dissociation with helium as the collision gas


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and normalized collision energy value set as 35.0%. The system control and data collection were

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achieved through Xcalibur software version 1.4 (Thermo).

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Database search and data process.

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The data derived from the ESI MS/MS analysis was searched by SEQUEST, against a composite

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database including both original and reversed human protein database of International protein

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Index (Combine.human.uniprot.sprot.090210.fasta).28 The relevant parameters were set to the

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following modifications: enzyme was selected as trypsin (partially enzymatic). A maximum of

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two missed cleavages (MCs) was allowed. Carboxamidomethylation (C, 57.02150) was set as

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fixed modification and the oxidation (M, 15.99492) as well as asparagine de-glycosylation (N,

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0.98402) were set as variable modifications. Precursor mass and fragment mass tolerance was 10

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ppm and ±0.6 Da for SEQUEST search. Mass value was set as monoisotopic.

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To statistically validate the accuracy of peptide assignments to tandem mass spectra from

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SEQUEST, Trans-Proteomic Pipeline (TPP) was applied to effectively compute the probability

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for the likelihood of each identification being correct in a data-dependent fashion.

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PeptideProphet would give high-confidence spectrum to peptide interpretation (score≥0.90), and

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only those peptides passed the peptide probability threshold 0.95 can be accepted for further data

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interpretation. The Asn modification that did not occur in N-X-S/T motif (X≠P) was eliminated

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to ensure the false positive rate below 1% for the identified glycosylation sites.

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Solid phase extraction of N-linked glycoprotein

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A mixture of model proteins containing RNase B (1mg) and BSA (1 mg) was directly oxidized

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and quenched in the same way as described above. After lyophilization, the incubation step was

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allowed to proceed at 37 ℃ for 12 h. Then gycoproteins attached to the solid phase were washed


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three times with a solution containing 8 M urea and 0.4 M NH4HCO3 (pH=8.3) to eliminate the

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non-specifically bounded impurities. The denaturing process was carried out through dispersing

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the nanoparticles in 8 M urea and 0.4 M NH4HCO3 buffer containing 10 mM DTT at 37 ℃ for 1

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h. Then the free thiols were alkylated with 12 mM iodoacetamide for 60 min at room

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temperature in the dark. Afterwards, the nanoparticles were washed twice with 8 M urea

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solution, H2O and 50 mM NH4HCO3 buffer successively. Finally, the deglycosylation was

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realized through the incubation of nanoparticles with 1 µL PNGase F overnight at 37 ℃.

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For comparison, conventional glycoproteomic analysis using hydrazide gels to capture

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glycoprotein was performed according to the standard protocol reported previously. Briefly, the

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same amount of oxidized mixture of the two model proteins was suspended in coupling buffer

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and incubated with the prewashed hydrazide gels (500 mL gel/mg protein) for 12 h at room

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temperature. Then glycoproteins attached to the gels were denatured in the same way as

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described above. Subsequent rinsing of the nanoparticles was performed by washing twice with

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each of the following solutions successively: 80% ACN/ 0.1% TFA, 8 M urea/ 0.4 M NH4HCO3/

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0.1% SDS, N,N-Dimethylformamide (DMF) and 100 mM NH4HCO3. The deglycosylation

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procedure was performed with 1 µL PNGase F overnight at 37 ℃.

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To analyze the deglycosylated proteins, 10 µL of the finally obtained supernatant obtained

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with each method was mixed and boiled with loading buffer separately to conduct the SDS-

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PAGE (12%) analysis. Afterwards, the gel was stained with Coomassie brilliant blue and

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bleached.

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RESULTS AND DISSCUSSION.

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Characterization of Fe3O4@SiO2@NH2


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The morphology and crystallography of the as-prepared silica-based magnetic nanoparticles were

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validated through various aspects. The transmission electron microscopy (TEM) image (shown

239

in Fig.1A) revealed the iron oxide microspheres were well encapsulated in a condensed,

240

amorphous silica shell, which was critical for preventing them from aggregation and beneficial

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for their rapid redispersion.27 Moreover, Fig.S1† presents a typical Scanning electron microscopy

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(SEM) image of the Fe3O4@SiO2@NH2 nanoparticles, which indicates the Fe3O4@SiO2@NH2

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nanoparticles possess the sizes of 200 nm with a narrow size distribution. The single crystal

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nature of the Fe3O4 magnetic nanoparticles is shown in Fig.S2†, with the relative intensity and

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position of the five main diffraction peaks matched well with those reported previously.29 The

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hysteresis loops shown in Fig. S3† demonstrates that the Fe3O4 and Fe3O4@SiO2@NH2 possess

247

the saturation values of about 55.6 emug-1 and 28.7 emug-1 respectively. Notably, the remanence

248

of these nanoparticles is zero once the external magnetic field is removed, implying that these

249

microspheres are superparamagnetic and possess high magnetic responsibility. The chemical

250

modification of the amine-functionalized nanoparticles was validated by Fourier-transform

251

infrared (FT-IR) spectroscopy. As shown in Fig.1(B)-c, the absorption peaks for

252

Fe3O4@SiO2@NH2 at 1087 cm-1 is ascribed to the Si-O-Si vibration, whereas the peaks around

253

1620 cm-1 and the wide peaks around 3400 cm-1 are attributed to the stretching and bending

254

vibrations of N-H. By comparing Fig.1 (B)-c and b, it’s obvious that the intensity of the band

255

corresponding to the vibration of Si-OH around 950 cm-1 after APTES grafting decreases to

256

some extent, which also evidences the successful grafting of APTES. In addition, the zeta

257

potential of Fe3O4@SiO2 and Fe3O4@SiO2@NH2 are -12.4 mV and 0.749 mV respectively at pH

258

7.0, reflecting the surfaces of the magnetic nanoparticles are positively charged after

259

modification, thus also implying the existence of protonated amino groups.


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Optimization of the enrichment performance of Fe3O4@SiO2@NH2

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The performance of Fe3O4@SiO2@NH2 for the selective enrichment of glycopeptides was

262

evaluated by capturing glycopeptides from the mixture of glycopeptides and nonglycopeptides.

263

As shown in scheme 1(B), after incubation, non-glycosylated peptides could be washed away

264

whereas glycopeptides remained immobilized on the surface of nanoparticles. Afterwards, with

265

the aid of specific release of N-glycopeptides by PNGase F, characterization of N-linked

266

glycosylation can be achieved. Scheme 1(C) demonstrates a stepwise enrichment procedure

267

involving imine formation followed by reduction with NaBH3CN. When the oxidized

268

glycopeptides were mixed with amine-functionalized nanoparticles, the methanol in the coupling

269

solution could facilitate reaction of aldehydes with amino groups to form carbinol amines, which

270

subsequently dehydrated in a reversible manner to form the intermediate imines. Afterwards, the

271

imines were efficiently converted into their corresponding amines through the reduction of

272

NaBH3CN.

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functionalized magnetic nanoparticles through the formation of stable C-N bond. Taking a

274

standard glycoprotein asialofetuin (ASF) as the model, we systematically investigated the

275

conditions that affected the enrichment performance of the glycopeptides. The influences of the

276

peptide concentration and temperature are shown in Fig. S4†, S5† and Fig. S6†, S7† respectively.

277

The optimized incubation condition could be defined as: the initial concentration of

278

glycopeptides was range from 500~2500 ng/µL and amine- functionalized nanoparticles were

279

added with the ratio of 10 mg Fe3O4@SiO2@NH2 per 1 mg proteins. The incubation process was

280

conducted for 4 h at 45 ℃ in an incubation buffer containing 70% methanol and 30% acetic acid.

281

Obviously, we could find the proposed method was capable of obtaining excellent performance

282

from tryptic ASF with the concentration of glycopeptides as low as 25 ng/µL, which indicated

26

Thus, glycopeptides could be well immobilized on the surface of amine-


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283

the high sensitivity of this protocol. It may be due to the fact that a desalting step after

284

glycopeptides oxidation, which may cause large amount of sample loss, was eliminated.30

285

Selective enrichment of N-linked glycopeptides/glycoproteins with Fe3O4@SiO2@NH2

286

The specificity of such strategy in the optimized condition was evaluated by effectively

287

separating glycopeptides from a digest mixture derived from ASF and a nonglycoprotein

288

myoglobin (MYO). MALDI-TOF mass spectra of the mixture of ASF and MYO digests at the

289

mole ratio of 1:1 before and after isolation by Fe3O4@SiO2@NH2 are illustrated in Fig.2. It

290

could be recognized that not any glycopeptide is observed before enrichment. While after

291

isolation, six dominant peaks of the ASF deglycosylated peptides are prominently identified with

292

a clean background. The peaks observed at m/z 1626.7, 1755.8, 1781.5, 2755.6, 3017.8 and

293

3558.8 are deglycosylatd peptides and relevant to [M+H]+ of LCPDCPLLAPLNDSR,

294

KLCPDCPLLAPLNDSR, NAESNGSYLQLVEISR, DIEIDTLETTCHVLDPTPLAN CSVR, as

295

well as RPTGEVYDIEIDTLETTCHVLDTPLANCSVR. These deglycosylatd peptides are

296

assigned

297

RPTGEVYDIEIDTLETTCHVLDTP-LANCSVR+(GlcNAc)2(Man)3(GlcNAc)2,

298

APLNDSR+(GlcNAc)2(Man)3(GlcNAc)2,

299

+(GlcNAc)2(Man)3(GlcNAc)2 respectively. Table S1† summarizes the molecular masses and

300

corresponding glycopeptides of each deglycoslylated glycopeptides from ASF digests after

301

enrichment with amine-functionalized magnetic nanoparticles. To further validate the selectivity,

302

the digests of nonglycoprotein MYO were mixed with the ASF digests at the mole ratio of 10:1,

303

remarkable separation performance could also be achieved with the six deglycopeptides existed

304

in mass spectra (shown in Fig. S8†).

to

the

corresponding

and

tryptic

glycopeptides

of

ASF:

LCPDCPLL-

VVHAVEVALATFNAESNGSYLQLVEISR


14

Page 15 of 24

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305

The potential of the method for isolation of glycoproteins was demonstrated by extracting

306

of glycoprotein from a mixture of model protein, which contained Ribonuclease B (RNase B, 1

307

mg) and Bovine Serum Albumin (BSA, 1 mg). For comparison, similar procedure for the

308

enrichment of glycoproteins with commercialized hydrazide gels was also conducted. As shown

309

in the SDS-PAGE in Fig. 3, two bands corresponding to BSA and RNase B were obviously

310

appeared in the lane of the mixture, whereas only deglycosylated RNase B could be detected

311

after enrichment (shown in figure 3 lane 2 and 4). By comparing the performance of these two

312

methods, it was suggested that similar enrichment selectivity and efficiency could be achieved

313

through both of them.

314

N-glycoproteome profiling of human serum by Fe3O4@SiO2@NH2

315

To intensively evaluate the feasibility of applying the amine-functionalized magnetic

316

nanoparticles for the profiling of glycosylation, solid phase extraction was further applied to

317

complex biological samples. Human serum, which is an attractive source for the discovery of

318

biomarker related to diseases, often dominated by a variety of high abundance proteins. In this

319

report, profiling of glycoproteins from colorectal cancer serum has been performed. The serum

320

sample from colorectal cancer patient was kindly provided by Fudan University Shanghai cancer

321

center. The research followed the tenets of the Declaration of Helsinki and was approved by the

322

Ethics Committee of Fudan University Shanghai cancer center. The pretreatment process was

323

carried out according to the procedure shown in scheme 1B. After reduction and alkylation, the

324

serum sample was digested into peptides, followed by oxidation and the quench of oxidation.

325

Then the lyophilized oxidized peptides were treated by Fe3O4@SiO2@NH2. Afterwards, the

326

harvested supernatants were lyophilized and send for nano-LC-MS/MS analysis. Combined with

327

the mass increment of 0.98402 Da of asparagine (N) transforms into aspartic acid (D), the


15


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Page 16 of 24

328

identification of N-linked glycosylation sites as well as glycopeptides could be clearly and

329

definitely realized by the existence of N-X-S/T (X≠P) sequences. In total, 111 unique N-

330

glycosylation sites were found in 108 glycopeptides, which were assigned to 60 glycoproteins.

331

The detail information is demonstrated in Table S2†. There are several other reports for

332

glycoproteome enrichment using 15-50 µL of human serum or plasma,23,32,33 and usually less

333

than 60 glycoproteins were identified. Our proposed method exhibited comparable performance

334

in the extraction of N-linked glycopeptides with only 5 µL human serum.

335

CONCLUSIONS

336

In summary, Fe3O4@SiO2@NH2 nanoparticles inherit the virtues of easy to prepare, cost-

337

effective and could be readily separated and dispersed, which makes this novel protocol easily

338

accessible. Compared with the traditional solid-phase extraction method based on hydrazide

339

resins which usually needs 12~16 h to couple the glycopeptides, this protocol could render

340

excellent enrichment performance within 4 h. In addition, different from the isolation realized by

341

hydrazide chemistry, the desalting step is no longer needed in our protocol after sample

342

oxidation. As a result, it turns out to be sample-saving with higher detection sensitivity.

343

Therefore, an easily accessible glycopeptide solid-phase extraction protocol through reductive

344

amination was established. This protocol is believed to be promising for the mapping of N-linked

345

glycosylation of complex biological samples and shed new light upon glycosylation

346

characterization.

347 348 349 350

ACKNOWLEDGMENT The

work

2012YQ12004409),

was NSF

supported

by

(21025519,

NST

(2012CB910602,

21005020

and

2012AA020203

31070732),


Shanghai

and

Projects

16

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351

(11XD1400800, Eastern Scholar and B109).

352

“Supporting information available: This material is available free of charge via the Internet at

353

http://pubs.acs.org.”

354

REFERENCES

355

(1)Xiong, Z.; Zhao, L.; Wang, F.; Zhu, J.; Qin, H.; Wu, R.; Zhang, W.; Zou, H. Chem. Commun.

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(4) Nilsson, J.; Halim, A.; Grahn, A.; Larson, G. Nat. Methods. 2009, 6. 809-811.

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(5) Ohtsubo, K.; Marth, J. D. Cell 2006, 126. 855-867.

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(6) Grewal, P. K.; Uchiyama, S.; Ditto, D.; Varki, N.; Le, D. T.; Nizet, V.; Marth, J. D. Nat.

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Takahashi, N.; Isobe, T. Nat. Biotechnol. 2003, 21. 667-672. (12) Ahn, Y. H.; Kim, Y. S.; Ji, E. S.; Lee, J. Y.; Jung, J. A.; Ko, J. H.; Yoo, J. S. Anal. Chem. 2010, 82. 4441-4447. (13) Xu, Y.; Wu, Z.; Zhang, L.; Lu, H.; Yang, P.; Webley, P. A.; Zhao, D. Anal. Chem. 2008, 81. 503-508. (14) Zhang, Q.; Tang, N.; Brock, J. W.; Mottaz, H. M.; Ames, J. M.; Baynes, J. W.; Smith, R. D.; Metz, T. O. J. Proteome. Res.2007, 6. 2323-2330

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Sawaki, H.; Yamauchi, Y.; Shinkawa, T. J. Proteome. Res. 2012,11,4553-4566.

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(21) Chen, Y.; Cao, J.; Yan, G.; Lu, H.; Yang, P. Talanta 2011, 85. 70-75.

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(22) Tian, Y.; Zhou, Y.; Elliott, S.; Aebersold, R.; Zhang, H. Nat. Protoc. 2007, 2. 334-339.

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Y. J. Proteome. Res. 2011, 74. 2159-2168. (33) Ueda, K.; Takami, S.; Saichi, N.; Daigo, Y.; Ishikawa, N.; Kohno, N.; Katsumata, M.; Yamane, A.; Ota, M.; Sato, T. A. Mol. Cell. Proteomics 2010, 9. 1819-1828.

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410 411

Scheme 1. (A) Illustration of consecutive steps for the synthesis of Fe3O4@SiO2@NH2. (B)

412

The flow chart of the enrichment process for glycoproteins/glycopeptides. (C) The

413

schematic overview of the chemical reactions involved in the enrichment procedure.

414 415 416

Fig. 1 (A) The typical TEM image of Fe3O4@SiO2, (B) The FT-IR spectra of (a) Fe3O4, (b) Fe3O4@SiO2, (c) Fe3O4@SiO2@NH2.

417 418 419

Fig. 2 MALDI-TOF mass spectra of tryptic digest mixture of asialofetuin and myoglobin

420

(with a mole ratio of ASF:MYO= 1:1) (A) direct analysis (B) analysis after isolation by

421

Fe3O4@SiO2@NH2 and deglycosylation by PNGase F. “ ✲ ”and “#”denote the

422

deglycosylated peptides and nonglycopeptides, respectively.

423 424

Fig. 3 Analysis of isolated glycoprotein from protein mixture by 12% SDS-PAGE. “M”

425

stands for protein marker. Lane 1 and lane 3 represent a protein mixture of BSA (2.5 µg)

426

and RNase B (2.5 µg), lane 2 and lane 4 represent the released deglycosylated RNase B

427

after enrichment with Fe3O4@SiO2@NH2 nanoparticles and hydrazide resin respectively.

428


19


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429 430 431

432 433 434 435 436

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For TOC only

A novel glycopeptides solid-phase extraction method based on reductive amination reaction by amine-functionalized magnetic nanoparticles was established and applied for profiling of Nglycoproteome from human colorectal cancer serum.

437


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Scheme 1 168x104mm (300 x 300 DPI)



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

Figure 1 90x35mm (300 x 300 DPI)


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Figure 2 284x331mm (300 x 300 DPI)



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

Figure 3 81x55mm (300 x 300 DPI)


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An Accessible Protocol for Solid-Phase Extraction of N-Linked

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