Preparation and characterization of hydrophilic hybrid monoliths via thiol-ene click polymerization and their applications in chromatographic analysis and glycopeptides enrichment
Abstract: A macroporous hydrophilic organic-silica hybrid monolithic column was synthesized via photoinitiated thiol-ene click polymerization reaction of 1-thioglycerol-modified polyhedral oligomeric vinylsilsesquioxane (vinylPOSS) and dithiothreitol (DTT) in a binary porogenic system consisting of tetrahydrofuran (THF) and dodecanol. 1-Thioglycerol was used to modify vinylPOSS in order to form a precursor with good solubility in the binary porogenic system. The influences of both the ratio of 1-thioglycerol/vinylPOSS and the porogenic solvents on the morphology and permeability of hybrid monoliths were studied in detail. The physical properties of hybrid monolith were characterized by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy and nitrogen adsorption/desorption measurement. The chromatographic performance was evaluated by separation of neutral polar compounds in capillary liquid chromatography (cLC). The resulting column possessed homogeneous macroporous structure and showed hydrophilic interaction liquid chromatography (HILIC) separation mechanism with high efficiency of 65,000 N m-1 for formamide. Ultimately, the hybrid matrix was grafted with hydrazine groups and then exhibited the ability of glycopeptides enrichment.
1Introduction
Monolithic columns have emerged in the late 1980s [1, 2] and been considered as an alternative to classic particle packed columns for chromatographic separations in capillary electrochromatography (CEC) and capillary liquid chromatography (cLC) [3-7] due to their inherently high permeability and fast analysis. Based on the nature of the matrix, monolithic columns can be mainly classified into three categories: organic polymer-based, silica-based and organic-silica hybrid monolithic columns [8-11]. Particularly, the organic-silica hybrid monoliths, which combine the advantages of the former two monoliths and overcome the inherent drawbacks like the pH sensitivity of silica-based monolith and the solvent instability of organic monolith, have attracted much attention and been widely applied in the field of immobilized enzyme reactors [12] and sample pretreatment of solid-phase microextraction [13].At present, a variety of monoliths were widely used for the separation of complex samples in reversed-phase liquid chromatography (RPLC) [14]. As a result of its poor ability in separation of polar compounds, hydrophilic interaction liquid chromatography (HILIC) provided an alternative approach [15-17], which was developed by Alpert in 1990 [18]. In recent years, the interest in HILIC has continued to grow on account of its broad application in the separation of polar compounds, including peptides [19-21], proteins [22-24], carbohydrates [25-27], pharmaceuticals[28] and polar contaminants in food and environmental samples [29]. Also, its use as a tool in selective enrichment of glycopeptides and phosphopeptides in proteomics application is of special interest [30, 31].
The acetonitrile (ACN)-rich eluents in HILIC could match with mass spectrometry and improve ionization efficiency, leading to a gain in detection sensitivity [32, 33]. In our previous work, a zwitterionic organic-silica hybrid monolithic capillary column was successfully prepared via a single-step thermal-treatment “one-pot” approach for HILIC, which exhibited high efficiency and satisfactory reproducibility for separation of neutral, basic, acidic analytes, as well as small peptides in cLC [33].The organic-silica hybrid monolithic columns were generally fabricated with threeapproaches, sol-gel process of trialkoxysilanes and tetraalkoxysilanes, “one-pot” process of alkoxysilanes and organic monomers, as well as free radical polymerization of monomers containing silanes [13, 34]. In the last several years, thiol-ene click reaction was attractive in the area of functionalization of surfaces and chromatographic stationary phases owing to its mild process, simplicity, compatibility with aqueous media and high reaction yield [35-37]. Yao et al. reported a new strategy for the preparation of monolithic trypsin microreactor by thiol-ene “click” chemistry [38]. Nischang’s group demonstrated a facile route to fabricate hybrid porous materials via thiol-ene “click” chemistry [39]. Our group successfully applied the photoinitiated thiol-ene polymerization to fabricate porous polymeric monoliths (PPMs) with high chromatographic performance [40]. Therefore, thiol-ene “click” reaction was supposed to prepare highly efficient hybrid monolith and applied for HILIC.Herein, we fabricated a macroporous hydrophilic hybrid monolithic column via photoinitiated thiol-ene “click” reaction. To address the insolubility of vinylPOSS in prepolymerization solution, the monomer of vinylPOSS was firstly modified with nonstoichiometric 1-thioglycerol, and then the resulting oligomers polymerized with dithiothreitol (DTT) for preparation of hybrid monolithic column (Fig. 1). The morphology, permeability and chromatographic properties of the resulting poly(vinylPOSS-co-DTT) monolithic column were systematically characterized. Ultimately, the hybrid material indicated hydrophilic separation performance and was successfully applied for enrichment of glycopeptides.
2Experimental
VinylPOSS (CH2CHSiO3/2)n (n = 8) (Mw = 633.05) with 8 vinyls was purchased from Hybrid Plastics (USA). 1-Thioglycerol (95%), DTT, dodecanol, vinyltrimethoxysilane (VTMS), trifluoroacetic acid (TFA), 2,5-dihydroxybenzoic acid (DHB), immunoglobulin G (IgG), trypsin, formic acid (FA, for mass spectrometry, 98%) andpolystyrene standards (Mw = 800, 4000, 13,200, 50,000, 90,000, 280,000 and 900,000) were purchased from Sigma (St. Louis, MO, USA) and used directly without further purification. 2,2-Dimethoxy-2-phenylacetophenone (DMPA) was purchased from Acros Organics (New Jersey, USA). Hydrazine hydrate (80%), methanol and ethanol were obtained from Tianjin Kermel Chemical Plant (Tianjin, China). Tetrahydrofuran (THF) and acetonitrile (ACN) were HPLC-grade and acquired from Yuwang Group (Shandong, China). Iodoacetamide (IAA) was purchased from Sino-American Biotechnology Corporation (Beijing, China). Thiourea, N,N-dimethylformamide (DMF), formamide, methylbenzene and dodecanol were of analytical grade, and obtained from Tianjin Kermel Chemical Plant (Tianjin, China). Deionized water was prepared with a Milli-Q system (Milli-pore, MA, USA). The flexible fused-silica capillary (UV-transparent coating) with inner dimension of 75 μm was purchased from Reafine Chromatography Ltd. (Hebei, China).Preparation of hybrid monolithic column (I) via photo-initiated polymerization Before preparing a hybrid monolithic column, the inner wall of UV-transparent fused-silica capillary was pretreated and modified with VTMS for anchoring monolithic matrix, as described in a previous report [41]. The capillary was rinsed with 0.1 mol L-1 NaOH (3 h), then flushed with water (1 h), followed by 0.1 mol L-1 HCl (3 h), water (1 h) and methanol (3 h), successively. After being dried under nitrogen stream, the capillary was adequately filled with VTMS solution in methanol (50%, v/v), and both ends were sealed with rubbers and sank in a water bath at 40 oC for 12 h.
Finally, the capillary was washed with methanol to flush out residual reagents and dried under a stream of nitrogen.Typically, two reactants of vinylPOSS and 1-thioglycerol (molar ratio of vinyl to thiol, 8/3) were dissolved concurrently in THF. Then the mixture was sonicated for 5 min to form a homogeneous solution. A certain amount of DMPA (10%, w/w, DMPA/1-butanol) (photoinitiator) was added to this sonicated solution. Whereafter, the solution was placed in a glass Petri dish and covered with plastic wrap, then put in a UV-curing instrument (XL-1500A, λ = 365 nm, Spectronics Corporation, New York,USA) for 1200 s. The solution was finally transferred to a round-bottom flask andremoved bulk solvent with rotary evaporation. The resulting solid was used as a precursor for preparation of hybrid monolithic column (I). DTT and the precursor were dissolved in a binary porogenic system consisting of THF and dodecanol in an Eppendorf tube, and the photoinitiator was subsequently added. The details of fabrication conditions were listed in Table 1. The mixed homogeneous prepolymerization solution was immediately introduced into the pretreated capillary with a syringe. With both ends of the capillary sealed with silicon septa, the capillary and the remanent prepolymerization solution in the Eppendorf tube were put into the UV-curing instrument for 300s. After irradiation, the hybrid monolithic column (I) was then flushed with methanol to remove residual reagents. Finally, both ends of hybrid monolithic column (I) were immersed in the water for further usage. Meanwhile, the bulk monolithic material was also formed in the Eppendorf tube for further characterization and modification.The above-mentioned bulk monolithic material was dipped into ethanol and extracted for one day to remove the residuals, and repeated 3 times. Finally, the obtained material was dried in vacuum drying oven at 60 oC for 12 h, and then ground to small pieces for ease of weighing. A certain amount of dried monolithic material was oxidized with NaIO4 solution (4 mmol L-1) for 0.5 h, and then extracted with ethanol for one day.
After adding the aqueous solution of hydrazine hydrate (1/5, v/v), the mixture was incubated at 25 oC for 24 h. In the end, the mixture was centrifuged and removed the supernatant, and the material was collected and dried under vacuum for glycopeptides enrichment, named hydrazine-modified material (II).The hydrazine density of hydrazine-modified material (II) was measured according to previous report with minor modification [42]. The hydrazine-modified material (II) (2 mg) was placed in a 1.5 mL Eppendorf tube, washed 3 times with 1mL of deionized water, and equilibrated 4 times with 1 mL of a coupling solution of 0.8% (v/v) glacial acetic acid in anhydrous methanol. After adding an excess amount of4-nitrobenzaldehyde, the mixture was incubated for 4 h with gentle end-over-endrotation at room temperature. The hydrazine-modified material (II) was collected by centrifugation and washed with 0.3 mL coupling solution for 3 times. All the supernatants were combined, and then its absorbance was measured at 265 nm. The amount of uncoupled 4-nitrobenzaldehyde was calculated with a calibration curve derived from a set of standard 4-nitrobenzaldehyde solutions.Fourier-transformed infrared spectroscopy (FT-IR) characterization was carried out on Thermo Nicolet 380 spectrometer with KBr pellets (Nicolet, Wisconsin, USA). The microscopic images of monolithic materials were obtained by scanning electron microscopy (SEM, JEOL JSM-5600, Tokyo, Japan). The elemental analysis was obtained by Elementar (Vario EL Ⅲ, Elementar Analysen Syetem GmbH, Germany). The permeability (B0) can be calculated according to Darcy’s law by the equation B0 =FηL/(πr2ΔP), where F (m3 s-1) is the flow rate of mobile phase, η is the viscosity of mobile phase (0.38×10-3 Pa s for ACN), L and r (m) are effective length and inner radius of the column, respectively, ΔP (Pa) is the pressure drop across the column. The data of ΔP and F were obtained on an ACQUITY UltraPerformance LC (Waters, USA). The flow rates of mobile phase were set at 0.1-1.5 μL min-1.The cLC experiments were performed on a LC system combining with an Agilent 1100 micropump, a 7725i injector with a 20 μL sample loop, and a K-2501 UV detector (Knauer, Berlin, Germany).
UV detector wavelength was set at 214 nm. The mobile phase was composed of ACN/water. Samples were injected through an injection valve with an internal 2 µL sample loop. A tee-piece was used as a splitter, one end of the monolithic column was connected with the tee-piece, and the other end was connected to a 15 cm long blank capillary (50 μm i.d.). The outlet of the monolithic column was connected with a Teflon tube to an empty fused-silica capillary (50 μm i.d.), where a detection window was made by removing a 2 mm length of the polyimide coating in a position of 5.5 cm from the separation monolithic column outlet. The actual flow rate through monolithic column was calculated by measuring the weight of mobile phase which was collected with a centrifuge tube for20 min. The retention factor (k) was defined as (tr-t0)/t0, where tr and t0 represent theretention times of the analytes and the void time marker, respectively. The chromatographic data were collected at room temperature and analyzed with a software program HW-2000 from Qianpu Software (Shanghai, China).The IgG tryptic digest was prepared according to the reported method with minor modification [43]. The hydrazine-modified material (II) was applied to enrich glycopeptides from IgG tryptic digest.IgG tryptic digest (20 μg) was dissolved in 80 μL oxidation solution (100 mmol L-1 sodium acetate, 150 mmol L-1 NaCl, pH=5.5) and 20 μL 4 mmol L-1 NaIO4 solution, then kept in the dark place for 1 h. The 20 μL aqueous solution of Na2S2O3 (100 mmol L-1) was added to terminate oxidation reaction for 10 min.
After that, II (2 mg) was washed by oxidation solution for 3 times and then added into above IgG tryptic digest solution. The mixture was incubated at 25 oC for 12 h. The material was washed 3 times by NaCl aqueous solution (1.5 mol L-1), methanol and NH4HCO3 solution (100 mmol L-1), successively. After removing the supernatant and adding 40 μL NH4HCO3 solution (10 mmol L-1), the glycopeptides were deglycosylated with 0.2 μg PNGase F at 37 oC for 8 h and then analyzed by MALDI-TOF-MS.IgG tryptic digest (10 μg) was diluted in different loading solutions (200 μL ACN/H2O/TFA, 80/19/1, 84/15/1, 88/11/1 and 92/7/1, v/v/v) containing 1.5 mg II. Then, the mixture was incubated for 50 min at 25 oC and centrifuged. After washing with the loading solution for 3 times, the elution solution (100 μL ACN/H2O/TFA, 30/69/1, v/v/v) was introduced to release glycopeptides. Finally, the glycopeptides were analyzed directly by MALDI-TOF-MS or deglycosylated with 0.2 μg PNGase F (pH = 8.0) at 37 oC for 12 h to remove the glycan moieties.MALDI-TOF-MS experiments were carried out by a 5800 Proteomics Analyzer (Applied Biosystems, USA) with a pulsed Nd/YAG laser at 355 nm in linear positive ion mode. The mixture of sample solution (0.5 μL) and DHB solution (0.5 μL, 25 mgmL−1 DHB in ACN/H2O/H3PO4, 70/29/1, v/v/v) was spotted on the MALDI plate for MS analysis.
3Results and discussion
VinylPOSS is a derivative of POSS with eight vinyls, which has been reported to prepare porous organic-inorganic monolithic materials via radical-mediated step-growth process [44, 45]. Unfortunately, vinylPOSS has poor solubility in a majority of organic solvents such as acetonitrile and methanol, which limits its utilization in conventional “one-pot” preparation. If only THF was used as porogenic solvent, the monolithic column with good permeability could not be obtained. A binary porogenic system would be selected, but vinylPOSS and DTT could not be simultaneously dissolved. As a consequence, vinylPOSS was modified by 1-thioglycerol with the purpose of overcoming this problem. As can be seen in Fig. 1, the proposed approach for preparation of hybrid monolithic column involves two steps: (1) 1-thioglycerol-modified vinylPOSS was synthesized as a precursor via thiol-ene “click” chemistry; (2) fast preparation of hybrid monolithic column via photo-initiated thiol-ene “click” polymerization reaction of the precursor and DTT in the presence of a binary porogenic system (THF and dodecanol).The molar ratio of vinyl/thiol groups had a giant effect on the solubility of modifiedvinylPOSS. Three kinds of 1-thioglycerol-modified vinylPOSS were synthesized with different molar ratio of vinyl/thiol groups, and then characterized by a MALDI-TOF-MS. The mass spectra of three precursors are shown in Fig. 2. It could be clearly seen that three mass spectra in the range of 700–1600 Da tended to present a Gaussian distribution, and the mass difference of repeating units was 108 Da corresponding to 1-thioglycerol molecule. The theoretical peak value of vinylPOSS, 1-thioglycerol and Na+ was 633, 108 and 23, respectively. Eight peaks from m/z 762 to 1520 represented 1-substituted to 8-substituted POSS, respectively. This result revealed that 1-thioglycerol-modified vinylPOSS was successfully prepared. It couldbe found that the highest peak was drifted with the increase of 1-thioglycerol content. Although the synthesized precursors were a mixture, they could be further used to prepare monoliths.In our case, we selected a mixture of THF and dodecanol as the porogenic system, the synthetic precursors as crosslinker and DTT as functional monomer.
After adding a photoinitiator (DMPA) and exposing under UV-light, the phase separation would emerge within 1 min by visual inspection. As shown in Fig. S1, the influence of polymerization time (3.0, 5.0, 10.0 and 15.0 min) on retention factor (k) and separation efficiency of hybrid monolithic column E was investigated. When the polymerization time was greater than 5 min, the retention factors (k) and plate heights(H) were nearly unchanged. Finally, the polymerization reaction time we chose was 5 min, which was much faster than common thermal-initiated mode for preparation of monoliths. The porous morphology and permeability of hybrid monolith, as is well-known, were affected by the contents of monomers and composition of porogenic solvents, which were investigated in detail (Table 1 and Fig. 3). It turned out that the solubility of synthetic precursors was different in the porogenic solvents. The precursor synthesized with relatively few 1-thioglycerol (8/2, molar ratio of vinyl/thiol functional groups) was not easily dissolved and barely formed a homogeneous solution to fill the capillary (column A), while the precursor synthesized with relatively much 1-thioglycerol (8/4, molar ratio of vinyl/thiol functional groups) was easily dissolved, resulting in a low permeability and becoming too hard to pump through for monolithic column (column F). Finally, we chose the precursor synthesized with the molar ratio of 8/3 (vinyl/thiol) to further optimize the preparation of POSS-based hybrid monolithic column (as columns B-E in Table 1). It was found that the permeability was increased from 2.74 to 12.6 × 10-14 m2 with a decrease of THF content in porogenic system.
It was deduced that THF served as microporogenic solvent (good solvent), while dodecanol served as macroporogenic solvent (poor solvent) in porogenic system. As shown in Fig. 3a, b and d, it could be obviously concluded that the composition of porogenic solvents had a great effect onmorphology of hybrid monolith. The monolithic network gradually formed, and thesize of the macropores became smaller with an increase of THF content in porogenic system.Fig. 3c and d show the SEM images of POSS-based hybrid monolithic column E. As illustrated in Fig. 3c, it was observed that the monolithic matrix was well anchored to the inner wall of the capillary. Meanwhile, poly(vinylPOSS-co-DTT) possessed a bicontinuous monolithic network, which contained a few macropores of around 1 μm and narrow framework of around 0.5 μm (Fig. 3d).Fig. 4 presents FT-IR spectra of vinylPOSS, 1-thioglycerol, 1-thioglycerol-modified vinylPOSS, DTT and the resulting hybrid monolithic column (I). The strong peaks at 1108 and 779 cm−1 were assigned to the Si-O-Si vibrations, meanwhile the peak at 1604 cm−1 signified the characteristic band of C=C stretch in Fig. 4a. In Fig. 4b, the peak at 2555 cm−1 was assigned as -SH adsorption. Compared to Fig. 4a and b, it could be found from Fig. 4c that the peak intensity of C=C band were remarkably decreased, and –SH adsorption disappeared, which confirmed that the precursor of 1-thioglycerol-modified vinylPOSS was successfully synthesized via thiol-ene additional reaction. The FT-IR spectrum of DTT (Fig. 4d) was similar to that of 1-thioglycerol. As shown in Fig. 4e, characteristic band of -SH (2552 cm−1) disappeared entirely, and the peak signal of C=C band was further decreased. This result indicated the almost complete reaction of thiol groups in DTT with alkenyl groups in 1-thioglycerol-modified vinylPOSS via thiol-ene “click” polymerization reaction.Moreover, the components of the precursor, which synthesized with the molar ratio of8/3 (vinyl/thiol), and the monolithic column E were characterized with elemental analysis. According to the theoretical calculation, the weight percentages of sulphur in precursor and monolith E were 10.0% and 24.3%, respectively.
However, the weight percentages of sulphur in precursor and monolith E were measured as 13.0% and 18.1%, respectively. There was a little deviation between the theoretical values and measurement values, which might be due to the steric hindrance during thepolymerization reaction. As a consequence, it could be confirmed the successfulintroduction of DTT into monolithic column.The porous property of hybrid column E was characterized by size exclusion chromatography (SEC). Based on the equation: R = 0.0246Mr0.588, where the pore diameter, R, is in nanometer, the hydrodynamic diameters were calculated [46, 47]. Connecting to our instance, benzene (Mr = 78) corresponded to a hydrodynamic diameter of 0.32 nm, while the highest molecular weight of polystyrene standard (Mr= 900,000) had a hydrodynamic diameter of 78.0 nm. The retention volume of benzene was considered as the total pore volume, which could penetrate into all pores larger than 0.32 nm as a result of the smallest molecular size. The equation (εT = VM/VC) could be applied for calculating the total porosity, where VM is the elution volume of benzene, and VC is the geometrical volume of the empty column. Simultaneously, the external porosity was calculated according to the formula (ε0 = V0/VC), which was anticipated to be excluded from pores smaller than 78.0 nm, where V0 is the elution volume of largest polystyrene standard. As seen in Fig. S2, εT and ε0 of column E were 66.61% and 48.61%, respectively, suggesting that the uniform POSS-based hybrid monolith exhibited high porosity, which facilitated fast mass transfer.Subsequently, the mechanical stability of column E was calculated by measuring the back pressure, and the result was shown in Fig. S3. The back pressure in the range of 2-32 MPa was linearly associated with flow rate (R = 0.998), suggesting that the hybrid monolithic column exhibited a satisfactory mechanical stability. What’s more, as shown in Table 1, the permeability of column E was measured at 2.74 ×10-14 m2 to point out a good permeability. Besides, the hybrid monolithic column E exhibited a low specific surface area (1.28 m2/g) due to sharp shrinkage of the bulk hybrid monolith, which was measured by nitrogen adsorption/desorption measurements.
Chromatographic performance in cLCThe poly(vinylPOSS-co-DTT) column was equilibrated with the mobile phase of ACN/H2O (95/5, v/v), and a mixture containing toluene, DMF, formamide and thiourea was used to evaluate the retention behavior in HILIC. As can be seen in Fig.5a, four neutral polar compounds were completely separated with narrow peak widthon column E at the flow rate of 415 nL min-1. The eluting peaks in sequence were toluene < DMF < formamide < thiourea in accordance with their polarity in order from low to high, reflecting a typical HILIC retention mechanism. The column efficiency of poly(vinylPOSS-co-DTT) monolith was evaluated by adjusting the flow rate of mobile phase in the range of 64-516 nL min-1 as shown in Fig. 5b. The lowest plate height reached 15.50-16.70 μm at a velocity of 0.55 mm s-1 corresponding to 60,000-65,000 N m-1 of column efficiencies. Plots of retention factor (k) on column E versus ACN content in the mobile phase over a range of 70-95% were shown in Fig. 5c. It could be observed that the corresponding retention factors of DMF, formamide, and thiourea increased with an increase of ACN content, also suggesting a HILIC retention mechanism at high ACN content (> 70%).The run-to-run reproducibility on column E was appraised through the relative standard deviation (RSD) for the k value of thiourea as model analyte while toluene was selected as void time marker. The RSD value of run-to-run reproducibility for chromatographic separation was 1.46% (n = 5). Both column-to-column and batch-to-batch reproducibility were also evaluated, which were 5.42% (n = 3) and 14.02% (n = 4), respectively. It was indicated that column E owned satisfactory reproducibility.Considering about hydrophilic property of hybrid monolith, we further applied it for enrichment of glycopeptides from IgG tryptic digest. At the outset, we attempted to directly use capillary monolith for enrichment of glycopeptides via HILIC method with different sample loading solutions (ACN/H2O/TFA, 80/19/1, 84/15/1, 88/11/1/ and 92/7/1, v/v/v). Unfortunately, the MS signals of glycopeptides could not be acquired by MALDI-TOF MS. So we modified the capillary monolith with hydrazine functional groups to enrich glycopeptides via both hydrazide chemistry and HILIC modes. However, satisfactory results were not obtained, which was possibly ascribed to the occurrence of disconnection between the inner wall and the monolithic matrix after oxidation reaction.
As a consequence, we tried to employ the bulkhydrazine-modified material (II) to enrich glycopeptides. The pristine hybrid matrixwas transformed into hydrazine-modified material (II) following by two steps (Fig. 1):(i) oxidation by NaIO4, converting the vicinal diols groups of monolith matrix to aldehydes and (ii) coupling with hydrazine. Both the intermediate and the product were characterized by ATR-FTIR. The emergence of the aldehyde group (the peak at 1714 cm−1) indicated the success of oxidation reaction (Fig. S4a), while the peak at 1634 cm−1 was assigned as absorption of C=N stretching vibration (Fig. S4b), demonstrating a success of the preparation of hydrazine-modified material (II). Through UV-vis spectroscopic method, the hydrazine density of hydrazine-modified material (II) was calculated to be 52.1 μmol/g, which was larger than that of previously reported magnetic silica particles (8 μmol/g) [48], but much less than that of the magnetic nanoparticles via reversible addition-fragmentation chain transfer (RAFT) polymerization (1442 μmol/g) [42].The approaches of glycopeptides enrichment mainly include lectin affinity chromatography, immobilized metal affinity chromatography (IMAC), HILIC and hydrazide chemistry [49]. The hydrazide chemistry method has the advantage of high enrichment specificity, which benefits from the formation of covalent bonds between glycans of glycopeptides and hydrazine groups on materials. Herein, we exploited hydrazide chemistry method for glycopeptides enrichment with hydrazine-modified material (II) from IgG digest, as can be seen in Fig. S5a. Initially, IgG digest was directly analyzed by MALDI-TOF MS without any enrichment, and the result was given in Fig. 6a. There were lots of non-glycopeptides peaks with high intensity, and the MS signals of the glycopeptides could be scarcely detected.
During the process of glycopeptide enrichment with hydrazide chemistry, the enriched glycopeptides were eluted by NH4HCO3 solution and then followed by deglycosylation with PNGase F to remove the N-linked glycans. As shown in Fig. 6b, two deglycosylation peptides at m/z 1158.5 and 1190.5 were identified, describing EEQFN#STFR and EQTN#STYR. However, the intensity of two peptides was low. Therefore, we employed HILIC enrichment method to enrich glycopeptides (Fig. S5b).As for HILIC-based enrichment of glycopeptides, different loading solutions(ACN/H2O/TFA, 80/19/1, 84/15/1, 88/11/1 and 92/7/1, v/v/v) were chosen to optimizethe enrichment conditions. When the content of ACN was relatively low (80% and 84%), there was hardly any enrichment effect. When the ACN concentration was increased to 88% (Fig. 7a) and 92% (Fig. 7b), the abundant non-glycosylated peptides in the digests were efficiently lessened. Meanwhile, N-linked glycopeptides could be clearly detected with high MS intensities. Additionally, 9 glycopeptides (S/N > 20) with m/z from 2600 to 3500 from IgG digests were detected by MALDI-TOF-MS (Table S1 and S2) at these conditions, but there were less non-glycosylated peaks when using 92% ACN as the loading solution. This result showed a relatively low glycopeptides enrichment efficiency than some polymeric particles [50]. After being treated with PNGase F, two released peptides in conformity to N-linked glycopeptides were detected distinctly with high intensity as shown in Fig. 7c and d. As a consequence, the POSS-based hybrid monolith matrix could be developed into a kind of hydrazine materials and exhibited the ability of enrichment of N-glycosylated peptides.
4Conclusions
In this work, a macroporous hydrophilic organic-silica hybrid monolith was successfully fabricated through two steps of photoinitiated thiol-ene reaction. 1-Thioglycerol-modified vinylPOSS was synthesized to improve its solubility and then exploited as the precursor to further polymerize with DTT in the existence of porogenic system. The molar ratio of thiol and vinyl groups had a notable influence on the solubility of precursor, as well as the morphology and permeability of hybrid monolithic column. The resulting POSS-based hybrid monolithic columns exhibited homogeneous macroporous structure, well permeability and good separation ability for neutral polar compounds in HILIC. In addition, the hybrid monolith matrix was successfully turned into a kind of hydrazine material for glycopeptides enrichment due to the existence of a lot of vicinal diols groups. This successful application implied the hydrophilic hybrid monolith would be taken as a promising material for the pretreatment of biological sample, 1-Thioglycerol material science and analytical science.