Preparation of Sequence-Controlled Triblock Copolymer-Grafted

Dec 11, 2014 - In this work, we prepared sequence controlled triblock copolymer grafted silica-microparticles (TCP-SMs) by sequential atom transfer ra...
1 downloads 9 Views 2MB Size
Download PDF
Article pubs.acs.org/ac

Preparation of Sequence-Controlled Triblock Copolymer-Grafted Silica Microparticles by Sequential-ATRP for Highly Efficient Glycopeptides Enrichment Yiting Pan,†,‡ Cheng Ma,† Wei Tong,§ Chao Fan,† Qian Zhang,† Wanjun Zhang,† Fang Tian,† Bo Peng,† Weijie Qin,*,† and Xiaohong Qian*,† †

National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 100850, China ‡ Chemical Engineering College, Beijing Institute of Petrochemical Technology, Beijing 102617, China § Tianjin Key Laboratory for Prevention and Control of Occupational and Environmental Hazards, Logistics College of CAPF, Tianjin 300162, China S Supporting Information *

ABSTRACT: As one of the most important subproteomes in eukaryote cells, N-glycoproteins play crucial roles in various of biological processes and have long been considered closely correlated with the occurrence, progression, and metastasis of cancer. Comprehensive characterization of protein N-glycosylation and association of their aberrant patterns to the corresponding cancer stage may provide a unique way to discover new diagnostic biomarkers and therapeutic drug targets. However, the extremely complex nature of biological samples and relatively low abundance of N-glycosylated proteins makes the enrichment of glycoprotein/glycopeptide a prerequisite for large scale N-glycosylation identification. In this work, we prepared sequence controlled triblock copolymer grafted silica-microparticles (TCP-SMs) by sequential atom transfer radical polymerization (sequential-ATRP) of monosaccharides and zwitterionic-ion monomers for highly efficient and selective glycopeptides enrichment. The triblock copolymer is composed of sequence defined poly zwitterionic-ion, poly-Nacetylglucosamine and poly mannose blocks. The glycopolymer blocks carrying densely packed pendent sugars are excellent mimics of the natural carbohydrate clusters and may induce multivalent carbohydrate-carbohydrate interaction (CCI) with the target glycopeptides. Therefore, increased retention of glycopeptides can be expected by the combination of CCI and zwitterionic−HILIC interaction. As a result, 1244 glycopeptides were identified after TCP-SMs enrichment from mouse liver, which are 65−120% higher than that obtained by homoglycopolymer or random-copolymer grafted silica microparticles prepared using the conventional free radical polymerization. These results demonstrate the critical role of sequence-defined block copolymer of TCP-SMs for obtaining enhanced affinity toward glycopeptides and the potential of this sequential-ATRP strategy to integrate different affinity moieties into one enrichment material to achieve deeper coverage in protein PTM mapping.

P

accuracy, mass spectrometry-based glycoproteomics analysis is currently the method of choice for large scale and in-depth protein N-glycosylation profiling.10−13 However, because of the low abundance of the N-glycosylated proteins/peptides compared with their counterparts without glycosylation, glycopeptide enrichment is a crucial step for achieving efficient and sensitive identification by mass spectrometry.14 Various of enrichment methods based on hydrazide chemistry,15−18 boronic acid,19−21 and lectin affinity22,23 have been developed and successfully applied in glycopeptides enrichment. However, due to the nontemplate based biosynthesis of glycans

rotein N-glycosylation has been considered as one of the most common and complex post-translational modification in eukaryotes. It plays critical roles in many key cellular processes including intracellular communication, cell adhesion/ migration, and immune response and has close association with the invasion and metastasis of cancer.1−4 Alterations of the Nglycosylation sites and the attached glycans are frequently observed in various types of cancer, such as ovarian, breast, prostate, liver, and lung cancers and involved in almost every aspect of tumor progression.5−7 Large-scale systematic profiling of these variations of protein N-glycosylation in complex biological samples not only largely facilitates the discovery new biomarkers for early diagnosis of cancer but also provides useful information for therapeutic treatment and prognosis study of the cancer patients.8,9 Because of its speed, resolution, and © XXXX American Chemical Society

Received: September 11, 2014 Accepted: November 24, 2014

A

dx.doi.org/10.1021/ac5034215 | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

ymer brushes carrying densely attached mannose and Nacetylglucosamine moieties may adopt varying orientations and overall architecture, which makes them excellent mimics to the natural carbohydrate clusters and induces retention of glycopeptides via CCI. Furthermore, the poly-ZIC block in the copolymer provides unique hydrophilicity and ensures strong HILIC retention of glycopeptides. The cooperation of these two kinds of interactions largely increases the efficiency and comprehensiveness of glycopeptides enrichment, as demonstrated by the successful application of TCP-SMs in proteins glycosylation mapping of mouse liver, in which 1244 glycopeptides corresponding to 577 glycoproteins were identified. Apart from efficient covering the majority (>85%) of the glycopeptides identified by different traditional HILIC materials, TCP-SMs enrichment leads to 65−120% increase in the scale of glycosylation mapping indicating strong potential of this block copolymer based strategy to synthesize materials with integrated and enhanced affinity for PTM enrichment and identification.

controlled by multiple glycosyltransferases, protein glycosylation is notorious for its high heterogeneity. The greatly diversified glycoforms lead to varying properties of the glycopeptides which makes the complete enrichment extremely challenging. Currently, the glycopeptides enrichment and identification is still far from completion, as indicated by the only ∼5% coverage of the theoretical glycosylation sites.24 Among the commonly used glycopeptides enrichment methods, hydrophilic interaction liquid chromatography (HILIC)-based solid phase extraction (SPE) is particularly advantageous, due to its high compatibility with mass spectrometry and nondestructive enrichment capability. Preservation of the glycan moieties for intact glycopeptide analysis is a prerequisite for site-specific glycan identification. Different types of HILIC materials have been developed and applied in glycopeptides enrichment, including zwitterion-ion,25,26 click maltose,27−29 amide10,30 and cotton.31 In HILIC based SPE, the retention of glycopeptides relies mainly on the hydrophilic interaction between the samples and the stationary phase. The greatly diversified glycoforms and the presence of hydrophobic amino acids in glycopeptides result in large variations in their hydrophilic properties, which make comprehensive glycopeptides enrichment solely by hydrophilic retention difficult. Some relatively hydrophobic glycopeptides may not be well retained by the typical HILIC-SPE materials. Furthermore, coelution of the hydrophilic nonglycopeptides leads to severe interference and signal suppression of the glycopeptides in mass spectrometry analysis. Therefore, there are urgent needs to develop new robust SPE materials that integrate multiple types of affinity/retention toward glycopeptides for achieving improved enrichment selectivity and comprehensiveness. Carbohydrate-carbohydrate interactions (CCI) displayed on cell surfaces widely exist in many critical biological events, including cell adhesion, migration, cell−cell interaction, and communication.32−34 This relatively weak interaction can be largely enhanced by cooperation of multiple CCIs between natural carbohydrate clusters, which is known as the “multivalent binding effect”.35,36 On the basis of this principle, we synthesized a new triblock copolymer grafted silica microparticles (TCP-SMs) based SPE material which combines CCI and HILIC for highly efficient and selective glycopeptides enrichment. TCP-SMs are prepared by sequence-controlled block copolymerization of zwitterion-ion and monosaccharide monomers on silica microparticles using sequential atom transfer radical polymerization (sequential-ATRP). Compared with the conventional “graft to” based surface coating methods, in which long polymer chains are attached to the surface, “graft from” based ATRP strategy offers significantly increased surface grafting density and well-controlled polymer structure and thickness,37−40 because it enables highly controllable in situ growth of polymer brushes from the initiator-capped surface. Three kinds of monomers including the highly hydrophilic zwitterionic-ion, N-acetylglucosamine, and mannose which are the two major components of natural carbohydrates were used for copolymer grafting on silica microparticles. By exploiting the advantage of sequential-ATRP, copolymer composed of three well-defined-blocks (poly-mannose, poly-N-acetylglucosamine, and poly-ZIC) with controlled ratio of monomers in each block can be easily obtained, even though the polymerization activities of the three monomers are quite different. Such characteristics are a challenge to obtain using conventional methods of monolayer-functionalization or free radical polymerization on silica microparticles. The block glycopol-

■

EXPERIMENTAL SECTION Materials and Reagents. Bovine serum albumin (BSA), bovine asialofetuin, chicken OVA, glycidyl methacrylate (GMA), acryloyl chloride, 2-bromoisobutyrate bromide, copper(II) chloride, cuprous chloride, D-glucosamine hydrochloride, D -mannosamine hydrochloride, and [2(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (MDSA) were obtained from Sigma (St. Louis, MO, USA). Porous silica microparticles (3 μm, 150 Å) were obtained from Agela Technologies (Tianjin, China). 3Aminopropyl-triethoxysilane and N,N,N′,N″,N″-pentamethyldiethylenetriamine were purchased from Acros (Belgium). FITC-labeled lectins (ConA and WGA) were purchased from Vector Laboratories (Burlingame, CA, USA). Sequencing grade porcine trypsin was received from Promega (Madison, WI, USA). PNGase F was obtained from New England Biolabs (Beverly, MA, U.S.A.). Mouse (C57BL/6J, males, aged 9−13 weeks) were received from Beijing HFK Bioscience company (Beijing, China). Deionized water used in all experiments (resistance >18 MΩ/cm) was prepared with a Millipore purification system (Billerica, MA, U.S.A.). Preparation of Triblock Copolymer Grafted SilicaMicroparticles (TCP-SMs) by Sequential-ATRP, PolyGMAM, Poly-AGA, Poly-MDSA Homopoloymer Grafted Silica Microparticles and Random Copolymer Grafted Silica Microparticles by ATRP. The procedures for preparing of N-acryloyl-glucosamine (AGA) and GMA modified mannosamine (GMAM) monomers were shown in the Supporting Information. The ATRP reaction initiator, 3-(2-bromoisobutyramido) propyl (triethoxy)-silane (BIBAPTES) was synthesized using the procedures reported in our previous work.41 After treatment of the silica microparticles with BIBAPTES (0.002 M) for 12 h for initiator immobilization on the surface of the microparticles, excess initiators were removed by repeated washing with methanol. Sequential-ATRP grafting was carried out by mixing the initiator immobilized silica microparticles with 0.01 M CuCl, 0.001 M CuCl2, 0.015 M N,N,N′,N″,N″pentamethyldiethylenetriamine and the first monomer, MDSA (2 M) in methanol. The reaction mixture was sonicated to form homogeneous solution, bubbled with nitrogen for 10 min to remove oxygen, sealed and put in a 37 °C water bath. After the preset reaction time and consumption of the MDSA monomers were reached, excess reagents were removed by repeated B

dx.doi.org/10.1021/ac5034215 | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

MALDI-TOF-MS Analysis. The obtained deglycosylated glycopeptides of asialofetuin were desalted by C18 Zip-Tip and redispersed in 5 μL of CHCA solution (5 mg/mL, 50% ACN, 0.1% TFA). The glycans released from the enriched glycopeptides of OVA were desalted by porous graphitic carbon column and redispersed in 5 μL of SDHB solution (10 mg/mL, 50% ACN, 0.1% TFA). A 1 μL sample was spotted on the target plate and air-dried. MALDI-TOF-MS analysis was carried out using a 4800 MALDI-TOF-TOF analyzer (AB Sciex, USA) equipped with a Nd:YAG laser at excitation wavelength 355 nm. All the mass spectra (1000 laser shots for every spectrum) were acquired in positive reflection mode after being calibrated using peptides originated from myoglobin and analyzed by Data Explorer (Version 4.5). LC-MS Analysis and Data Processing. The LC-MS/MS analysis was carried out using an EASY-nLC 1000 system coupled with a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA). The spray voltage was set at 2.0 kV. All MS and MS2 spectra were acquired in data-dependent acquisition mode. The mass spectrometer was set to a scan range of 300− 1400 m/z and the 20 most intense precursor ions were selected for MS/MS analysis by higher-energy collision dissociation (HCD). For data processing, all MS/MS spectra were searched against UniProt database (version 201204206, 65,493 entries) using Protein Discoverer software (version 2.0, Thermo Scientific). Trypsin was chosen as the proteolytic enzyme and up to two miss cleavages were allowed. Carbamidomethyl (Cys) was set as the fixed modification and Oxidation (Met) was set as the variable modification. The mass tolerance of the precursor ion was set to 15 ppm and that of the fragment ions was set to 20 mmu. The peptide false discovery rate (FDR) was set to 1%. The localization of the N-glycosylation site was determined by a mass increase of 0.984 Da on the N-X-S/T (X*P) sequon after deamidation of asparagine residue into aspartic acid by PNGase F deglycosylation.

washing with methanol. The second monomer, AGA was polymerized to the chain end of poly-MDSA using the polyMDSA grafted silica microparticles as the macromolecular initiators. At last, the third monomer, GMAM was polymerized to the poly-MDSA-AGA block copolymer using the same way. The molar ratio of GMAM, AGA and MDSA monomers used for sequential-ATRP is 1:1:2. The obtained TCP-SMs were dried by nitrogen flow and stored in 4 °C. For the preparation of poly-GMAM, poly-AGA, and poly-MDSA homopoloymer modified silica microparticles, ATRP grafting was carried out using similar conditions as that used in sequential-ATRP. For the preparation of random copolymer modified silica microparticles, mixture of GMAM, AGA, and MDSA at 1:1:2 molar ratio was used for ATRP grafting. Characterization of TCP-SMs. The surface morphology of bare silica microparticles without grafting and TCP-SMs was characterized by scanning electron microscope (SEM) using a XL30 ESEM-TMP scan electron microscope (Philips-FEI Corporation, Holland) operated with acceleration voltage of 120 keV. Thermogravimetric analysis (TGA) performed on a SDT Q600 instrument (TA Instruments, USA) was used to demonstrate the core−shell structure of TCP-SMs and highly controllable polymer grafting by sequential-ATRP. All samples were dried in a vacuum oven at 60 °C prior to each TGA measurement to remove residue water or solvent. The samples were heated from 20 to 700 °C at a heating rate of 5 °C/min under a flow of nitrogen. The fluorescence images showing the interaction pattern of FITC-ConA and FITC-WGA toward TCP-SMs were acquired using an IX71 fluorescence microscope (OLYMPUS, Japan). Protein Sample Preparation. Mouse live tissues were taken out and frozen in liquid nitrogen. The frozen liver tissue was grinded and homogenized using a Polytron homogenizer in 2 mL of denaturing buffer, containing 50 mM ammonium bicarbonate (pH 8.2) and 8 M urea. Proteins were extracted by sonicating the homogenized liver tissue. After the mixture was centrifuged at 12000g for 15 min at 10 °C, the supernatant was recovered, and the concentration of the obtained protein extracts was determined with the Bradford assay (Bio-Rad). Next, 50 μLof protein extracts from mouse liver, bovine asialofetuin, or OVA dissolved in 25 mM ammonium bicarbonate and 8 M urea were denatured by DTT reduction and IAA alkylation. After the protein solution was diluted with 25 mM ammonium bicarbonate to reduce the urea concentration below 1 M, trypsin was introduced at protein substrate to trypsin ratios of 25:1, and the mixture was incubated at 37 °C for 16 h for digestion. Finally, 4 μL of formic acid (10%) was added to the solution to terminate the digestion. Glycopeptide Enrichment. For glycopeptide enrichment, 2 mg of TCP-SMs were packed into a 200 μL of pipet tip. The packed TCP-SMs were activated and conditioned with 3 × 100 μL water containing 0.5% FA and 3 × 100 μL of 60% ACN containing 0.5% FA, respectively. After that, 100 μL of protein digests dispersed in 60% ACN, 5% FA and 10 mM Ca2+ were loaded to the packed TCP-SMs. Next, 3 × 100 μL of 60% ACN with 5% FA and 10 mM Ca2+ was used to remove the nonspecifically adsorbed nonglycopeptides. The retained glycopeptides were eluted by 3 × 100 μL of water with 0.5% FA. The obtained glycopeptides were freeze-dried and resuspended in 25 mM ammonium bicarbonate containing PNGase F (100 Unit) and incubated in 37 °C overnight for deglycosylation.

■

RESULTS AND DISCUSSION Preparation and Characterization of the Triblock Copolymer Grafted-Silica Microparticles (TCP-SMs) by Sequential-ATRP. To achieve the combined retention by zwitterionic-ion based HILIC and CCI, three types of monomer including the highly hydrophilic zwitterionic MDSA and two kinds of monosaccharide (mannose and Nacetylglucosamine) which broadly exist in natural carbohydrates are chosen for surface grafting on silica microparticles. The preparation procedure of TCP-SMs is illustrated in Scheme 1. Briefly, 3-(2-bromoisobutyramido) propyl (triethoxy)-silane (the ATRP initiator) is immobilized on the silica microparticles via Si−O bond. To obtain sequence-controlled grafting of block copolymer on the silica microparticles, sequential-ATRP is carried out, in which the three monomers (MDSA, AGA, and GMAM) are introduced to the polymerization system in a consecutive way. In this method, the subsequent monomer is not added until the consumption of the previous one. Benefiting from the unique characteristics of sequential-ATRP living polymerization, the surface grafted polymer chains with “active” alkyl halide at their ends serve as macromolecule initiators for the subsequent grafting of the next monomer. In this way, sequence-controlled poly(MDSA)-(AGA)-(GMAM) triblock copolymer containing unique zwitterion region (polyMDSA) and well-defined glycopolymer regions (poly-AGA and poly-GMAM) are obtained. Furthermore, the ratio of each block can be easily tuned by simply varying the molar ratio of C

dx.doi.org/10.1021/ac5034215 | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

triblock copolymer was determined by TGA analysis via plotting the weight loss versus temperature as shown in Figure 2. TCP-SMs consist of both thermally stable silica core that

Scheme 1. Schematic Overview of the Preparation of TCPSMs by Sequential-ATRP

each kind of monomers added to the polymerization system without the need of optimizing reaction conditions, even though the polymerization activity of the monomers used varies significantly. Such characteristics are difficult to obtain by conventional free radical polymerization methods. The obtained copolymer chains may adopt diversified conformations which result in uniform charge distribution in the zwitterion region and oligosaccharide clusters with varying density, structure and orientation. In contrast to the slightly negatively charged commercial ZIC-HILIC materials because of the more exposed SO3− groups,14 the neutrally charged TCPSMs are advantageous in reducing the electrostatic adsorption of the nonglycopeptides. Furthermore, the mannose and Nacetylglucosamine clusters in the glycopolymer regions may induce enhanced CCI with glycopeptides by “multivalent binding”, which can be further increased by the facilitation of divalent cations.42 Therefore, stronger and more specific binding toward glycopeptides can be expected by the combination of HILIC and CCI. Next, the surface morphology of the prepared TCP-SMs was characterized by scanning electron microscopy (SEM). As shown in Figure 1, TCP-SMs is fully covered by a polymer shell

Figure 2. TGA analysis of silica microparticles (a), poly-MDSA grafted silica microparticles (b), poly-MDSA-AGA grafted silica microparticles (c), and poly-MDSA-AGA-GMAM grafted silica microparticles (d).

remains in the residue and decomposable polymer shell that contribute to the weight loss after thermo treatment. Figure 2 are the TGA curves of TCP-SMs at different stage of sequential-ATRP block copolymerization, that is, bare silica microparticles without grafting (a), poly-MDSA-SMs (b), polyMDSA-AGA-SMs (c), and poly-MDSA-AGA-GMAM-SMs (d). As shown in the figure, only 3% weight loss is found for bare silica microparticles in the region below 100 °C which can be attributed to the evaporation of physically adsorbed water. In contrast, higher weight loss is observed for the polymer grafted silica microparticles and the weight loss increases roughly proportional to the feed in amount of the corresponding monomers after each step of block copolymerization. In total, ∼22% weight loss of surface bound polymer is found in Figure 2. Therefore, highly efficient surface grafting of block copolymer with controlled ratio between each block is obtained by the sequential-ATRP method. Next, the surface composition of TCP-SMs is characterized by X-ray photoelectron spectroscopy (XPS) and fluorescent lectin labeling analysis. XPS is a sensitive surface analysis technique that measures the surface elemental composition of about 5 nm in depth. As shown in Supporting Information Figure S-1, clear absorption peak of S 2p in vicinity of 168 eV representing the sulfonate group in MDSA is shown in the XPS spectrum. In contrast, no corresponding peak can be found in the bare silica microparticles demonstrating successful polymerization of MDSA on the surface of the silica microparticles. Next, fluorescent lectin labeling analysis was conducted to confirm successful grafting of AGA and GMAM. FITC tagged ConA and WGA which specifically recognize high mannose and N-acetyl-glucosamine were used. As revealed by the fluorescent micrographs in Supporting Information Figure S-2 (a and c), TCP-SMs display strong adsorption toward both FITC-WGA and FITC-ConA. Intense fluorescent signal is observed from TCP-SMs which clearly distinguished them from the background even after repeated washing to remove nonspecific adsorption. However, almost no fluorescent signal is observed in Supporting Information Figure S-2 (b and d) from silica microparticles grafted with only MDSA indicating little nonspecific adsorption of the fluorescent lectins. A competition

Figure 1. SEM images of TCP-SMs (a) and the bare silica microparticles without grafting (b).

composed of nanoparticles of about 50−100 nm in size and no bare silica is left without coating. Taking advantage of the surface initiations by the immobilized initiators, the polymer nanoparticles are solely grown from the surface of the silica microparticle with no bulk structure formation or cross-linking of the silica microparticles. More importantly, plenty of voids among the surface bound polymer nanoparticles can be found and the porous surface is beneficial for achieving high binding capacity. Next, the amount of each block of the surface grafted D

dx.doi.org/10.1021/ac5034215 | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

Figure 3. MALDI-TOF-MS spectra of direct analysis (a) and after TCP-SMs enrichment (b) of tryptic digested asialofetuin and BSA (1:10).

glycosites of asialofetuin are easily identified with high S/N (Figure 3b). To further evaluate whether TCP-SMs have biased retention for glycopeptides with specific glycoforms, the glycans of the TCP-SMs enriched glycopeptides from OVA were analyzed by MALDI-TOF-MS. To facilitate glycoform identification in MALDI-TOF-MS, the enriched glycopeptides were treated with PNGase F to release the glycan moieties. As shown in Figure S-3, 19 glycoforms with molecular weight ranging from 932.4 to 2311.9 Da are found in the MS spectrum. Even for the smallest glycan structure (932.4 Da), strong signal is observed in the MS spectrum indicating efficient enrichment is achieved. The large variety of the enriched glycoforms indicates nondiscriminated retention of glycopeptides by TCP-SMs, which is critical for achieving comprehensive coverage in protein glycosylation mapping. The enrichment efficiency of TCP-SMs for real biological sample was evaluated using mouse liver protein extracts. After TCP-SMs enrichment, 891, 805, and 1031 deglycosylated Nglycopeptides were identified in three replicates with 70.43% average enrichment specificity (Supporting Information Table S-1), which is obviously higher than that obtained after enrichment by commercially available ZIC-HILIC materials (233 deglycosylated N-glycopeptides and 43.4% specificity).43 The improved selectivity reduces the competition of nonglycopeptides in MS analysis and therefore facilitates glycopeptides identification and protein glycosylation mapping with deeper coverage. To evaluate the spontaneous deamidation caused false-discovery of N-glycopeptides, a control experiment was conducted using conditions exactly the same as that used in the TCP-SMs based enrichment and LC-MS analysis, except that the enriched peptides were not treated with PNGase F. In three replicates, we found 10, 13, and 17 peptides with 0.984 Da shift at Asn and the N−X−S/T/C (X ≠ P) motif (Supporting Information Table S-2), corresponding to

binding experiment was also performed by incubating FITCWGA or FITC-ConA with TCP-SMs in the presence of 500 mM N-Acetyl-glucosamine or α-Methyl D-mannoside. The presence of the two competition binding inhibitors efficiently inhibits the binding between FITC-WGA/ConA and TCPSMs. As shown in Supporting Information Figure S-2 (e and f), almost no fluorescent signal of FITC-WGA/ConA was found left on TCP-SMs. This result confirms the binding between FITC-WGA/ConA and TCP-SMs is via lectin-carbohydrate interaction. The strong XPS signal of sulfonate group and specific labeling of FITC-WGA/ConA on the surface of TCPSMs not only further demonstrates successful incorporation of the three monomers in the triblock copolymer by sequentialATRP, but also indicates that all of three polymer blocks are exposed on the surface of TCP-SMs and are capable of interacting with glycopeptides via HILIC and CCI. The ability to specifically enrich low abundant glycopeptides from complex mixture is a key issue for glycosylation mapping for real biological samples. Efficient depletion of nonglycopeptides largely enhances the detection sensitivity of low abundant glycopeptides because of the reduced signal suppression in mass spectrometer. To evaluate the enrichment selectivity of TCP-SMs, a complex sample containing a large fraction of nonglycopeptides was used. MALDI-TOF-MS spectra obtained from direct analysis and after TCP-SMs enrichment of a mixture of tryptic digest of 10 fmol of asialofetuin and 100 fmol of BSA are displayed in Figure 3. Without enrichment, the signals of the deglycosylated Nglycopeptides of asialofetuin are overwhelmed by the highly abundant nonglycopeptides and cannot be identified in the spectrum (Figure 3a). While, for the enriched sample, the signals from nonglycopeptides are almost completely removed and leaves a very clear background in the spectrum. Three deglycosylated N-glycopeptides that cover all the theoretical E

dx.doi.org/10.1021/ac5034215 | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

a 1.46% average false-discovery rate of N-glycopeptides in our experiment, which is consistent with the literature reported value.44 This result suggests that spontaneous deamidation is a minor issue in the TCP-SMs based glycopeptides enrichment and thus will not increase the FDR in the N-glycopeptide assignment in this research. The reproducibility of the TCPSMs based enrichment is also crucial for obtaining reliable glycopeptides identification and quantification. As shown in Figure 4, 70.8% deglycosylated N-glycopeptides and 82.0% N-

Furthermore, TCP-SMs display broader affinity toward different glycopeptides, as revealed by the high coverage of the identification results obtained by TCP-SMs to those by different kinds of HP-SMs. As shown in Figure 5b, more than 85% of the deglycosylated N-glycopeptides identified by the three types of HP-SMs was covered by TCP-SMs enrichment. For N-glycoproteins, even higher coverage (>90%) was achieved by TCP-SMs enrichment. The above results demonstrate the feasibility of polymerizing zwitterionicion and monosaccharide monomers into a hybrid stationary phase for achieving more comprehensive enrichment and deeper protein glycosylation coverage. Finally, we compared the enrichment capability of TCP-SMs with that of the hydrophilic copolymer grafted silica microparticles prepared by random copolymerization using the same three monomers (GMAM, AGA, and MDSA), in which the three monomers are randomly distributed in the copolymer chains. 754 deglycosylated N-glycopeptides and 413 Nglycoproteins were identified after enrichment by the random copolymer grafted silica microparticles in three replicates, which reach only 60.6% (deglycosylated N-glycopeptide)/71.6. % (N-glycoprotein) of that obtained by TCP-SMs enrichment indicating the critical role of forming sequence-defined and ratio-controlled block copolymer for achieving highly efficient glycopeptide enrichment. In TCP-SMs, the highly hydrophilic zwitterionic layer ensures strong hydrophilic retention of glycopeptides. More importantly, the pendent mannose and N-acetylglucosamine moieties in the poly-GMAM and polyAGA blocks may form higher-order oligosaccharide clusters with various density, structures and orientations. According to the “zipper” CCI model, such a “sugar code” is more advantageous in forming complementary conformations with the glycans of the target glycopeptides under the facilitation of Ca2+ that functions as the “zipper puller” providing driving force to stabilize the complementary carbohydrate-carbohydrate interactions.42 Therefore, broader and stronger binding affinity toward glycopeptides via cooperative HILIC and CCI can be expected. Such characteristic is difficult to obtain using random copolymer, due to the lack of control on the ratio and sequence of each kind of monomer that incorporated in the copolymer chain.

Figure 4. Reproducibility evaluation of TCP-SMs based enrichment, identified deglycosylated N-glycopeptides (a) and identified Nglycoproteins (b) in three replicates.

glycoprotein were identified in at least two replicates and 48.4% deglycosylated N-glycopeptides and 64.8% N-glycoproteins were identified in all three tests demonstrating good reliability of this enrichment method. To further evaluation the glycopeptides enrichment efficiency of TCP-SMs, the glycosylation identification of mouse liver proteins after enrichment was analyzed and compared with that obtained by homopolymer modified silica microparticles (HP-SMs) prepared using a single type of monomer (GMAM, AGA or MDSA). 1244 deglycosylated Nglycopeptides corresponding to 577 N-glycoproteins were identified using TCP-SMs enrichment (Supporting Information Table S-3), which is about 80−120% and 50−90% higher than the number identified after poly-GMAM-SM, poly-AGA-SM, or poly-MDSA-SM enrichment indicating the advantage of using TCP-SMs for more efficient enrichment (Figure 5a).

■

CONCLUSION In conclusion, we demonstrated the preparation of TCP-SMs by sequence controlled grafting of zwitterionic ion and monosaccharide on silica microparticles via sequential-ATRP for highly efficient and selective glycopeptides enrichment. Successful application of TCP-SMs in the enrichment of mouse liver glycoproteome was achieved by the combination of CCI and HILIC which results in enhanced glycosylation identification scale. Further applications of this facile strategy to prepare enrichment materials with combined affinity are expected to provide deeper coverage in protein PTM mapping.

■

ASSOCIATED CONTENT

S Supporting Information *

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

■

Figure 5. (a) Number of the deglycosylated N-glycopeptides and Nglycoproteins identified by TCP-SMs or HP-SMs enrichment. (b) Percentage of deglycosylated N-glycopeptides/N-glycoproteins identified by HP-SMs enrichment that covered by TCP-SMs enrichment.

AUTHOR INFORMATION

Corresponding Authors

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

dx.doi.org/10.1021/ac5034215 | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

Notes

(29) Xiong, Z. C.; Zhao, L.; Wang, F. J.; Zhu, J.; Qin, H. Q.; Wu, R. A.; Zhang, W. B.; Zou, H. F. Chem. Commun. 2012, 48, 8138. (30) Wohlgemuth, J.; Karas, M.; Eichhorn, T.; Hendriks, R.; Andrecht, S. Anal. Biochem. 2009, 395, 178. (31) Selman, M. H. J.; Hemayatkar, M.; Deelder, A. M.; Wuhrer, M. Anal. Chem. 2011, 83, 2492. (32) Sudibya, H. G.; Ma, J. M.; Dong, X. C.; Ng, S.; Li, L. J.; Liu, X. W.; Chen, P. Angew. Chem., Int. Ed. 2009, 48, 2723. (33) Ferrara, C.; Grau, S.; Jager, C.; Sondermann, P.; Brunker, P.; Waldhauer, I.; Hennig, M.; Ruf, A.; Rufer, A. C.; Stihle, M.; Umana, P.; Benz, J. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 12669. (34) Altamore, T. M.; Fernandez-Garcia, C.; Gordon, A. H.; Hubscher, T.; Promsawan, N.; Ryadnov, M. G.; Doig, A. J.; Woolfson, D. N.; Gallagher, T. Angew. Chem., Int. Ed. 2011, 50, 11167. (35) Lorenz, B.; de Cienfuegos, L. A.; Oelkers, M.; Kriemen, E.; Brand, C.; Stephan, M.; Sunnick, E.; Yuksel, D.; Kalsani, V.; Kumar, K.; Werz, D. B.; Janshoff, A. J. Am. Chem. Soc. 2012, 134, 3326. (36) Jayaraman, N.; Maiti, K.; Naresh, K. Chem. Soc. Rev. 2013, 42, 4640. (37) Barbey, R.; Lavanant, L.; Paripovic, D.; Schuwer, N.; Sugnaux, C.; Tugulu, S.; Klok, H. A. Chem. Rev. 2009, 109, 5437. (38) Matyjaszewski, K.; Tsarevsky, N. V. Nat. Chem. 2009, 1, 276. (39) Ping, Y. A.; Liu, C. D.; Tang, G. P.; Li, J. S.; Li, J.; Yang, W. T.; Xu, F. J. Adv. Funct. Mater. 2010, 20, 3106. (40) Xu, F. J.; Wang, Z. H.; Yang, W. T. Biomaterials 2010, 31, 3139. (41) Qin, W.; Song, Z.; Fan, C.; Zhang, W.; Cai, Y.; Zhang, Y.; Qian, X. Anal. Chem. 2012, 84, 3138. (42) Luo, P. J. G.; Wang, H. F.; Gu, L. R.; Lu, F. S.; Lin, Y.; Christensen, K. A.; Yang, S. T.; Sun, Y. P. ACS Nano 2009, 3, 3909. (43) Huang, G.; Xiong, Z. C.; Qin, H. Q.; Zhu, J.; Sun, Z.; Zhang, Y.; Peng, X. J.; Ou, J. J.; Zou, H. F. Anal. Chim. Acta 2014, 809, 61. (44) Zhu, J.; Sun, Z.; Cheng, K.; Chen, R.; Ye, M. L.; Xu, B.; Sun, D. G.; Wang, L. M.; Liu, J.; Wang, F. J.; Zou, H. F. J. Proteome Res. 2014, 13, 1713.

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by National Key Program for Basic Research of China (2013CB911204, 2011CB910603, 2012CB910603), National High-Tech Research and Development Program (2012AA020202, 2012AA020203, 2014AA020906), National Key Scientific Instrument Development Program of China (2011YQ09000504), National Natural Science Foundation of China (21275005, 21235001).

■

REFERENCES

(1) Nothaft, H.; Szymanski, C. M. Nat. Rev. Microbiol. 2010, 8, 765. (2) Hart, G. W.; Copeland, R. J. Cell 2010, 143, 672. (3) Service, R. F. Science 2012, 338, 321. (4) Tharakaraman, K.; Jayaraman, A.; Raman, R.; Viswanathan, K.; Stebbins, N. W.; Johnson, D.; Shriver, Z.; Sasisekharan, V.; Sasisekharan, R. Cell 2013, 153, 1486. (5) Kufe, D. W. Nat. Rev. Cancer 2009, 9, 874. (6) Perego, P.; Gatti, L.; Beretta, G. L. Nat. Rev. Cancer 2010, 10, 523. (7) Slawson, C.; Hart, G. W. Nat. Rev. Cancer 2011, 11, 678. (8) Marino, K.; Bones, J.; Kattla, J. J.; Rudd, P. M. Nat. Chem. Biol. 2010, 6, 713. (9) Song, X. Z.; Lasanajak, Y.; Xia, B. Y.; Heimburg-Molinaro, J.; Rhea, J. M.; Ju, H.; Zhao, C. M.; Molinaro, R. J.; Cummings, R. D.; Smith, D. F. Nat. Methods 2011, 8, 85. (10) Wang, X.; Emmett, M. R.; Marshall, A. G. Anal. Chem. 2010, 82, 6542. (11) Selman, M. H. J.; McDonnell, L. A.; Palmblad, M.; Ruhaak, L. R.; Deelder, A. M.; Wuhrer, M. Anal. Chem. 2010, 82, 1073. (12) Mysling, S.; Palmisano, G.; Hojrup, P.; Thaysen-Andersen, M. Anal. Chem. 2010, 82, 5598. (13) Christiansen, M. N.; Kolarich, D.; Nevalainen, H.; Packer, N. H.; Jensen, P. H. Anal. Chem. 2010, 82, 3500. (14) Zhang, H. M.; Guo, T. N.; Li, X.; Datta, A.; Park, J. E.; Yang, J.; Lim, S. K.; Tam, J. P.; Sze, S. K. Mol. Cell. Proteomics 2010, 9, 635. (15) Tian, Y.; Esteva, F. J.; Song, J.; Zhang, H. Mol. Cell. Proteomics 2012, 11, No. M111.011403. (16) Wang, L.; Aryal, U. K.; Dai, Z. Y.; Mason, A. C.; Monroe, M. E.; Tian, Z. X.; Zhou, J. Y.; Su, D.; Weitz, K. K.; Liu, T.; Camp, D. G.; Smith, R. D.; Baker, S. E.; Qian, W. J. J. Proteome Res. 2012, 11, 143. (17) Chen, J.; Shah, P.; Zhang, H. Anal. Chem. 2013, 85, 10670. (18) Zhang, L. J.; Jiang, H. C.; Yao, J.; Wang, Y. L.; Fang, C. Y.; Yang, P. Y.; Lu, H. J. Chem. Commun. 2014, 50, 1027. (19) Qi, D. W.; Zhang, H. Y.; Tang, J.; Deng, C. H.; Zhang, X. M. J. Phys. Chem. C 2010, 114, 9221. (20) Wang, H. Y.; Bie, Z. J.; Lu, C. C.; Liu, Z. Chem. Sci. 2013, 4, 4298. (21) Wang, Y. L.; Liu, M. B.; Xie, L. Q.; Fang, C. Y.; Xiong, H. M.; Lu, H. J. Anal. Chem. 2014, 86, 2057. (22) Kaji, H.; Shikanai, T.; Sasaki-Sawa, A.; Wen, H. L.; Fujita, M.; Suzuki, Y.; Sugahara, D.; Sawaki, H.; Yamauchi, Y.; Shinkawa, T.; Taoka, M.; Takahashi, N.; Isobe, T.; Narimatsu, H. J. Proteome Res. 2012, 11, 4553. (23) Mann, B. F.; Mann, A. K. P.; Skrabalak, S. E.; Novotny, M. V. Anal. Chem. 2013, 85, 1905. (24) Khoury, G. A.; Baliban, R. C.; Floudas, C. A. Sci. Rep 2011, 1, No. 90, DOI: 10.1038/srep00090. (25) Shen, A. J.; Guo, Z. M.; Yu, L.; Cao, L. W.; Liang, X. M. Chem. Commun. 2011, 47, 4550. (26) Liu, J.; Wang, F. J.; Lin, H.; Zhu, J.; Bian, Y. Y.; Cheng, K.; Zou, H. F. Anal. Chem. 2013, 85, 2847. (27) Yu, L.; Li, X. L.; Guo, Z. M.; Zhang, X. L.; Liang, X. M. Chem. Eur. J. 2009, 15, 12618. (28) Qu, Y. Y.; Xia, S. M.; Yuan, H. M.; Wu, Q.; Li, M.; Zou, L. J.; Zhang, L. H.; Liang, Z.; Zhang, Y. K. Anal. Chem. 2011, 83, 7457. G

dx.doi.org/10.1021/ac5034215 | Anal. Chem. XXXX, XXX, XXX−XXX