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Fabrication of diverse pH-sensitive functional mesoporous silica for selective removal or depletion of highly abundant proteins from biological samples
Author’s Accepted Manuscript Fabrication of diverse pH–sensitive functional mesoporous silica for selective removal or depletion of highly abundant proteins from biological samples Jiaojiao Wang, Jingfeng Lan, Huihui Li, Xiaoyan Liu, Haixia Zhang www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(16)30758-5 http://dx.doi.org/10.1016/j.talanta.2016.10.003 TAL16923
To appear in: Talanta Received date: 18 June 2016 Revised date: 16 September 2016 Accepted date: 2 October 2016 Cite this article as: Jiaojiao Wang, Jingfeng Lan, Huihui Li, Xiaoyan Liu and Haixia Zhang, Fabrication of diverse pH–sensitive functional mesoporous silica for selective removal or depletion of highly abundant proteins from biological samples, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fabrication of diverse pH–sensitive functional mesoporous silica for selective removal or depletion of highly abundant proteins from biological samples Jiaojiao Wang, Jingfeng Lan, Huihui Li, Xiaoyan Liu*, Haixia Zhang
State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous, Metals Chemistry and Resources Utilization of Gansu Province and College of, Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
Corresponding author Tel.: +86 931 8912510; fax: +86–931–8912582.
[email protected] Abstract In proteomic studies, poor detection of low abundant proteins is a major problem due to the presence of highly abundant proteins. Therefore, the specific removal or depletion of highly abundant proteins prior to analysis is necessary. In response to this problem, a series of pH–sensitive functional mesoporous silica materials composed of 2–(diethylamino)ethyl methacrylate and methacrylic acid units were designed and synthesized via atom transfer radical polymerization. These functional mesoporous silica materials were characterized and their ability for adsorption and separation of proteins was evaluated. Possessing a pH–sensitive feature, the synthesized functional materials showed selective adsorption of some proteins in aqueous or buffer solutions at certain pH values. The specific removal of a particular protein from a mixed protein solution was subsequently studied. The analytical results confirmed that all the target 1
proteins (bovine serum albumin, ovalbumin, and lysozyme) can be removed by the proposed materials from a five–protein mixture in a single operation. Finally, the practical application of this approach was also evaluated by the selective removal of certain proteins from real biological samples. The results revealed that the maximum removal efficiencies of ovalbumin and lysozyme from egg white sample were obtained as 99% and 92%, respectively, while the maximum removal efficiency of human serum abumin from human serum sample was about 80% by the proposed method. It suggested that this treatment process reduced the complexity of real biological samples and facilitated the identification of hidden proteins in chromatograms.
Keywords: pH–sensitive; functional mesoporous silica; atom transfer radical polymerization; highly abundant proteins
1. Introduction
The identification and quantification of proteins in a complex biological system are important issues in current proteomics. However, with regard to current analytical technologies, such as two dimensional electrophoresis, high–performance liquid chromatography (HPLC) coupled to mass spectrometry and on–line multidimensional
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liquid chromatography, it is difficult to separate all proteins completely in a single operation due to the enormous amount and dynamic concentration range of proteins in complex biological samples, which leads to the suppression of information on low abundant proteins (LAP) by highly abundant proteins (HAP). To minimize the complexity of samples and improve the identification of LAP, various technologies and methodologies have been developed to deplete HAP based on their biochemical and biophysical features, such as molecular weight (MW) [1–6], hydrophobicity [7– 8], surface charge [3, 6, 9–10], isoelectric point (pI) [11–14], metal ion affinity effect [15–16], basic structure differences [17–18], amino acid sequence (epitope) [19–21], and immunoaffinity effect [22–23]. Among them, affinity chromatography [15–16, 22–23] is the most common method and possesses unique advantages for the depletion of HAP [22, 24–26]. However, commercial available immunaffinity columns and adsorbent media are costly and limited, which cannot meet the various demands for practical applications. Besides, ionic exchange [27] and hydrophobic interaction [7–8] materials are also widely used as adsorbents for separation of proteins. Nevertheless, when the different media and different synthesized methods were employed to prepare the adsorbents, protein interactions with the adsorbents would be different due to the present of multiple interactions between proteins and the adsorbent bead or spacer–linker structure carrying the nominal ligand [28]. Therefore, it is still essential to develope a facile and easily scalable technique for fabrication of
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various adsorbent with potential application in the removal of HAPfrom a real biological sample.
Recently, we reported several approaches for the immobilization or selective removal of target proteins using fabricated functional mesoporous silica materials based on Santa Barbara Amorphous–15 (SBA–15) with various anchor groups [3, 6, 9]. These functional materials have strong anionic or cationic exchange ability and their adsorption of protein samples were only investigated in aqueous and neutral phosphate–buffered saline (PBS). “Smart” polymers with stimuli based on changes in pH are attractive materials for potential application in various fields [29–30]. In general, pH–sensitive polymers were formed by block copolymers with amines, carboxylic acid or hydroxy acid, which became charged species by protonation or deprotonation at different pH solutions [18, 31]. This resulted in a pronounced change in the adsorption of proteins by these materials compared with those containing strong anionic or cationic groups [3, 6, 9]. Encouraged by the above results, in the present study, we synthesized diverse pH–sensitive functionalized SBA–15 materials by atom transfer radical polymerization (ATRP). In addition, the adsorption of protein by these functionalized materials, which depend on the composition of the polymer as well as the pH value, was investigated. Subsequently, the use of these materials for the removal or depletion of HAP from real biological samples was developed.
2. Experimental
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2.1 Materials and methods
All the materials, reagents and instrumental analysis were listed in the electronic supplementary information.
2.2 Preparation of diverse pH–sensitive functionalized SBA–15
The ATRP initiator was immobilized on SBA–15 (SBA–15–NH–Br) according to our previous work [6] and the details are described in the Electronic Supplementary Information Fig. S1. The functionalized SBA–15 was prepared by ATRP as shown in Fig. 1. Details of these procedures are given below.
2.2.1 Surface modification of SBA–15 with poly(2–(diethylamino)ethyl methacrylate) (PDEAM) or poly(methacrylic acid) (PMAA) brush by surface– initiated ATRP
PDEAM (or PMAA) brush–grafted SBA–15 was prepared by SBA–15–NH–Br in dry toluene. A typical polymerization procedure was as follows: the obtained SBA– 15–NH–Br was immersed in 20.0 mL of dry toluene and dry nitrogen gas was then bubbled through the reaction to expel oxygen. The catalyst of CuBr and the ligand of N,N,N′,N′′,N′′–pentamethyldiethylenetriamine (PMDETA) (the molar ratio of CuBr and PMDETA to ATRP–initiator was kept at 1:1:1) were then added under a nitrogen atmosphere. The degassed 2–(diethylamino) ethyl methacrylate (DEAM) (or Methacrylic acid (MAA)) monomer solution (the molar ratio of monomer to initiator
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was kept at 45:1) was added via a syringe and the solution was further degassed by three freeze–pump–thaw cycles. The flask was then placed in a preheated oil bath at 100 ℃ for 40 min. The ATRP reaction was then stopped and the polymer–grafted SBA–15 was washed by ultrasonication using toluene followed by centrifugation to remove unreacted monomers. The resulting polymer–grafted SBA–15 was further washed with acetone, distilled water and ethanol until the supernatant was colorless. Finally, PDEAM (or PMAA) brush–grafted SBA–15 was dried under reduced pressure at 40 ℃ overnight and the resulting material was denoted as Material I (or Material II).
2.2.2 Surface modification of SBA–15 with amphoteric polymer brush by surface–initiated ATRP
Amphoteric polymer grafted SBA–15 was synthesized via ATRP polymerization as described above. In brief, the above PDEAM brush–grafted SBA–15 (Material I) was reacted with MAA monomer (the molar ratio of monomer to initiator was also kept at 45:1) using the aforementioned approach. The resulting material was denoted as SBA–15–(DEAM)x–(MAA)y–Br (Material III).
2.3 Characterization
Elemental analysis was measured with a VarioEl element analyzer, Elemental Analysis system (Hanau, Germany). Fourier Transform Infrared (FT–IR) Spectra of
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mesoporous materials before and after functionalization were observed by using Vertex 70 FT–IR (Bruker Optics, Ettlingen, Germany). Thermogravimetric analysis (TGA) of the original and functionalized SBA–15 was carried out with a Pyris Diamond TGA (Perkin–Elmer, Woodland, California, USA) up to 800 ℃ with a heating rate of 10 K/min under nitrogen. The narrow–angle powder X–ray diffraction patterns of the original and functionalized materials from 0.50 to 4.00 were recorded with a D/max–2400 (Rigaku, Japan) using Cu Ka radiation. Transmission electron microscopy (TEM) was performed on a Tecnai G2 TF20 (FEI, Hillsboro, Oregon, USA) at 200 kV. Nitrogen adsorption–desorption measurements were carried out at 77 K on a Tristar 3000 Surface Area and Porosimetry analyzer (Micromeritics Instrument Corp., Atlanta, Georgia, USA). The surface–area measurement was based on the Brunauer–Emmett–Teller (BET) method, and the pore–size distribution was calculated from the Barrett–Joyner–Halenda (BJH) formula. The zeta potentials of the diverse functionalized SBA–15 were measured using Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) at 25 ℃ in ultrapure water and B–R buffer solution at different pH values (3.0–8.0), respectively.
2.4 Removal of proteins from a standard protein mixture
2.4.1 Static adsorption test of the original and functionalized SBA–15 for different proteins
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Batch adsorption experiments were carried out by adding 40.0 mg of the original or functionalized SBA–15 (Material I, II or III) to 20.0 mL of bovine serum albumin (BSA, 1.0 × 10–6 mol L–1), transferrin (Trf, 1.0 × 10–6 mol L–1), myoglobin (Mb, 1.0 × 10–6 mol L–1), lysozyme (Lyz, 6.9 × 10–6 mol L–1) and ovalbumin (OVA, 6.9 × 10–6 mol L–1) aqueous solution, respectively. The concentration of each protein was designated to obtain detectable signals before and after treatment with different functionalized materials. The adsorbent and protein solution were shaken in a SHA–C constant–temperature shaker (Guohua Co., Changzhou, Jiangsu Province, China) at 25 ℃ for different lengths of time. The mixture was collected (700 μL) and centrifuged at 6580 g for 5 min at 4 ℃ (H 2050R, Xiang YI, Xiangtan, Hunan Province, China), then 500 μL of supernatant were assayed using the Bradford method by adding to 2.5 mL of the color reagent. The absorbance at 595 nm was measured by a spectrophotometer and the amount of adsorbed protein was calculated by the standard curve method using the corresponding determined proteins as reference standards, respectively.
Furthermore, the same concentrations of BSA, Trf, Mb, Lyz, and OVA as above procedure were prepared in Britton–Robinson (B–R) buffer solution at different pH values, respectively. Similarly, the protein solution and adsorbent were shaken at 25 ℃ for 200 min, then centrifuged at 6580 g for 5 min at 4 ℃. Considering that proteins might be unsteady or denatured at low or high pH value, some interference might be present if spectrophotometry was used to analyze the concentration of proteins. The 8
above supernatant and the original sample solution were filtered through a 0.22 μm nylon membrane and 20 μL of these solutions were separated and analyzed by the HPLC system, respectively. At the same time, the corresponding standard protein was determined by HPLC and used to calculate the adsorbed amount of materials for each protein using standard curve method, respectively.
All measurements were repeated three times and the average was obtained.
2.4.2 Removal or depletion of the target proteins from a five–protein mixture in aqueous or B–R buffer solution
In this section, selective removal or depletion of HAP such as BSA, OVA, and Lyz from protein mixture was investigated. Based on the results of the section 2.4.1, Material III and SBA–15 were selected to perform these tasks. Details of the procedure were described as follows: 5.0 mL of the five–protein mixture in aqueous solution (BSA 1.0 × 10–6 mol L–1, Trf 1.0 × 10–6 mol L–1, Mb 1.0× 10–6 mol L-1, Lyz 6.9 × 10–6 mol L–1, and OVA 6.9 × 10–6 mol L–1) was placed in a 10 mL centrifuge tube, then 40.0 mg of Material III were added and the mixture was shaken at 25 ℃ for 10 min to facilitate adsorption of the target protein (BSA and OVA). The mixture was then centrifuged and 20 μL of the filtered supernatant and the original sample solutions were injected into the HPLC system, respectively. Furthermore, Material III was subsequently replaced by the original SBA–15 to remove of the target protein (Lyz) from the protein mixture in B–R buffer solution (pH 7.0) at 25 ℃ for 200 min. 9
2.5 Removal or depletion of HAP from real biological samples
First, egg white was separated from a fresh egg purchased from a local supermarket. Then it was diluted two–fold with B–R buffer solution (pH 7.0) and centrifuged (10,280 g) at 4 ℃ for 30 min. The supernatant was collected and diluted 50 times with ultrapure water and B–R buffer solution (pH 7.0), respectively. And 5.0 mL of the diluted sample were shaken with 40.0 mg of SBA–15, Material I, Material II, Material III, and 80.0 mg of the mixture of SBA–15 (40.0 mg) and Material I (40.0 mg) at 25 ℃ for 200 min, respectively. Finally, 20 μL of the diluted and treated egg white sample were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the stained gels were imaged and processed using Image Processing and Analysis in Java software (downloaded from http://imagej.net/Downloads).
In addition, crude human serum (100 mL) was obtained from a serum bank and immediately frozen at – 20 ℃, and then thawed at 4 ℃ prior to analysis. The serum sample was diluted 50 times with ultrapure water and pH 7.0 B–R buffer solution, respectively. Five milliliters of the diluted serum samples treated with SBA–15, Material I, Material II and Material III at 25 ℃ for 200 min, respectively. Then 20 μL of these serum samples were determined by SDS–PAGE and processed the images of the stained gels as above technique.
3. Results and discussion 10
3.1 Characterization of functionalized SBA–15
The successful synthesis of functionalized SBA–15 (Material I, II and III) was confirmed by FT–IR and element analysis. The FT–IR spectra of the original and functionalized materials (Electronic Supplementary Information, Fig. S2) show that the adsorption bands at around 1084 and 465 cm–1 corresponded to the stretching vibration of the Si–O–Si band. The stretching vibration and out–of–plane bending of free silicon–OH groups were observed at around 3436 and 801 cm–1, respectively. These two bands were present in both the original and functionalized SBA–15. For functionalized SBA–15 (Material I, II and III), the C–H stretching vibration at around 2960 and 2925 cm–1, and the C–H bending vibration at around 1460 cm–1, indicated the incorporation of alkyl groups. In addition, the νc=o band at 1735 cm–1 confirmed the presence of carbonyl groups, which suggested the successful functionalization of SBA–15.
Element analyses of the C, H and N content of the original and functionalized SBA–15 (Material I, II and III) are listed in Table 1. Compared with the reactive material, an increase in C, N and H content in each resulting material also indicated successful grafting of functional groups onto SBA–15. The number of amino groups in SBA–15–NH2 and SBA–15–NH–Br were calculated using the results of element analysis, respectively, which provided the basis for the amount of initiator, catalyst and monomer added to the ATRP reactive system.
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The thermogravimetric curves of the original and functionalized SBA–15 materials are shown in Electronic Supplementary Information Fig. S3. The profiles show that SBA–15 had a relatively high thermal stability. Material I and Material II had similar mass losses (inflexion) within the range of 200 to 650 ℃ but with distinct thermogravimetric profiles. The weight loss region may have been due exclusively to the removal of organic functional groups, and it was calculated that the mass ratios of the grafted monomers of Material I and Material II to SBA–15–NH–Br were 4.91% and 2.58%, corresponding to 0.27 and 0.30 mmol g–1 of monomer, respectively. This was in accordance with the results of element analysis (0.24 and 0.25 mmol/g, respectively). For Material III, its weight loss to SBA–15–NH–Br was the total amount of the above monomers.
Typical powder X–ray diffraction patterns of the original and the functionalized SBA–15 materials in the 2θ range from 0.50 – 4.00 are illustrated in Electronic Supplementary Information Fig. S4. As shown in Fig. S4, SBA–15 exhibited diffraction patterns with well–resolved peaks at 0.90, 1.50 and 1.80 which were attributed to the (100), (110) and (200) Bragg diffractions, respectively, indicating the existence of the highly ordered hexagonal structure of SBA–15. However, for Material I, II and III, the diffraction peaks at 1.50 and 1.80 (2θ) disappeared, suggesting that the ordered hexagonal structure of SBA–15 was changed or blocked after functionalization.
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TEM images of SBA–15 before and after functionalization are depicted in Fig. S5 (Electronic Supplementary Information). Fig. S5 (b and c) show the pore channels in Material I and Material II, which were indistinct compared with those of the original SBA–15. This may have been due to the modified polymer occupying or blocking some pore channels in the material and the polymer chains were outside the pores of the material matrix. Furthermore, for Material III, the pore channels (Fig. S5d) were more indistinct than those in Material I and II, suggesting that the polymerization reaction was carried out on the surface of the material.
Nitrogen gas adsorption–desorption isotherm tests were performed to further investigate the changes in pore structure and size between the original and functionalized SBA–15. The surface areas, adsorption and desorption cumulative pore volumes and pore sizes of the original and functionalized materials are listed in Table 2. It can be seen that the surface area and pore volume of the synthesized materials (Material I, II and III) were markedly decreased. According to TEM analysis (Fig. S5), this was attributed to a large occupied surface area on the polymer functionalized SBA–15 support. Furthermore, a significant difference in the distribution of pore diameter in the original and functionalized SBA–15 was observed (Electronic Supplementary Information, Fig. S6) and the diameters of Material I, II and III were smaller than that of SBA–15 in the mesoporous region (Table 2 and Fig. S6). It could be speculated that the tight grafting of the groups in the mesopores of SBA–15 led to
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some channels being occupied or blocked (Fig. S5). In addition, large polymer pores were formed on the surface of the materials (Fig. S6).
The relative curves of the zeta potential for SBA–15, Material I, II and III in B– R buffer solution with the pH range of 3.0–8.0 were measured. As shown in Fig. 2, the relative curve of the zeta potential for SBA–15 was similar to the reported reference values [32]. Fig. 2 also exhibits that the relative curve of the zeta potential for Material I was different from the values obtained for Material II and III, indicating that the grafted polymer in Material I was different from that in Material II and III. It can be seen from Fig. 1 that Material III was synthesized by grafting carboxylic groups onto Material I. Therefore, the surface charge of Material III should be the same as that of Material II. The similar curves of the zeta potentials for Material II and III demonstrated that the grafting of carboxylic groups on the Material III and Material II was successful. In addition, the zeta potentials of SBA–15, Material I, II and III in ultrapure water were –16.7, 2.62, –17.0 and –6.91 mV, respectively.
3.2 Protein adsorption
3.2.1 Static adsorption test of the original and functionalized SBA–15 for different proteins
Fig. 3 shows the adsorption–time profiles obtained by the materials at certain concentrations of the protein aqueous solution for different time periods. It was
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observed that SBA–15 showed primary adsorption of Lyz and Mb, which was mainly attributed to the weak electrostatic interaction [33–34]. It also adsorbed BSA and Trf based on the size selectivity between SBA–15 and the proteins. According to the zeta potential results (Fig. 2), the surface of Material I was positively charged in aqueous solution. Therefore, the negatively charged proteins BSA (pI of 4.9) [35], Trf (pI of 5.4) [36] and OVA (pI of 4.5) [37] were adsorbed based on an electrostatic interaction mechanism. It was in agreement with the experimental results. Similarly, the surface of Material II was negatively charged and it should adsorb the positively charged Lyz (pI of 10.7) [37] and repel the negatively charged proteins such as BSA, Trf and OVA. However, the results showed that a certain amount of BSA and Trf was adsorbed on the Material II. It could be due to the size matching interaction between the proteins and the grafted polymer of material. With regard to Material III, Fig. 3 shows that it adsorbed BSA and OVA, but did not adsorb Trf, Mb and Lyz. Moreover, it is noteworthy that the adsorption capacity of Material III for BSA was initially high but decreased slowly with increased adsorption time. The reason for this may be that the adsorption derived from the grafted polymer of matrices in the initial stages. Although the pore size distributions of the grafted polymer of Material I, Material II, and Material III were similar (Electronic Supplementary Information, Fig. S6), the amount and pore shape of the grafted polymer of different materials should be different, which led to different adsorption behavior for different proteins. Compared with Material I and Material II, the greater amount of grafted polymer of Material III 15
resulted in the faster transfer velocity and higher adsorption capacity for the size matching proteins (BSA). Subsequently, the interior grafting positively charged polymer retained the interior BSA and the outer negatively charged polymer repelled and released the outer BSA from Material III during shaking process. In addition, Fig. 3 also shows that the adsorption equilibria of all the materials for the most of proteins in aqueous solution were reached within 200 min. Therefore, 200 min was selected to investigate the effect of pH on the adsorption of the investigated proteins by the above materials.
In theory, adsorption depends on the surface charge of materials and proteins at different pH values if electrostatic interaction is the only interaction mechanism. Fig. 2 shows that the surface of all the materials was negatively charged at pH greater than 5.5. The acidic proteins BSA, Trf and OVA were also negatively charged and a repulsive interaction occurred between the proteins and materials at pH>6.0. Therefore, the adsorption of BSA and OVA by these materials decreased at pH 6.0– 8.0 (Fig. 4). However, Fig. 4 shows an increase in adsorbed Trf at pH>5.0 for most of materials, especially for Material I and II. It is tempting to speculate that the proteins configuration was correlated with the change of pH value (or medium) of solution. The Trf configuration would be gradually matched with the pore size/shape of the grafted polymer of Material I and II in buffer solution at pH>5.0. In respect of the neutral protein Mb (pI of 7.3) [38], it is partially or fully positively charged at pH<7.0. SBA–15, Material II and III, whose surface charges were negative in the pH 16
rang of 3.5–7.0, should have high adsorption ability for them at pH<7.0. Similarly, the surface charge of Material I was negative within the pH ranged of 5.5–7.0. Higher adsorption of Mb by Material I should be achieved within this pH range. However, this did not totally agree with the experimental observations (Fig. 4). It may be that hydrophobic or size matching interaction also occurred under the investigated conditions. Moreover, Fig. 4 shows that the amount of the basic protein Lyz adsorbed onto SBA–15 markedly increased from pH 5.0 to 7.0 and then showed a slight change when the pH increased from 7.0 to 8.0. This was due to the surface of SBA–15 being negatively charged at a pH greater than 3.7 (Fig. 2) and Lyz was positively charged at a pH less than its pI of 10.7 [37]. Therefore, Lyz molecules strongly interacted with negatively charged silanol groups by Coulomb’s force at pH values ranging from 4.0 to 7.0.
3.2.2 Removal or depletion of the target proteins from the five–protein mixture
In the chromatographic analysis of complex biological sample, significant peak overlap is always encountered and some special techniques should be required to resolve this problem. Therefore, the most desired result is that the proteins (especially for the low abundant proteins) with a simiar chromatographic retention time were not depleted in the process of removal of the HAP. Fig. 5A (a) (including OVA, Mb, Lyz and BSA) and Fig. 5B (a) (including OVA, Mb, Lyz and Trf) show the chromatograms of four–protein mixture, respectively. It can be seen from Fig. 5 (A
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and B) that the retention time of BSA was the same as that of Trf. To avoid the depletion of Trf, Material III was selected to remove BSA (HAP in serum sample) and OVA (HAP in egg white sample) according to the results of Fig. 3. Fig. 5A (b) and 5B (b) exhibit the chromatograms of four–protein mixture after treatment with Material III in aqueous solution for 10 min, which revealed that Trf was not adsorbed but almost all BSA was removed by Material III from the four–protein mixture Finally, the removal of BSA and OVA from the five tested proteins in aqueous solution was achieved by treatment with Material III for 10 min (Fig. 5C). Fig. 5C (a) shows the chromatographic peaks of BSA and Trf overlapped in the original protein mixture solution. However, after the protein mixture was treated with Material III, the chromatographic peak of Trf was still existed and can be detected by HPLC–UV (Fig. 5C (b)).
Subsequently, removal of the basic protein Lyz (HAP in egg white sample) was accomplished using the original SBA–15 in B–R buffer solution at pH of 7.0 for 200 min. Fig. 6 shows the chromatograms of the five–protein mixture in B–R buffer solution (pH 7.0) before (a) and after (b) treatment with the original SBA–15 for 200 min. It can be seen from Fig. 6, all of Lyz was removed by 40.0 mg of SBA–15 (Fig. 6 (b)). The result showed that SBA–15 has potential application in the removal of basic proteins from complex biological samples at a specific pH.
Fig. 6 is near here
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3.3 Removal or depletion of proteins from real biological samples
In order to verify the practical application of this approach, egg white and serum samples were selected as the real biological samples to verify the removal of the HAP (OVA, Lyz, and BSA) using the above procedure, respectively. Fig. 7a and Fig. 7b show the results of SDS–PAGE analysis for the selective adsorption of proteins from the egg white sample by Material I, Material II, Material III, SBA–15, and the mixture of SBA–15 and Material I in aqueous and B–R buffer solution (pH of 7.0), respectively. It is known that the major HAP in egg white are OVA (45 kDa, pI of 4.5), ovotransferrin (76 kDa, pI of 6.0), ovomucin (5500–8300 kDa, pI of 4.5–5.0) and Lyz (14.4 kDa, pI of 10.7) [37]. The bands corresponding to these proteins were observed following SDS–PAGE of the egg white sample diluted with ultrapure water (lane 1 in Fig. 7a) or B–R buffer solution at pH 7.0 (lane 1 in Fig. 7b), with the exception of the large molecular weight protein ovomucin. According to the results of Fig. 3 and Fig. 4, OVA can be removed or depleted by Material I and III in aqueous soltuion, but the efficient adsorbents for Lyz were SBA–15 and Material II, no matter in aqueous or in buffer solution (pH of 7.0). Fig. 7a demonstrate that the removal efficiencies of Material I (lane 2) and III (lane 4) for OVA from the real egg white sample were up to approximately 73% and 70% (calculations were performed using Image Processing and Analysis in Java software), respectively. Fig. 7a and 7b show that the color of the Lyz bands faded or disappeared after the egg white sample was treated with Material II (lane 3) or SBA–15 (lane 5) in aqueous and B–R buffer (pH 19
of 7.0) solution, respectively. At the same time, it can be clearly observed that the band of Lyz treated with Material II (lane 3) and SBA–15 (lane 5) faded more significant in B–R buffer solution (pH of 7.0) than in aqueous soltuion. Image Processing and Analysis in Java technique calculated that approximately 90% and 92% of Lyz were removed by Material II and SBA–15 from egg white sample in B–R buffer solution (pH of 7.0), respectively. Furthermore, the mixture of Material I and SBA–15 was selected to remove or deplete OVA and Lyz simulaneously from the egg white sample in aqueous and B–R buffer (pH of 7.0) soltuion, respectively. Fig. 7a (lane 6) shows that approximately 99% of OVA and 90% of Lyz can be effectively removed from egg white sample in aqueous solution. Meanwhile, we also observed that the aforementioned HAP ovotransferrin can be adsorbed from egg white sample in aqueous by Material II (lane 3 in Fig. 7a) and SBA–15 (lane 5 in Fig. 7a), respectively. This can be attributed to that ovotransferrin (pI of 6.0) is partially positively charged in aqueous solution (The pH value of ultrapure water is 6.0–6.5 in our laboratory), so it can be adsorbed by negatively charged Material II and SBA–15 in ultrapure water (see section 3.1, zeta potential). In any case, all above results revealed that we can select or combine suitable adsorbents from the above materials to remove or deplete a kind of HAP or all the HAP from egg white sample.
Fig. 8 exhibits the results of SDS–PAGE analysis for the removal or depletion of the HAP from serum samples diluted with ultrapure water (Fig. 8a) and B–R buffer solutions (Fig. 8b), respectively. It can be seen that the gel electrophoregram of the 20
original serum sample (lane 1 in Fig. 8a and 8b) showed the band of 66 kDa, 45.0– 66.2 kDa and 25–35 kDa corresponding to highly abundant human serum albumin (HSA, pI of 5.0), and heavy and light chain of immunoglobulin G (IgG, pI of approximately 5.0), respectively [39–42]. Based on the above researches (Fig. 3), all of the original and functionalized SBA–15 could be the promising adsorbents for treatment of highly abundant HSA from human serum sample in aqueous soltuion. Indeed, Fig. 8a displays that the desired result was obtained by Material III (lane 4 in Fig. 8a, approximately 80% of HAS was removed). However, Fig. 8a also shows that other materials cannot remove HSA from human serum sample in aqueous solution. It may be due to the less amount of the grafted polymer of the materials and the highly complexity of the serum sample. As for the highly abundant protein IgG, the most effecitive adsorbent still was Material III. Therefore, Material III can be selected while the HAP needed to be removed from serum sample.
Based on the above results, SBA–15 and Material II can remove or deplete HAP ovotransferrin and Lyz from the egg white samples, which depended on the prevailing electrostatic interaction. Material III depleted the HAP (OVA, HSA and IgG) from egg white or human serum samples in aqueous solution by a synergistic interaction. The experimental results also confirm that a rational design for improving the signal of chromatogram or gel electrophoregram can be conducted based on the above researches. Certainly, these materials may also adsorb other LAP, as this is a common problem in all similar techniques. When the proposed techniques were compared with 21
the affinity system, different mechanisms were involved in the adsorption of proteins. Thus, they have different advantages and limitations. For example, affinity systems are process specific in capturing proteins. However, some of the bound HAP may also bind with many species, such as some peptide hormones, cytokines, and chemokines, and cause nonspecific removal of some other proteins [25]. Our experimental results showed that the proposed materials had selective adsorption of some proteins and can be used to treat real biological samples. This treatment process will reduce the complexity of electrophoregram or chromatograms to some extent. In addition, this approach can also be combined with affinity systems or other techniques to compensate for loss of information in real biological samples during the removal of HAP.
4. Conclusion
In this study, a series of pH–sensitive grafted SBA–15 materials were successfully synthesized via ATRP and used as adsorbents for the selective removal or depletion of target proteins. The design concept was confirmed by the adsorption results of the materials for standard proteins in B–R buffer solutions at different pH values. The removal or depletion of target proteins from the standard protein mixture was also achieved using this approach. The results showed that the removal or depletion of certain proteins and a reduction in the complexity of real biological samples prior to the separation steps were validated. However, although being
22
validated for targeted study, this approach is still inadequate for untargeted proteomic analysis and more research is required to extend the application of the proposed process.
Acknowledgements
The authors thank the National Natural Science Foundation of China (NSFC) Fund (No. 21105039 and No. 21375052) for supporting the project.
23
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Figure captions
Fig. 1. Synthesis protocol of pH–sensitive polymer brush–grafted mesoporous silica.
Fig. 2. Zeta potential of the original and diverse functional SBA–15 as a function of pH. The zeta potential was measured in B–R buffer solution.
Fig. 3. Effect of time on adsorption of the original and functionalized SBA–15 (Material I, II, and III) for BSA, Trf, Mb, Lyz, and OVA in aqueous solution, respectively. The standard protein solution: BSA (1.0 × 10–6 mol L–1), Trf (1.0 × 10–6 mol L–1), Mb (1.0× 10–6 mol L–1), Lyz (6.9 × 10–6 mol L–1), and OVA (6.9 × 10–6 mol L–1). Each point represented the mean value of three measurements.
Fig. 4. Effect of pH on adsorption of the original and functionalized SBA–15 (Material I, II, and III) for BSA, Trf, Mb, Lyz, and OVA in B–R buffer solution, respectively. The concentrations of standard proteins were the same as Fig. 4. Each measuring point represented the mean value of three specimens.
Fig. 5. The chromatograms of the aqueous solution of protein mixture before (a) and after (b) treatment with Material III for 10 min. The protein mixtures A, B included 32
four standard proteins (A: BSA, Mb, Lyz, and OVA; B: Trf, Mb, Lyz, and OVA). The protein mixture C contained above five standard proteins (BSA, Trf, Mb, Lyz, and OVA). The concentrations of standard proteins are as follows: BSA (1.0 × 10–6 mol L–1), Trf (1.0 × 10–6 mol L–1), Mb (1.0× 10–6 mol L–1), Lyz (6.9 × 10–6 mol L–1) and OVA (6.9 × 10–6 mol L–1). Chromatographic conditions were described in Electronic Supplementary Information.
Fig. 6. Chromatograms of protein mixtures before (a) and after (b) treated with SBA– 15. The mixture protein solution: BSA (1.0 × 10–6 mol L–1), Trf (1.0 × 10–6 mol L–1), Mb (1.0× 10–6 mol L–1), Lyz (6.9 × 10–6 mol L–1) and OVA (6.9 × 10–6 mol L–1). Chromatographic conditions were described in Electronic Supplementary Information.
Fig. 7. SDS–PAGE analysis of egg white diluted 100 times with ultrapure water (a) and pH of 7.0 B–R buffer solution (b) before (lane 1) and after treatment with the Material I (lane 2), Material II (lane 3), Material III (lane 4), SBA–15 (lane 5), and the mixture of SBA–15 and Material I (lane 6). The processes of sample treatment and electrophoresis were performed as outlined in Section 2.2 and Section 2.6. Loading amount of sample: 20 μL.
33
Fig. 8. SDS–PAGE analysis of human serum sample diluted 50 times with ultrapure water (a) and pH of 7.0 B–R buffer solution (b) before (lane 1) and after treatment with the Material I (lane 2), Material II (lane 3), Material III (lane 4), and SBA–15 (lane 5). The processes of sample treatment and electrophoresis were performed as outlined in Section 2.5 and Electronic Supplementary Information. Loading amount of sample: 20 μL.
Table 1. Element analysis (N, C and H) of the original and functionalized SBA-15
Materials
N (%)
C (%)
H (%)
SBA-15
0.00
0.93
1.02
SBA-15-NH2
2.72
8.37
2.13
SBA-15-NH-Br
2.52
11.62
2.42
Material I
2.86
16.01
3.01
Material II
2.50
12.81
2.61
Material III
2.70
18.17
3.21
34
Table 2. Physicochemical properties of original and functional SBA-15
Sample
SBET (m2/g)a
SBJH (m2/g)b
VBJH (cm3/g)c
DBJHd (nm)
Adsorption
Desorption
Adsorption
Desorption
Adsorption
Desorption
SBA-15
379
282
394
0.49
0.55
6.90
5.58
Material I
54
74
74
0.09
0.09
5.02
5.03
Material II
42
56
56
0.07
0.07
4.90
4.94
Material III
25
36
37
0.05
0.05
5.24
5.23
a
BET surface area.
b
BJH adsorption and desorption cumulative surface area of pores.
c
BJH adsorption and desorption cumulative volume of pores.
d
BJH adsorption and desorption average pore width (4V/A).
35
A series of pH sensitive groups grafted SBA-15 was synthesized via ATRP.
The proposed materials had selective adsorption for some proteins at a certain pH.
The selective removal of the target protein from protein mixtures can be achieved.
The complexity of real biological samples can be reduced by the proposed process.
36
37
38
39
40
41
42
43
44
45
46
47
48
PII: DOI: Reference:
S0039-9140(16)30758-5 http://dx.doi.org/10.1016/j.talanta.2016.10.003 TAL16923
To appear in: Talanta Received date: 18 June 2016 Revised date: 16 September 2016 Accepted date: 2 October 2016 Cite this article as: Jiaojiao Wang, Jingfeng Lan, Huihui Li, Xiaoyan Liu and Haixia Zhang, Fabrication of diverse pH–sensitive functional mesoporous silica for selective removal or depletion of highly abundant proteins from biological samples, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fabrication of diverse pH–sensitive functional mesoporous silica for selective removal or depletion of highly abundant proteins from biological samples Jiaojiao Wang, Jingfeng Lan, Huihui Li, Xiaoyan Liu*, Haixia Zhang
State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous, Metals Chemistry and Resources Utilization of Gansu Province and College of, Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
Corresponding author Tel.: +86 931 8912510; fax: +86–931–8912582.
[email protected] Abstract In proteomic studies, poor detection of low abundant proteins is a major problem due to the presence of highly abundant proteins. Therefore, the specific removal or depletion of highly abundant proteins prior to analysis is necessary. In response to this problem, a series of pH–sensitive functional mesoporous silica materials composed of 2–(diethylamino)ethyl methacrylate and methacrylic acid units were designed and synthesized via atom transfer radical polymerization. These functional mesoporous silica materials were characterized and their ability for adsorption and separation of proteins was evaluated. Possessing a pH–sensitive feature, the synthesized functional materials showed selective adsorption of some proteins in aqueous or buffer solutions at certain pH values. The specific removal of a particular protein from a mixed protein solution was subsequently studied. The analytical results confirmed that all the target 1
proteins (bovine serum albumin, ovalbumin, and lysozyme) can be removed by the proposed materials from a five–protein mixture in a single operation. Finally, the practical application of this approach was also evaluated by the selective removal of certain proteins from real biological samples. The results revealed that the maximum removal efficiencies of ovalbumin and lysozyme from egg white sample were obtained as 99% and 92%, respectively, while the maximum removal efficiency of human serum abumin from human serum sample was about 80% by the proposed method. It suggested that this treatment process reduced the complexity of real biological samples and facilitated the identification of hidden proteins in chromatograms.
Keywords: pH–sensitive; functional mesoporous silica; atom transfer radical polymerization; highly abundant proteins
1. Introduction
The identification and quantification of proteins in a complex biological system are important issues in current proteomics. However, with regard to current analytical technologies, such as two dimensional electrophoresis, high–performance liquid chromatography (HPLC) coupled to mass spectrometry and on–line multidimensional
2
liquid chromatography, it is difficult to separate all proteins completely in a single operation due to the enormous amount and dynamic concentration range of proteins in complex biological samples, which leads to the suppression of information on low abundant proteins (LAP) by highly abundant proteins (HAP). To minimize the complexity of samples and improve the identification of LAP, various technologies and methodologies have been developed to deplete HAP based on their biochemical and biophysical features, such as molecular weight (MW) [1–6], hydrophobicity [7– 8], surface charge [3, 6, 9–10], isoelectric point (pI) [11–14], metal ion affinity effect [15–16], basic structure differences [17–18], amino acid sequence (epitope) [19–21], and immunoaffinity effect [22–23]. Among them, affinity chromatography [15–16, 22–23] is the most common method and possesses unique advantages for the depletion of HAP [22, 24–26]. However, commercial available immunaffinity columns and adsorbent media are costly and limited, which cannot meet the various demands for practical applications. Besides, ionic exchange [27] and hydrophobic interaction [7–8] materials are also widely used as adsorbents for separation of proteins. Nevertheless, when the different media and different synthesized methods were employed to prepare the adsorbents, protein interactions with the adsorbents would be different due to the present of multiple interactions between proteins and the adsorbent bead or spacer–linker structure carrying the nominal ligand [28]. Therefore, it is still essential to develope a facile and easily scalable technique for fabrication of
3
various adsorbent with potential application in the removal of HAPfrom a real biological sample.
Recently, we reported several approaches for the immobilization or selective removal of target proteins using fabricated functional mesoporous silica materials based on Santa Barbara Amorphous–15 (SBA–15) with various anchor groups [3, 6, 9]. These functional materials have strong anionic or cationic exchange ability and their adsorption of protein samples were only investigated in aqueous and neutral phosphate–buffered saline (PBS). “Smart” polymers with stimuli based on changes in pH are attractive materials for potential application in various fields [29–30]. In general, pH–sensitive polymers were formed by block copolymers with amines, carboxylic acid or hydroxy acid, which became charged species by protonation or deprotonation at different pH solutions [18, 31]. This resulted in a pronounced change in the adsorption of proteins by these materials compared with those containing strong anionic or cationic groups [3, 6, 9]. Encouraged by the above results, in the present study, we synthesized diverse pH–sensitive functionalized SBA–15 materials by atom transfer radical polymerization (ATRP). In addition, the adsorption of protein by these functionalized materials, which depend on the composition of the polymer as well as the pH value, was investigated. Subsequently, the use of these materials for the removal or depletion of HAP from real biological samples was developed.
2. Experimental
4
2.1 Materials and methods
All the materials, reagents and instrumental analysis were listed in the electronic supplementary information.
2.2 Preparation of diverse pH–sensitive functionalized SBA–15
The ATRP initiator was immobilized on SBA–15 (SBA–15–NH–Br) according to our previous work [6] and the details are described in the Electronic Supplementary Information Fig. S1. The functionalized SBA–15 was prepared by ATRP as shown in Fig. 1. Details of these procedures are given below.
2.2.1 Surface modification of SBA–15 with poly(2–(diethylamino)ethyl methacrylate) (PDEAM) or poly(methacrylic acid) (PMAA) brush by surface– initiated ATRP
PDEAM (or PMAA) brush–grafted SBA–15 was prepared by SBA–15–NH–Br in dry toluene. A typical polymerization procedure was as follows: the obtained SBA– 15–NH–Br was immersed in 20.0 mL of dry toluene and dry nitrogen gas was then bubbled through the reaction to expel oxygen. The catalyst of CuBr and the ligand of N,N,N′,N′′,N′′–pentamethyldiethylenetriamine (PMDETA) (the molar ratio of CuBr and PMDETA to ATRP–initiator was kept at 1:1:1) were then added under a nitrogen atmosphere. The degassed 2–(diethylamino) ethyl methacrylate (DEAM) (or Methacrylic acid (MAA)) monomer solution (the molar ratio of monomer to initiator
5
was kept at 45:1) was added via a syringe and the solution was further degassed by three freeze–pump–thaw cycles. The flask was then placed in a preheated oil bath at 100 ℃ for 40 min. The ATRP reaction was then stopped and the polymer–grafted SBA–15 was washed by ultrasonication using toluene followed by centrifugation to remove unreacted monomers. The resulting polymer–grafted SBA–15 was further washed with acetone, distilled water and ethanol until the supernatant was colorless. Finally, PDEAM (or PMAA) brush–grafted SBA–15 was dried under reduced pressure at 40 ℃ overnight and the resulting material was denoted as Material I (or Material II).
2.2.2 Surface modification of SBA–15 with amphoteric polymer brush by surface–initiated ATRP
Amphoteric polymer grafted SBA–15 was synthesized via ATRP polymerization as described above. In brief, the above PDEAM brush–grafted SBA–15 (Material I) was reacted with MAA monomer (the molar ratio of monomer to initiator was also kept at 45:1) using the aforementioned approach. The resulting material was denoted as SBA–15–(DEAM)x–(MAA)y–Br (Material III).
2.3 Characterization
Elemental analysis was measured with a VarioEl element analyzer, Elemental Analysis system (Hanau, Germany). Fourier Transform Infrared (FT–IR) Spectra of
6
mesoporous materials before and after functionalization were observed by using Vertex 70 FT–IR (Bruker Optics, Ettlingen, Germany). Thermogravimetric analysis (TGA) of the original and functionalized SBA–15 was carried out with a Pyris Diamond TGA (Perkin–Elmer, Woodland, California, USA) up to 800 ℃ with a heating rate of 10 K/min under nitrogen. The narrow–angle powder X–ray diffraction patterns of the original and functionalized materials from 0.50 to 4.00 were recorded with a D/max–2400 (Rigaku, Japan) using Cu Ka radiation. Transmission electron microscopy (TEM) was performed on a Tecnai G2 TF20 (FEI, Hillsboro, Oregon, USA) at 200 kV. Nitrogen adsorption–desorption measurements were carried out at 77 K on a Tristar 3000 Surface Area and Porosimetry analyzer (Micromeritics Instrument Corp., Atlanta, Georgia, USA). The surface–area measurement was based on the Brunauer–Emmett–Teller (BET) method, and the pore–size distribution was calculated from the Barrett–Joyner–Halenda (BJH) formula. The zeta potentials of the diverse functionalized SBA–15 were measured using Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) at 25 ℃ in ultrapure water and B–R buffer solution at different pH values (3.0–8.0), respectively.
2.4 Removal of proteins from a standard protein mixture
2.4.1 Static adsorption test of the original and functionalized SBA–15 for different proteins
7
Batch adsorption experiments were carried out by adding 40.0 mg of the original or functionalized SBA–15 (Material I, II or III) to 20.0 mL of bovine serum albumin (BSA, 1.0 × 10–6 mol L–1), transferrin (Trf, 1.0 × 10–6 mol L–1), myoglobin (Mb, 1.0 × 10–6 mol L–1), lysozyme (Lyz, 6.9 × 10–6 mol L–1) and ovalbumin (OVA, 6.9 × 10–6 mol L–1) aqueous solution, respectively. The concentration of each protein was designated to obtain detectable signals before and after treatment with different functionalized materials. The adsorbent and protein solution were shaken in a SHA–C constant–temperature shaker (Guohua Co., Changzhou, Jiangsu Province, China) at 25 ℃ for different lengths of time. The mixture was collected (700 μL) and centrifuged at 6580 g for 5 min at 4 ℃ (H 2050R, Xiang YI, Xiangtan, Hunan Province, China), then 500 μL of supernatant were assayed using the Bradford method by adding to 2.5 mL of the color reagent. The absorbance at 595 nm was measured by a spectrophotometer and the amount of adsorbed protein was calculated by the standard curve method using the corresponding determined proteins as reference standards, respectively.
Furthermore, the same concentrations of BSA, Trf, Mb, Lyz, and OVA as above procedure were prepared in Britton–Robinson (B–R) buffer solution at different pH values, respectively. Similarly, the protein solution and adsorbent were shaken at 25 ℃ for 200 min, then centrifuged at 6580 g for 5 min at 4 ℃. Considering that proteins might be unsteady or denatured at low or high pH value, some interference might be present if spectrophotometry was used to analyze the concentration of proteins. The 8
above supernatant and the original sample solution were filtered through a 0.22 μm nylon membrane and 20 μL of these solutions were separated and analyzed by the HPLC system, respectively. At the same time, the corresponding standard protein was determined by HPLC and used to calculate the adsorbed amount of materials for each protein using standard curve method, respectively.
All measurements were repeated three times and the average was obtained.
2.4.2 Removal or depletion of the target proteins from a five–protein mixture in aqueous or B–R buffer solution
In this section, selective removal or depletion of HAP such as BSA, OVA, and Lyz from protein mixture was investigated. Based on the results of the section 2.4.1, Material III and SBA–15 were selected to perform these tasks. Details of the procedure were described as follows: 5.0 mL of the five–protein mixture in aqueous solution (BSA 1.0 × 10–6 mol L–1, Trf 1.0 × 10–6 mol L–1, Mb 1.0× 10–6 mol L-1, Lyz 6.9 × 10–6 mol L–1, and OVA 6.9 × 10–6 mol L–1) was placed in a 10 mL centrifuge tube, then 40.0 mg of Material III were added and the mixture was shaken at 25 ℃ for 10 min to facilitate adsorption of the target protein (BSA and OVA). The mixture was then centrifuged and 20 μL of the filtered supernatant and the original sample solutions were injected into the HPLC system, respectively. Furthermore, Material III was subsequently replaced by the original SBA–15 to remove of the target protein (Lyz) from the protein mixture in B–R buffer solution (pH 7.0) at 25 ℃ for 200 min. 9
2.5 Removal or depletion of HAP from real biological samples
First, egg white was separated from a fresh egg purchased from a local supermarket. Then it was diluted two–fold with B–R buffer solution (pH 7.0) and centrifuged (10,280 g) at 4 ℃ for 30 min. The supernatant was collected and diluted 50 times with ultrapure water and B–R buffer solution (pH 7.0), respectively. And 5.0 mL of the diluted sample were shaken with 40.0 mg of SBA–15, Material I, Material II, Material III, and 80.0 mg of the mixture of SBA–15 (40.0 mg) and Material I (40.0 mg) at 25 ℃ for 200 min, respectively. Finally, 20 μL of the diluted and treated egg white sample were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the stained gels were imaged and processed using Image Processing and Analysis in Java software (downloaded from http://imagej.net/Downloads).
In addition, crude human serum (100 mL) was obtained from a serum bank and immediately frozen at – 20 ℃, and then thawed at 4 ℃ prior to analysis. The serum sample was diluted 50 times with ultrapure water and pH 7.0 B–R buffer solution, respectively. Five milliliters of the diluted serum samples treated with SBA–15, Material I, Material II and Material III at 25 ℃ for 200 min, respectively. Then 20 μL of these serum samples were determined by SDS–PAGE and processed the images of the stained gels as above technique.
3. Results and discussion 10
3.1 Characterization of functionalized SBA–15
The successful synthesis of functionalized SBA–15 (Material I, II and III) was confirmed by FT–IR and element analysis. The FT–IR spectra of the original and functionalized materials (Electronic Supplementary Information, Fig. S2) show that the adsorption bands at around 1084 and 465 cm–1 corresponded to the stretching vibration of the Si–O–Si band. The stretching vibration and out–of–plane bending of free silicon–OH groups were observed at around 3436 and 801 cm–1, respectively. These two bands were present in both the original and functionalized SBA–15. For functionalized SBA–15 (Material I, II and III), the C–H stretching vibration at around 2960 and 2925 cm–1, and the C–H bending vibration at around 1460 cm–1, indicated the incorporation of alkyl groups. In addition, the νc=o band at 1735 cm–1 confirmed the presence of carbonyl groups, which suggested the successful functionalization of SBA–15.
Element analyses of the C, H and N content of the original and functionalized SBA–15 (Material I, II and III) are listed in Table 1. Compared with the reactive material, an increase in C, N and H content in each resulting material also indicated successful grafting of functional groups onto SBA–15. The number of amino groups in SBA–15–NH2 and SBA–15–NH–Br were calculated using the results of element analysis, respectively, which provided the basis for the amount of initiator, catalyst and monomer added to the ATRP reactive system.
11
The thermogravimetric curves of the original and functionalized SBA–15 materials are shown in Electronic Supplementary Information Fig. S3. The profiles show that SBA–15 had a relatively high thermal stability. Material I and Material II had similar mass losses (inflexion) within the range of 200 to 650 ℃ but with distinct thermogravimetric profiles. The weight loss region may have been due exclusively to the removal of organic functional groups, and it was calculated that the mass ratios of the grafted monomers of Material I and Material II to SBA–15–NH–Br were 4.91% and 2.58%, corresponding to 0.27 and 0.30 mmol g–1 of monomer, respectively. This was in accordance with the results of element analysis (0.24 and 0.25 mmol/g, respectively). For Material III, its weight loss to SBA–15–NH–Br was the total amount of the above monomers.
Typical powder X–ray diffraction patterns of the original and the functionalized SBA–15 materials in the 2θ range from 0.50 – 4.00 are illustrated in Electronic Supplementary Information Fig. S4. As shown in Fig. S4, SBA–15 exhibited diffraction patterns with well–resolved peaks at 0.90, 1.50 and 1.80 which were attributed to the (100), (110) and (200) Bragg diffractions, respectively, indicating the existence of the highly ordered hexagonal structure of SBA–15. However, for Material I, II and III, the diffraction peaks at 1.50 and 1.80 (2θ) disappeared, suggesting that the ordered hexagonal structure of SBA–15 was changed or blocked after functionalization.
12
TEM images of SBA–15 before and after functionalization are depicted in Fig. S5 (Electronic Supplementary Information). Fig. S5 (b and c) show the pore channels in Material I and Material II, which were indistinct compared with those of the original SBA–15. This may have been due to the modified polymer occupying or blocking some pore channels in the material and the polymer chains were outside the pores of the material matrix. Furthermore, for Material III, the pore channels (Fig. S5d) were more indistinct than those in Material I and II, suggesting that the polymerization reaction was carried out on the surface of the material.
Nitrogen gas adsorption–desorption isotherm tests were performed to further investigate the changes in pore structure and size between the original and functionalized SBA–15. The surface areas, adsorption and desorption cumulative pore volumes and pore sizes of the original and functionalized materials are listed in Table 2. It can be seen that the surface area and pore volume of the synthesized materials (Material I, II and III) were markedly decreased. According to TEM analysis (Fig. S5), this was attributed to a large occupied surface area on the polymer functionalized SBA–15 support. Furthermore, a significant difference in the distribution of pore diameter in the original and functionalized SBA–15 was observed (Electronic Supplementary Information, Fig. S6) and the diameters of Material I, II and III were smaller than that of SBA–15 in the mesoporous region (Table 2 and Fig. S6). It could be speculated that the tight grafting of the groups in the mesopores of SBA–15 led to
13
some channels being occupied or blocked (Fig. S5). In addition, large polymer pores were formed on the surface of the materials (Fig. S6).
The relative curves of the zeta potential for SBA–15, Material I, II and III in B– R buffer solution with the pH range of 3.0–8.0 were measured. As shown in Fig. 2, the relative curve of the zeta potential for SBA–15 was similar to the reported reference values [32]. Fig. 2 also exhibits that the relative curve of the zeta potential for Material I was different from the values obtained for Material II and III, indicating that the grafted polymer in Material I was different from that in Material II and III. It can be seen from Fig. 1 that Material III was synthesized by grafting carboxylic groups onto Material I. Therefore, the surface charge of Material III should be the same as that of Material II. The similar curves of the zeta potentials for Material II and III demonstrated that the grafting of carboxylic groups on the Material III and Material II was successful. In addition, the zeta potentials of SBA–15, Material I, II and III in ultrapure water were –16.7, 2.62, –17.0 and –6.91 mV, respectively.
3.2 Protein adsorption
3.2.1 Static adsorption test of the original and functionalized SBA–15 for different proteins
Fig. 3 shows the adsorption–time profiles obtained by the materials at certain concentrations of the protein aqueous solution for different time periods. It was
14
observed that SBA–15 showed primary adsorption of Lyz and Mb, which was mainly attributed to the weak electrostatic interaction [33–34]. It also adsorbed BSA and Trf based on the size selectivity between SBA–15 and the proteins. According to the zeta potential results (Fig. 2), the surface of Material I was positively charged in aqueous solution. Therefore, the negatively charged proteins BSA (pI of 4.9) [35], Trf (pI of 5.4) [36] and OVA (pI of 4.5) [37] were adsorbed based on an electrostatic interaction mechanism. It was in agreement with the experimental results. Similarly, the surface of Material II was negatively charged and it should adsorb the positively charged Lyz (pI of 10.7) [37] and repel the negatively charged proteins such as BSA, Trf and OVA. However, the results showed that a certain amount of BSA and Trf was adsorbed on the Material II. It could be due to the size matching interaction between the proteins and the grafted polymer of material. With regard to Material III, Fig. 3 shows that it adsorbed BSA and OVA, but did not adsorb Trf, Mb and Lyz. Moreover, it is noteworthy that the adsorption capacity of Material III for BSA was initially high but decreased slowly with increased adsorption time. The reason for this may be that the adsorption derived from the grafted polymer of matrices in the initial stages. Although the pore size distributions of the grafted polymer of Material I, Material II, and Material III were similar (Electronic Supplementary Information, Fig. S6), the amount and pore shape of the grafted polymer of different materials should be different, which led to different adsorption behavior for different proteins. Compared with Material I and Material II, the greater amount of grafted polymer of Material III 15
resulted in the faster transfer velocity and higher adsorption capacity for the size matching proteins (BSA). Subsequently, the interior grafting positively charged polymer retained the interior BSA and the outer negatively charged polymer repelled and released the outer BSA from Material III during shaking process. In addition, Fig. 3 also shows that the adsorption equilibria of all the materials for the most of proteins in aqueous solution were reached within 200 min. Therefore, 200 min was selected to investigate the effect of pH on the adsorption of the investigated proteins by the above materials.
In theory, adsorption depends on the surface charge of materials and proteins at different pH values if electrostatic interaction is the only interaction mechanism. Fig. 2 shows that the surface of all the materials was negatively charged at pH greater than 5.5. The acidic proteins BSA, Trf and OVA were also negatively charged and a repulsive interaction occurred between the proteins and materials at pH>6.0. Therefore, the adsorption of BSA and OVA by these materials decreased at pH 6.0– 8.0 (Fig. 4). However, Fig. 4 shows an increase in adsorbed Trf at pH>5.0 for most of materials, especially for Material I and II. It is tempting to speculate that the proteins configuration was correlated with the change of pH value (or medium) of solution. The Trf configuration would be gradually matched with the pore size/shape of the grafted polymer of Material I and II in buffer solution at pH>5.0. In respect of the neutral protein Mb (pI of 7.3) [38], it is partially or fully positively charged at pH<7.0. SBA–15, Material II and III, whose surface charges were negative in the pH 16
rang of 3.5–7.0, should have high adsorption ability for them at pH<7.0. Similarly, the surface charge of Material I was negative within the pH ranged of 5.5–7.0. Higher adsorption of Mb by Material I should be achieved within this pH range. However, this did not totally agree with the experimental observations (Fig. 4). It may be that hydrophobic or size matching interaction also occurred under the investigated conditions. Moreover, Fig. 4 shows that the amount of the basic protein Lyz adsorbed onto SBA–15 markedly increased from pH 5.0 to 7.0 and then showed a slight change when the pH increased from 7.0 to 8.0. This was due to the surface of SBA–15 being negatively charged at a pH greater than 3.7 (Fig. 2) and Lyz was positively charged at a pH less than its pI of 10.7 [37]. Therefore, Lyz molecules strongly interacted with negatively charged silanol groups by Coulomb’s force at pH values ranging from 4.0 to 7.0.
3.2.2 Removal or depletion of the target proteins from the five–protein mixture
In the chromatographic analysis of complex biological sample, significant peak overlap is always encountered and some special techniques should be required to resolve this problem. Therefore, the most desired result is that the proteins (especially for the low abundant proteins) with a simiar chromatographic retention time were not depleted in the process of removal of the HAP. Fig. 5A (a) (including OVA, Mb, Lyz and BSA) and Fig. 5B (a) (including OVA, Mb, Lyz and Trf) show the chromatograms of four–protein mixture, respectively. It can be seen from Fig. 5 (A
17
and B) that the retention time of BSA was the same as that of Trf. To avoid the depletion of Trf, Material III was selected to remove BSA (HAP in serum sample) and OVA (HAP in egg white sample) according to the results of Fig. 3. Fig. 5A (b) and 5B (b) exhibit the chromatograms of four–protein mixture after treatment with Material III in aqueous solution for 10 min, which revealed that Trf was not adsorbed but almost all BSA was removed by Material III from the four–protein mixture Finally, the removal of BSA and OVA from the five tested proteins in aqueous solution was achieved by treatment with Material III for 10 min (Fig. 5C). Fig. 5C (a) shows the chromatographic peaks of BSA and Trf overlapped in the original protein mixture solution. However, after the protein mixture was treated with Material III, the chromatographic peak of Trf was still existed and can be detected by HPLC–UV (Fig. 5C (b)).
Subsequently, removal of the basic protein Lyz (HAP in egg white sample) was accomplished using the original SBA–15 in B–R buffer solution at pH of 7.0 for 200 min. Fig. 6 shows the chromatograms of the five–protein mixture in B–R buffer solution (pH 7.0) before (a) and after (b) treatment with the original SBA–15 for 200 min. It can be seen from Fig. 6, all of Lyz was removed by 40.0 mg of SBA–15 (Fig. 6 (b)). The result showed that SBA–15 has potential application in the removal of basic proteins from complex biological samples at a specific pH.
Fig. 6 is near here
18
3.3 Removal or depletion of proteins from real biological samples
In order to verify the practical application of this approach, egg white and serum samples were selected as the real biological samples to verify the removal of the HAP (OVA, Lyz, and BSA) using the above procedure, respectively. Fig. 7a and Fig. 7b show the results of SDS–PAGE analysis for the selective adsorption of proteins from the egg white sample by Material I, Material II, Material III, SBA–15, and the mixture of SBA–15 and Material I in aqueous and B–R buffer solution (pH of 7.0), respectively. It is known that the major HAP in egg white are OVA (45 kDa, pI of 4.5), ovotransferrin (76 kDa, pI of 6.0), ovomucin (5500–8300 kDa, pI of 4.5–5.0) and Lyz (14.4 kDa, pI of 10.7) [37]. The bands corresponding to these proteins were observed following SDS–PAGE of the egg white sample diluted with ultrapure water (lane 1 in Fig. 7a) or B–R buffer solution at pH 7.0 (lane 1 in Fig. 7b), with the exception of the large molecular weight protein ovomucin. According to the results of Fig. 3 and Fig. 4, OVA can be removed or depleted by Material I and III in aqueous soltuion, but the efficient adsorbents for Lyz were SBA–15 and Material II, no matter in aqueous or in buffer solution (pH of 7.0). Fig. 7a demonstrate that the removal efficiencies of Material I (lane 2) and III (lane 4) for OVA from the real egg white sample were up to approximately 73% and 70% (calculations were performed using Image Processing and Analysis in Java software), respectively. Fig. 7a and 7b show that the color of the Lyz bands faded or disappeared after the egg white sample was treated with Material II (lane 3) or SBA–15 (lane 5) in aqueous and B–R buffer (pH 19
of 7.0) solution, respectively. At the same time, it can be clearly observed that the band of Lyz treated with Material II (lane 3) and SBA–15 (lane 5) faded more significant in B–R buffer solution (pH of 7.0) than in aqueous soltuion. Image Processing and Analysis in Java technique calculated that approximately 90% and 92% of Lyz were removed by Material II and SBA–15 from egg white sample in B–R buffer solution (pH of 7.0), respectively. Furthermore, the mixture of Material I and SBA–15 was selected to remove or deplete OVA and Lyz simulaneously from the egg white sample in aqueous and B–R buffer (pH of 7.0) soltuion, respectively. Fig. 7a (lane 6) shows that approximately 99% of OVA and 90% of Lyz can be effectively removed from egg white sample in aqueous solution. Meanwhile, we also observed that the aforementioned HAP ovotransferrin can be adsorbed from egg white sample in aqueous by Material II (lane 3 in Fig. 7a) and SBA–15 (lane 5 in Fig. 7a), respectively. This can be attributed to that ovotransferrin (pI of 6.0) is partially positively charged in aqueous solution (The pH value of ultrapure water is 6.0–6.5 in our laboratory), so it can be adsorbed by negatively charged Material II and SBA–15 in ultrapure water (see section 3.1, zeta potential). In any case, all above results revealed that we can select or combine suitable adsorbents from the above materials to remove or deplete a kind of HAP or all the HAP from egg white sample.
Fig. 8 exhibits the results of SDS–PAGE analysis for the removal or depletion of the HAP from serum samples diluted with ultrapure water (Fig. 8a) and B–R buffer solutions (Fig. 8b), respectively. It can be seen that the gel electrophoregram of the 20
original serum sample (lane 1 in Fig. 8a and 8b) showed the band of 66 kDa, 45.0– 66.2 kDa and 25–35 kDa corresponding to highly abundant human serum albumin (HSA, pI of 5.0), and heavy and light chain of immunoglobulin G (IgG, pI of approximately 5.0), respectively [39–42]. Based on the above researches (Fig. 3), all of the original and functionalized SBA–15 could be the promising adsorbents for treatment of highly abundant HSA from human serum sample in aqueous soltuion. Indeed, Fig. 8a displays that the desired result was obtained by Material III (lane 4 in Fig. 8a, approximately 80% of HAS was removed). However, Fig. 8a also shows that other materials cannot remove HSA from human serum sample in aqueous solution. It may be due to the less amount of the grafted polymer of the materials and the highly complexity of the serum sample. As for the highly abundant protein IgG, the most effecitive adsorbent still was Material III. Therefore, Material III can be selected while the HAP needed to be removed from serum sample.
Based on the above results, SBA–15 and Material II can remove or deplete HAP ovotransferrin and Lyz from the egg white samples, which depended on the prevailing electrostatic interaction. Material III depleted the HAP (OVA, HSA and IgG) from egg white or human serum samples in aqueous solution by a synergistic interaction. The experimental results also confirm that a rational design for improving the signal of chromatogram or gel electrophoregram can be conducted based on the above researches. Certainly, these materials may also adsorb other LAP, as this is a common problem in all similar techniques. When the proposed techniques were compared with 21
the affinity system, different mechanisms were involved in the adsorption of proteins. Thus, they have different advantages and limitations. For example, affinity systems are process specific in capturing proteins. However, some of the bound HAP may also bind with many species, such as some peptide hormones, cytokines, and chemokines, and cause nonspecific removal of some other proteins [25]. Our experimental results showed that the proposed materials had selective adsorption of some proteins and can be used to treat real biological samples. This treatment process will reduce the complexity of electrophoregram or chromatograms to some extent. In addition, this approach can also be combined with affinity systems or other techniques to compensate for loss of information in real biological samples during the removal of HAP.
4. Conclusion
In this study, a series of pH–sensitive grafted SBA–15 materials were successfully synthesized via ATRP and used as adsorbents for the selective removal or depletion of target proteins. The design concept was confirmed by the adsorption results of the materials for standard proteins in B–R buffer solutions at different pH values. The removal or depletion of target proteins from the standard protein mixture was also achieved using this approach. The results showed that the removal or depletion of certain proteins and a reduction in the complexity of real biological samples prior to the separation steps were validated. However, although being
22
validated for targeted study, this approach is still inadequate for untargeted proteomic analysis and more research is required to extend the application of the proposed process.
Acknowledgements
The authors thank the National Natural Science Foundation of China (NSFC) Fund (No. 21105039 and No. 21375052) for supporting the project.
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Figure captions
Fig. 1. Synthesis protocol of pH–sensitive polymer brush–grafted mesoporous silica.
Fig. 2. Zeta potential of the original and diverse functional SBA–15 as a function of pH. The zeta potential was measured in B–R buffer solution.
Fig. 3. Effect of time on adsorption of the original and functionalized SBA–15 (Material I, II, and III) for BSA, Trf, Mb, Lyz, and OVA in aqueous solution, respectively. The standard protein solution: BSA (1.0 × 10–6 mol L–1), Trf (1.0 × 10–6 mol L–1), Mb (1.0× 10–6 mol L–1), Lyz (6.9 × 10–6 mol L–1), and OVA (6.9 × 10–6 mol L–1). Each point represented the mean value of three measurements.
Fig. 4. Effect of pH on adsorption of the original and functionalized SBA–15 (Material I, II, and III) for BSA, Trf, Mb, Lyz, and OVA in B–R buffer solution, respectively. The concentrations of standard proteins were the same as Fig. 4. Each measuring point represented the mean value of three specimens.
Fig. 5. The chromatograms of the aqueous solution of protein mixture before (a) and after (b) treatment with Material III for 10 min. The protein mixtures A, B included 32
four standard proteins (A: BSA, Mb, Lyz, and OVA; B: Trf, Mb, Lyz, and OVA). The protein mixture C contained above five standard proteins (BSA, Trf, Mb, Lyz, and OVA). The concentrations of standard proteins are as follows: BSA (1.0 × 10–6 mol L–1), Trf (1.0 × 10–6 mol L–1), Mb (1.0× 10–6 mol L–1), Lyz (6.9 × 10–6 mol L–1) and OVA (6.9 × 10–6 mol L–1). Chromatographic conditions were described in Electronic Supplementary Information.
Fig. 6. Chromatograms of protein mixtures before (a) and after (b) treated with SBA– 15. The mixture protein solution: BSA (1.0 × 10–6 mol L–1), Trf (1.0 × 10–6 mol L–1), Mb (1.0× 10–6 mol L–1), Lyz (6.9 × 10–6 mol L–1) and OVA (6.9 × 10–6 mol L–1). Chromatographic conditions were described in Electronic Supplementary Information.
Fig. 7. SDS–PAGE analysis of egg white diluted 100 times with ultrapure water (a) and pH of 7.0 B–R buffer solution (b) before (lane 1) and after treatment with the Material I (lane 2), Material II (lane 3), Material III (lane 4), SBA–15 (lane 5), and the mixture of SBA–15 and Material I (lane 6). The processes of sample treatment and electrophoresis were performed as outlined in Section 2.2 and Section 2.6. Loading amount of sample: 20 μL.
33
Fig. 8. SDS–PAGE analysis of human serum sample diluted 50 times with ultrapure water (a) and pH of 7.0 B–R buffer solution (b) before (lane 1) and after treatment with the Material I (lane 2), Material II (lane 3), Material III (lane 4), and SBA–15 (lane 5). The processes of sample treatment and electrophoresis were performed as outlined in Section 2.5 and Electronic Supplementary Information. Loading amount of sample: 20 μL.
Table 1. Element analysis (N, C and H) of the original and functionalized SBA-15
Materials
N (%)
C (%)
H (%)
SBA-15
0.00
0.93
1.02
SBA-15-NH2
2.72
8.37
2.13
SBA-15-NH-Br
2.52
11.62
2.42
Material I
2.86
16.01
3.01
Material II
2.50
12.81
2.61
Material III
2.70
18.17
3.21
34
Table 2. Physicochemical properties of original and functional SBA-15
Sample
SBET (m2/g)a
SBJH (m2/g)b
VBJH (cm3/g)c
DBJHd (nm)
Adsorption
Desorption
Adsorption
Desorption
Adsorption
Desorption
SBA-15
379
282
394
0.49
0.55
6.90
5.58
Material I
54
74
74
0.09
0.09
5.02
5.03
Material II
42
56
56
0.07
0.07
4.90
4.94
Material III
25
36
37
0.05
0.05
5.24
5.23
a
BET surface area.
b
BJH adsorption and desorption cumulative surface area of pores.
c
BJH adsorption and desorption cumulative volume of pores.
d
BJH adsorption and desorption average pore width (4V/A).
35
A series of pH sensitive groups grafted SBA-15 was synthesized via ATRP.
The proposed materials had selective adsorption for some proteins at a certain pH.
The selective removal of the target protein from protein mixtures can be achieved.
The complexity of real biological samples can be reduced by the proposed process.
36
37
38
39
40
41
42
43
44
45
46
47
48