Highly Selective and Ultra Fast Solid-Phase Extraction of N

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Highly Selective and Ultra Fast Solid-Phase Extraction of N‑Glycoproteome by Oxime Click Chemistry Using AminooxyFunctionalized Magnetic Nanoparticles Ying Zhang,†,‡,§ Meng Yu,∥,§ Cheng Zhang,‡ Wanfu Ma,∥ Yuting Zhang,∥ Changchun Wang,*,∥ and Haojie Lu*,†,‡ †

Shanghai Cancer Center and Key Laboratory of Glycoconjuates Research Ministry of Public Health, Fudan University, Shanghai, 200032, People’s Republic of China ‡ Institutes of Biomedical Sciences and Department of Chemistry, Fudan University, Shanghai, 200032, People’s Republic of China ∥ State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai, 200433, People’s Republic of China S Supporting Information *

ABSTRACT: For the highly efficient extraction of the Nglycoproteome, a novel solid-phase extraction method based on oxime click chemistry has been developed. With the use of a newly synthesized aminooxy-functionalized magnetic nanoparticle, the oxidized glycan chains on glycopeptides readily react with the aminooxy groups through oxime click chemistry, resulting in the highly selective extraction of glycopeptides. Compared to the traditional hydrazide chemistry-based method, which takes 12−16 h of coupling time, this new method renders excellent enrichment performance within 1 h. Furthermore, the enrichment sensitivity (fmol level), selectivity (extracting glycopeptides from mixtures of nonglycopeptides at a 1:100 molar ratio), and reproducibility (CVs < 20%) are also dramatically improved. We have successfully profiled the N-glycoproteome from only 1 μL of human colorectal cancer serum using this innovative protocol, which offers a more efficient alternative N-glycoproteome extraction method.

A

Therefore, there is a strong demand to develop a simple, fast, and universal enrichment protocol to selectively extract glycoproteins for glycoproteome profiling. In a previous study, we reported an easily accessible protocol for the solid-phase extraction of N-glycoproteome through reductive amination by amine-functionalized magnetic nanoparticles, which shortened the coupling time to 4 h.9 However, to form a stable conjugate of glycopeptides and nanoparticles, the reductive amination reaction of aldehydes with primary amino groups requires a reducing reagent, which should be removed before MS analysis. The removal of the reducing reagent may cause sample loss, especially for glycoproteins at low concentration. To further shorten the coupling time and find more moderate reaction conditions, we began to investigate alternative routes. It is well documented that the reaction between the aldehyde group and aminooxy group (oxime click reaction) usually proceeds under mild conditions and produces stable oxime bonds because the aminooxy is more nucleophilic than hydrazide due to its α-effect.10,11 In addition, the product is very stable in a wide pH range even without adding any reducing reagent. These features would benefit glycoprotein purification because there is no need to

s one of the most important posttranslational modifications of proteins in eukaryotes, aberrant glycosylation is implicated in various diseases.1 The analysis of glycoprotein expression has facilitated the discovery of many novel potential biomarkers.2 The mass spectrometry (MS)-based strategy is currently the most sensitive and high-throughput analytical method for glycoproteome analysis.3,4 To perform a successful MS analysis, it is critical to derive a well-separated sample of glycoproteins/glycopeptides from extremely complex biological mixtures such as serum, which consists of tens of thousands of proteins spanning a dynamic range of 12 orders of magnitude. Selective enrichment of the glycoproteome significantly reduces the complexity of the sample, substantially increasing the sensitivity and the dynamic range of analysis.5 Among various enrichment methods,6 hydrazide chemistry-based solid-phase extraction has been employed extensively for the purification of glycoproteins/glycopeptides because of its remarkable selectivity.7,8 It is mediated by the binding of the oxidized glycoproteins/glycopeptides to a solid support through hydrazide chemistry. However, this protocol typically requires more than 12 h to complete the coupling reaction between the glycopeptides and the hydrazide resin. In addition, although commercially available, these hydrazide-functionalized solid supports are cumbersome to use due to the difficulties of separating them from the liquid phase and redispersing, consequently reducing the sensitivity and reproducibility. © 2014 American Chemical Society

Received: May 19, 2014 Accepted: July 21, 2014 Published: July 21, 2014 7920

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remove any additional substances and cause sample loss. Although this oxime click reaction has been extensively studied for decades in glycomics research, for example, labeling the reductive end of a glycan12 and introducing the isotope tag on sialic acid,13 it is rarely employed for N-glycoprotein enrichment except in recently reported work.14 In this strategy, an oxime reaction between the aldehyde group and the aminooxy group is employed to efficiently introduce an aminooxyfunctionalized biotin on the glycoproteins with specific glycans (i.e., terminated with sialic acid or galactose) to biotinylate the corresponding glycoproteins. These biotinylated glycoproteins are then captured on streptavidin-coated beads, thus enabling the targeted glycoproteins to be obtained with high specificity. However, the complete process is an involved, complex operation with many steps and is very cumbersome, which limits the throughput and sensitivity. In addition, the selectivity is somewhat limited by the relatively weak interaction between biotin and streptavidin. These disadvantages prompted us to design novel functionalized nanoparticles and create an extraction method based on this click reaction for the highly selective capture of the N-glycoproteome. Reported herein is a highly selective and ultra fast glycoprotein solid-phase extraction method exploiting the oxime click reaction between glycoprotein and an aminooxy group immobilized on the solid phase, which significantly reduces the enrichment time to 1 h. On the basis of the easy and rapid separation of the nanoparticles from the liquid phase, we designed novel aminooxy-functionalized nanoparticles with a magnetic core that could function as a versatile tool in protein separation due to its paramagnetism. After being functionalized with aminooxy groups through a silylation reaction, the magnetic nanoparticles (Fe3O4@SiO2-ONH2) were added to the sample solution. The attached aminooxy groups on the nanoparticles exclusively reacted with the oxidized glycan chain from glycoproteins/glycopeptides; therefore, glycopeptides were selectively bound to the nanoparticles, while other unbound peptides were washed away later (Scheme S1, Supporting Information). In this process, with the aid of the acid, the aldehydes from the oxidized glycan bind to the anilines in the solution, followed by dehydration to form a phenyl imine intermediate, which has a higher reactivity to trap aminooxy than aldehyde. Then, the aminooxys attack the phenyl imine intermediates and replace the anilines to form the oxime15,16 (Figure 1).

Figure 1. (a) Illustration of the oxime click reaction for glycoprotein enrichment. (b) Proposed main reaction pathways of the oxime formation.

Shanghai Chemical Reagent Co., Ltd., which were used as received without further purification. The water used in the experiments was ultrapure water obtained from a Milli-Q Water System (Millipore, Bedford, MA). Synthesis of BOC-Protected ONH2 Functional Group Silane Coupling Agent (BOC-AoTES). (3-Aminopropyl)triethoxysilane (442 mg, 2.00 mmol), (BOC-aminooxy)acetic acid (392 mg, 2.00 mmol), EDC (392 mg, 2.00 mmol), and ACN (30 mL) were combined in a 50 mL round-bottom flask and stirred at room temperature overnight. The mixture was concentrated under reduced pressure and purified by flash chromatography on silica gel using ethyl acetate to afford pure BOC-AoTES as a colorless oil (645 mg, 79%): 1H NMR (400 MHz, ClD3): δ 4.32 (s, 2H), 3.82 (q, 6H), 3.31 (dd, 2H), 1.68 (m, 2H), 1.48 (s, 9H), 1.23 (t, 9H), 0.68 (t, 2H). 13C NMR: 169.26 (NHCO), 157.77 (NHCO), 82.50 (CH2), 76.02 (C), 58.24 (CH2), 41.29 (CH2), 28.07 (CH3), 22.74 (CH2), 18.24 (CH3), 7.54 (CH2). ESI-MS (m/z): [BOC-AoTES + H+] calcd. for: C16H34N2O7Si, 395.54; found, 395.83. Preparation of Fe3O4 Nanoparticles. The Fe3O4 nanoparticles were prepared through a modified solvothermal reaction. Typically, 1.35 g of FeCl3·6H2O, 3.85 g of NH4Ac, and 0.40 g of sodium citrate were dissolved in 70 mL of ethylene glycol. The mixture was stirred vigorously for 1 h at 170 °C to form a homogeneous black solution and then transferred into a Teflon-lined stainless-steel autoclave (100 mL capacity). The autoclave was heated to 200 °C and maintained for 16 h and then cooled to room temperature. The black product was washed twice with ethanol, collected with the help of a magnet, and finally dried in the vacuum oven overnight at 40 °C. Preparation of Fe3O4@SiO2 Core−Shell Nanoparticles. The Fe3O4@SiO2 core−shell nanoparticles were prepared through a modified Stöber method. Typically, Fe3O4 nanoparticles (0.10 g) were dispersed in ethanol (80 mL) with 0.5 h sonication followed by the sequential addition of ammonia (25%, 1.00 mL), water (20.0 mL), and TEOS (1.0 mL). The resulting mixture was stirred for 12 h at room temperature. The



EXPERIMENTAL SECTION Materials and Chemicals. Asialofetuin from fetal calf serum (ASF) and myoglobin from horse heart (MYO) as well as (BOC-aminooxy)acetic acid, (3-aminopropyl)triethoxysilane (APTES), dithiothreitol (DTT), acetonitrile (ACN), ammonium bicarbonate (NH4HCO3), urea, MALDI matrix (α-cyano4-hydroxycinnamic acid, CHCA), and trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich. Sequencing grade modified trypsin was from Promega. The glyceol free peptideN-glycosidase (PNGase F, 500 units/μL) was from New England Biolabs. Sep-Pak C18 columns were from Waters. Bradford reagent together with Affi-Gel Hz hydrazide gel were from Bio-Rad. Human serum from colorectal cancer patients were provided by Fudan University Shanghai cancer center and stored at −80 °C before analysis. Ammonia, FeCl3·6H2O, NH4Ac, sodium citrate, ethylene glycol, and TEOS were obtained from Sinoreagent Chemical Reagent Co. Ltd. Other chemical reagents were of analytical grade and obtained from 7921

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nanoparticles (Fe3O4@SiO2-ONH2) is very easy. Compared to the tedious synthesis process of hydrazide functionalized resin, the Fe3O4@SiO2-ONH2 can be obtained simply by modifying the Fe3O4@SiO2 core−shell nanoparticles with an aminooxyfunctionalized silane coupling agent, as shown in Figure 2a. In

product was washed several times with ethanol and water and collected with the help of a magnet. The final product was dispersed in isopropanol for further use. Preparation of Fe3O4@SiO2-ONH2 Nanoparticles. The Fe3O4@SiO2-ONH2 nanoparticles were prepared by modifying Fe3O4@SiO2 nanoparticles with BOC-AoTES and then treated briefly with TFA. Two hundred milligrams of Fe3O4@SiO2 was dispersed in 60 mL of isopropanol, and then, 100 mg of BOCAoTES was added to the suspension. After the reaction solution was mechanically stirred for 24 h, 6 mL of TFA was added to the suspension. The products were collected by magnetic separation after 1 h and washed with ethanol and water and then finally dried in the vacuum oven overnight at 40 °C. Enrichment of N-Glycopeptides with Fe3O4@SiO2ONH2 Nanoparticles and MALDI MS Analysis. The lyophilized glycopeptides were redissolved in oxidation buffer (pH5.5) containing 100 mM sodium acetate and 150 mM NaCl. Then, the cis-diols of the carbohydrate groups on the glycopeptides were oxidized by 10 mM sodium periodate (NaIO4) at room temperature for 1 h in the dark under constant shaking. Afterward, to remove the excess NaIO4, 20 mM sodium sulfite was used to quench the oxidation by incubating for another 10 min at room temperature. Prior to the conjugation step, the oxidized products were lyophilized to guarantee the buffer exchange procedure. Then, a stock solution (pH4.6) containing 10 mM ammonium acetate was applied as a coupling buffer, followed by the addition of aminooxy end-functionalized nanoparticles that had been prewashed twice with coupling solution. Finally, 100 mM aniline was introduced to the above mixture in the dark and incubated at different temperatures for a specified time under constant shaking. The removal of impurities was achieved with the aid of a magnet by washing the nanoparticles twice with each of the following solutions sequentially: pure water, 80% ACN/20% H2O (v/v), and 50 mM NH4HCO3. Afterward, the glycans and peptide moieties were separated through the incubation of a mixture containing fresh 50 mM NH4HCO3 and 1 μL of PNGase F (500 units per μL) together with the nanoparticles at 37 °C overnight. The supernatant containing the released peptides was collected through magnetic separation for MS analysis. MALDI MS (Applied Biosystems, Framingham, MA) was applied for the analysis of model proteins. The automated acquisition of 5800 MALDI-TOF mass spectra was accomplished through the average of 1000 laser shots and performed in the positive ion mode. While operating, the laser was set to a 400 Hz repetition rate, and the wavelength was 355 nm. Typically, equivalent amounts of analytes together with matrix were sequentially spotted onto a MALDI target plate (AB SCIEX). The matrix employed in this work is the commonly used CHCA (α-cyano-4-hydroxycinnamic acid), which was freshly prepared by dissolving 10 mg/ mL CHCA in 50% ACN (v/v) containing 0.1% TFA. The mass instrument was calibrated with MYO digests as an internal calibration mode before each analysis. The enrichment of Nglycoproteome with Fe 3 O 4 @SiO 2 -ONH 2 nanoparticles, nano1DLC-MS/MS analysis, and data analysis can be found in the Supporting Information.

Figure 2. (a) Synthesis scheme for Fe3O4@SiO2-ONH2. (b) FT-IR spectra of (i) Fe3O4, (ii) BOC-AoTES, (iii) Fe3O4@SiO2-ONH-Boc, and (iv) Fe3O4@SiO2-ONH2. TEM image of (c) Fe3O4 and (d) Fe3O4@SiO2-ONH2. All scale bars are 100 nm.

detail, a silane coupling agent with a Boc-protected aminooxy functional group (BOC-AoTES) was synthesized and chromatographically purified and then used to modify Fe3O4@SiO2 nanoparticles through a one-step silylation reaction to obtain Fe3O4@SiO2−ONH-Boc. The final product of Fe3O4@SiO2ONH2 was obtained by treating the Fe3O4@SiO2-ONH-Boc with 10% trifluoroacetic acid (TFA) to remove the protecting group. The NMR spectra demonstrate the successful synthesis of the silane coupling agent (Figure S1, Supporting Information), and detailed attribution of the peaks can be found in the Experimental Section. To further provide direct proof of the aminooxy functionalization, Fourier transform infrared (FT-IR) spectroscopy was used to characterize the Fe3O4, BOC-AoTES Fe3O4@SiO2-ONH-Boc, and Fe3O4@ SiO2-ONH2 particles (Figure 2b). The FT-IR spectrum of Fe3O4 shows the asymmetric stretching vibrations of COO− at 1630 cm−1, the symmetric stretching vibrations of COO− at 1400 cm−1, and the stretching vibrations of Fe−O at 586 cm−1, indicating the formation of Fe3O4 nanoparticles. The spectrum of Boc-AoTES shows strong absorption peaks at approximately 2850−2960 and 1720 cm−1, corresponding to the C−H and CO stretching vibrations from the tert-butoxycarbonyl structure. The FT-IR spectrum of the Fe3O4@SiO2-ONHBoc particles displays the adsorption peak at 1087 cm−1 for the Si−O−Si vibration and the characteristic bands at approximately 2850−2960 and 1720 cm−1, which are in agreement with that of Boc-AoTES. The above results indicate the successful formation of the SiO2 shell and the modification of Boc-AoTES on the SiO2 shell. Furthermore, the disappearance of the peaks at approximately 2850−2960 and 1720 cm−1 in the FT-IR spectrum of the final product Fe3O4@SiO2-ONH2 implies Boc cleavage by trifluoroacetic acid. The transmission electron microscopy (TEM) images (Figure 2c,d) reveal that the iron oxide microspheres are well encapsulated in a condensed, amorphous silica shell, and both the functionalized



RESULTS AND DISCUSSION The Fe3O4@SiO2-ONH2 nanoparticles exhibit several remarkable features that are important in glycoproteome extraction. First, the synthesis of the aminooxy-functionalized magnetic 7922

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Fe3O4@SiO2 and Fe3O4@SiO2-ONH2 maintain the same shape as the unmodified counterpart Fe3O4 microspheres. Moreover, the superparamagnetic core of Fe3O4 (Figure S2, Supporting Information) facilitates the separation of the Fe3O4@SiO2ONH2 material from the solution within 30 s with the help of a magnet. After removal of the magnetic field, the nanoparticles can reversibly redisperse into the solution. This excellent magnetic response and redispersibility of the Fe3O4@SiO2ONH2 material plays an important role in the rapid separation process. The second advantage of the solid-phase extraction method is its simplicity and fast coupling speed. The proposed novel method for the isolation and analysis of N-glycoproteome is simple (Scheme S1, Supporting Information): (1) oxidation of glycopeptides by NaIO4; (2) coupling oxidized glycopeptides onto Fe3O4@SiO2-ONH2 through the oxime click reaction; (3) washing away nonspecifically adsorbed proteins/peptides and other impurities; (4) release of peptides by deglycosylation enzyme PNGase F and finally MS analysis. However, in the hydrazide chemistry-based enrichment procedure, a desalting step is necessary before step 2. Therefore, the omission of the desalting process simplified the overall process of this new method. With the exceptional click reaction in mind, we particularly investigated the coupling time in step 2 between the Fe3O4@SiO2-ONH2 nanoparticles and the glycopeptides. The intensity of the deglycosylated peptide showed significant enhancement from 30 min to 1 h, and a slight enhancement was observed between 1 and 4 h, while no significant change was observed when the reaction time varied from 4 to 8 h (Figure S3, Supporting Information). This result indicated that the enrichment could be performed in 1 h, which is nominal compared to the traditional hydrazide chemistry-based solidphase extraction that can range from 12 to 16 h. For complex sample analysis, a 4 h coupling time is suggested, as the S/N of enriched glycopeptides reaches saturation when the coupling time is 4 h (Figure S3, Supporting Information). Also, the reaction temperature is determined as 45 °C, while higher temperature did not result in better enrichment performance (Figure S4, Supporting Information). That may be caused by the thermal decomposition of oxime bonds in high temperature and form iminyl radicals.17 Importantly, in contrast to our recently developed method based on a reductive amination reaction,9 this click reaction can be performed under milder conditions in an ammonium acetate buffer. The mild reaction buffer is also important, because nonenzymatic asparagine deamidation could be eliminated. The third advantage of the proposed novel method for the isolation and analysis of N-glycoproteome is its specificity. To investigate the enrichment selectivity toward glycopeptides, the Fe3O4@SiO2-ONH2 nanoparticles were used to capture glycopeptides from the digests of a glycoprotein asialofetuin (ASF) containing both glycopeptides and nonglycopeptides. Before the selective enrichment, the spectrum was dominated by nonglycopeptides (Figure 3a), and their presence led to a low signal-to-noise ratio of glycopeptides, with only one glycopeptide detectable at m/z 5004.2. After selective enrichment, eight dominant peaks of ASF deglycosylated glycopeptides could be prominently identified with a clean background (Figure 3b). The peaks observed at m/z 1627.7, 1754.8, 1780.8, 1933.8, 1951.8, 2755.6, 3017.5, and 3558.8 represented deglycosylated peptides from the tryptic glycopeptides of ASF. These peaks included all of the three known Nglycosylated sites of ASF at N99CS, N176GS, and N156DS. The

Figure 3. MALDI-TOF mass spectra of tryptic digest of ASF (a) before isolation and (b) after isolation by Fe3O4@SiO2-ONH2 and deglycosylation by PNGase F. The symbols “∗”and “#” represent the nonglycopeptides and glycopeptides; “@” represents the deglycosylated glycopeptides, and “∧” represents fragments from deglycosylated glycopeptides.

detailed amino acid composition of each deglycosylated glycopeptide and the corresponding tryptic glycopeptides are listed in Table S1, Supporting Information. To further evaluate their ability to capture glycopeptides in complex samples, the Fe3O4@SiO2-ONH2 nanoparticles were applied to trap glycopeptides in a mixture of a tryptic digest of ASF and myoglobin (Myo, a nonglycoprotein) with the molar ratio of ASF/Myo at 1:100. No glycopeptides could be detected before enrichment due to the presence of large amounts of nonglycopeptides from the Myo (Figure S5a, Supporting Information). After Fe3O4@SiO2-ONH2 enrichment, deglycosylated peptides could be observed in the mass spectra (Figure S5b, Supporting Information), indicating that notable separation was achieved. In comparison, the selective enrichment was also performed using commercial hydrazine resin (Figure S5c, Supporting Information). The efficiency of the enrichment using the commercially available hydrazine resin was inferior, with lower deglycosylated peptide intensities, and nonglycopeptide peaks dominated the spectrum. Overall, the Fe3O4@SiO2ONH2 materials showed significantly higher specificity and efficiency for glycopeptide enrichment than the commercial hydrazine resin-based enrichment method. The fourth advantage of this method is its sensitivity. The ability to capture glycopeptides with high sensitivity is especially important for glycoproteome profiling because many glycopeptides are present at relatively low abundance. Therefore, we investigated the sensitivity of this novel method using standard glycopeptides with different initial concentrations from 500 to 10 ng·μL−1. Interestingly, when the concentration of the digest product was decreased by nearly 2 orders of magnitude, the deglycosylated peptide peaks, including all three glycosylated sites of ASF, remained observable in the mass spectrum after the selective trapping of the glycopeptides using Fe3O4@SiO2-ONH2 nanoparticles 7923

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Author Contributions

(Figure S6, Supporting Information). These results indicate that the nanoparticles can sensitively extract glycopeptides from a dilute solution, and the detection limit for the glycopeptides is in the low-fmol regime. We propose that these improvements may be due to the elimination of the desalting step after glycopeptide oxidation, which may cause a significant amount of sample loss. In addition, this method of enrichment has good reproducibility because the magnetic properties allow fast separation and ease of operation. This feature is important because, during the traditional resin wash steps, inadvertently aspirated resin tends to interfere with reproducibility. We found that the CVs between three replicates of three representative glycopeptides isolated from the model glycoprotein ASF were 37.0% (N99CS), 15.8% (N176GS), and 13.9% (N156DS) (calculated from Figure S6, Supporting Information); the CV for N99CS is relatively high because the signal of this glycopeptide is much lower than the other two. These data demonstrate that good reproducibility in glycopeptide isolation has been achieved using the magnetic Fe3O4@SiO2-ONH2. Moreover, the recovery of this method was evaluated to be 68.45% (Figure S7, Supporting Information). Because the described enrichment process has been proven to be effective for glycoproteomic analysis, we further tested this method with a serum sample provided by Fudan University Shanghai Cancer Center. After reduction and alkylation, 1 μL of the human serum sample was tryptic-digested into peptides, followed by oxidation, oxidation quenching, and lyophilization. Then, the oxidized peptides were redissolved and treated with Fe3O4@SiO2-ONH2. Afterward, the glycopeptides captured on the Fe3O4@SiO2-ONH2 were harvested and then released by PNGase F. Finally, the deglycosylated peptides were sent for nano-LC-MS/MS analysis. Combined with the mass increment of 0.98402 Da of asparagine (N) being transformed into aspartic acid (D), the identification of N-linked glycosylation sites and glycopeptides could be unambiguously assigned by the existence of N-X-S/T (X ≠ P) sequences. In total, 100 unique N-glycosylation sites were found in 143 unique glycopeptides corresponding to 61 glycoproteins (Table S2, Supporting Information). The number of identified glycoproteins from 1 μL of serum exhibited comparable results to several other reports that profiled the N-glycoproteome using 15−50 μL of human serum or plasma through hydrazide chemistry-based solid-phase extraction.18,19

§

Ying Zhang and Meng Yu contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the National Science and Technology Key Project of China (2012CB910602, 2012AA020204, and 2012CB910103), the National Science Foundation of China (Grant Nos. 21025519, 21335002, 51073040, and 21375026), and Shanghai Projects (Grants Eastern Scholar, 11XD1400800, 13520720200, and B109).





CONCLUSIONS In summary, this novel method achieved the highly efficient separation of N-glycoproteins with excellent sensitivity and proved to be able to effectively analyze a small sample. The process of enrichment is very quick and specific, opening up the potential exploitation of aminooxy-functionalized materials for practical applications in the MS-based glycoproteomic study of complex biological samples.



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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. 7924

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