Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Adenosine Phosphate Functionalized Magnetic Mesoporous Graphene Oxide Nanocomposite for Highly Selective Enrichment of Phosphopeptides Jie Su,† Xiwen He,† Langxing Chen,*,†,‡ and Yukui Zhang†,§ †
College of Chemistry, Tianjin Key Laboratory of Biosensing and Molecular Recognition, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China ‡ Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, China § Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, P. R. China S Supporting Information *
ABSTRACT: Developing an efficient strategy to enrich the low abundance phosphopeptides before mass spectrometry detection is a vital preprocessing step in phosphoproteomics. In this work, we synthesized an adenosine phosphate-Ti4+ functionalized magnetic mesoporous graphene oxide nanocomposite (denoted as MG@ mSiO2-ATP-Ti4+) to selectively extract phosphorylated peptides from complex biological samples based on the immobilized metal ion affinity chromatography (IMAC). Mesoporous silica was coated on the substrate material of magnetic graphene oxide and then the ATP containing three phosphate groups was grafted on the inwall of mesoporous channels as chelating ligands to immobilize the Ti4+ cations. With favorable properties, such as large surface area and good hydrophilicity and size-exclusion effect, the MG@mSiO2-ATPTi4+ exhibited excellent sensitivity and selectivity toward phosphopeptides whether in low concentration of β-casein digest (20 amol μL−1, 4 fmol) or the digest mixture of β-casein and bovine serum albumin (with molar ratio of 1:1000) as well as good reusability. Furthermore, MG@mSiO2-ATP-Ti4+ could also be applied in the selective enrichment of phosphorylated peptides from nonfat milk digest and human saliva and serum. KEYWORDS: Magnetic graphene oxide, Mesoporous silica, Adenosine triphosphate, Titanium ion, Phosphopeptide enrichment
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INTRODUCTION Dynamic and reversible protein phosphorylation plays crucial roles in biosystems, such as signal transduction, cell growth and apoptosis, and enzymatic activity.1−3 Research on phosphorylated proteins/peptides contributes to understanding the biological system and finding the latent biomarkers of diseases. However, at present, the conventional mass spectrometry (MS) method in phosphoproteomics still faces challenges because of the low content of phosphopeptides in samples and the signal suppression from high abundance nonphosphopeptides.4 Hence it is imperative to enrich phosphopeptides selectively before MS detection.5−7 To date, numerous strategies based on different mechanisms have been developed to selectively capture phosphorylated peptides, such as immunoaffinity,8,9 chemical modification,10 metal oxide affinity chromatography (MOAC),11−17 and immobilized metal ion affinity chromatography (IMAC).18−26 Among them, IMAC was used widely to design the extraction materials toward phosphopeptides due to the simple preparation process and the easily available metal cations and chelating ligands. Unfortunately, the nonspecific adsorption to peptides containing acidic amino acid residues is © XXXX American Chemical Society
the main drawback of this method. So, lots of efforts have been spent to enhance the performance of IMAC materials. Research has indicated that Ti4+-IMAC and Zr4+-IMAC materials exhibit higher selectivity toward phosphopeptides than other metal cations (e.g., Cu2+, Zn2+, Fe3+, Ga3+, Al3+, etc.) due to specific coordination of metal(IV) cations toward phosphate.27 In the choice of chelating ligand, adenosine triphosphate (ATP) is an excellent candidate28 owing to superiorly strong and active metal phosphonate sites offered by its three phosphate groups. Compared with the conventional phosphate-ligands containing only one phosphate group,22 ATP not only can reduce the loss of the immobilized metal cations but also increase the utilization rate of ligands to immobilize metal cations.28,29 Besides, the good hydrophilicity offered by the pentose sugar and purine base of ATP might contribute to reducing the nonspecific adsorption in extraction of phosphorylated peptides from complicated samples.30 Received: October 6, 2017 Revised: November 23, 2017 Published: December 11, 2017 A
DOI: 10.1021/acssuschemeng.7b03607 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
external magnetic force and washed by deionized water and ethanol successively. Then 100 mg of the obtained MGNs and 500 mg CTAB were dispersed in 450 mL H2O for ultrasonication for 30 min. Then, 10 mmol L−1 NaOH (50 mL) was added into the mixture. Afterward, 5 mL of TEOS/ethanol (v/v = 1:4) was added dropwise after the mixed solution was stirred for 30 min at 60 °C, and subsequently, the solution was continuously heated at 60 °C overnight. After being washed twice with ethanol, the obtained solids were redispersed in 60 mL of ethanol containing ammonium nitrate (6 g L−1) for refluxing to remove the CTAB. The resulting MG@mSiO2 was isolated by a magnet, washed with deionized water and ethanol, respectively, and dried at 50 °C under vacuum overnight for the future use. Preparation of MG@mSiO2-ATP-Ti4+ Nanocomposites. First, 2.5 g ATP was added in 50 mL of Na2CO3 (0.5 mol L−1, pH > 8). Afterward, the solution was stirred in an ice bath for 10 min before 0.8 mL of GLYMO was added into the solution drop by drop. After stirring for 30 min, the solution reacted at 65 °C under mechanical agitation for 12 h. The pH of the obtained GLYMO-ATP solution was tuned to 6 with concentrated HCl after naturally cooling. Second, 50 mg MG@mSiO2 was dispersed in the GLYMO-ATP solution, and then, the suspension was stirred at 95 °C for 2 h. After being separated by a magnet and washed, the obtained nanocomposites (denoted as MG@mSiO2-ATP) were incubated for 2 h in 40 mL of Ti(SO4)2 solution (100 mmol L−1 containing 0.1% (v/v) FA) at room temperature to immobilize titanium(IV) ions. Then, the prepared MG@mSiO2-ATP-Ti4+ nanocomposites were washed by 0.1% (v/v) FA several times for future use. Sample Preparation. The preparation procedure of tryptic digest of β-casein and BSA was followed from previous works.28,46 In brief, β-casein (1 mg) was dissolved in NH4HCO3 buffer (50 mmol L−1, 1 mL). Trypsin digest was carried out at a weight ratio of 50/1 (protein/enzyme) at 37 °C for 18 h. BSA (1 mg) was first denatured for 15 min in NH4HCO3 buffer (50 mmol L−1, 0.1 mL) containing urea (8 mol L−1) at 56 °C. Afterward, DTT (100 mmol L−1, 200 μL) was added in the mixture which then was incubated for 1 h at 56 °C. After adding IAA (200 mmol L−1, 200 μL), the mixture was incubated for 30 min in darkness at 37 °C. Finally, the treated BSA solution was diluted to 1 mL with 50 mmol L−1 of NH4HCO3 buffer and digested with the protein/enzyme ratio of 50:1 (w/w) for 18 h at 37 °C. Following the method in previous work,47 50 μL of nonfat milk was diluted 10-fold with NH4HCO3 (25 mmol L−1) and then denatured for 20 min with urea (8 mol L−1, 100 μL) at 56 °C. Afterward, the mixture was reduced with DTT (100 mmol L−1, 20 μL) at 56 °C for 1 h and alkylated with IAA (200 mmol L−1, 20 μL) for 30 min in darkness at 37 °C. At last, the obtained solution was diluted to 1 mL with 25 mmol L−1 NH4HCO3 and digested with trypsin (40 μg) for 16 h at 37 °C. Human serum was diluted to 10% by deionized water. Human saliva was collected from a healthy volunteer according to previous work.23 All prepared samples were stored at −20 °C before use. Enrichment of Phosphopeptides. The digest of standard proteins was first diluted to a certain concentration by loading buffer (50% ACN, 1% TFA). A 100 μg portion of MG@mSiO2-ATP-Ti4+ nanocomposites were incubated in 200 μL of the digest with vibration of 30 min. After that, the nanocomposites were separated by a magnet from supernatant and washed with above buffer for three times to remove nonphosphopeptides. The captured phosphopeptides were eluted by 80 μL of 10% ammonia aqueous solution for 25 min. Finally, the eluent was analyzed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). As a control, the enrichment performance of commercial TiO2 beads was investigated in digest mixture of BSA and β-casein (molar ratio of 100:1). The enrichment procedure was the same as those used for MG@mSiO2-ATP-Ti4+, except that TiO2 beads with trapped peptides were collected by centrifugation. The enrichment of the phosphopeptides from nonfat milk digest and human serum and saliva by MG@mSiO2-ATP-Ti4+ was performed on the same procedure.
Due to the uniform mesoporous channels, narrow pore size distribution, and large surface area,31,32 mesoporous silica nanomaterials have been widely used in extraction of small-size targets, such as peptides,33−37 and glycans,38,39 where it can capture the target analytes selectively instead of the large molecular weight interfering substances by the size-exclusion effect. Meanwhile, the affinity composites combined magnetic materials with mesoporous structure have attracted immense interest because of the excellent magnetic response.40−44 In this work, a novel strategy for synthesis of an ATP-Ti4+ functionalized magnetic mesoporous graphene oxides nanocomposite (MG@mSiO2-ATP-Ti4+) was developed. The MG@mSiO2-ATP-Ti4+ nanocomposite exhibited several attractive advantages. First, the graphene oxide and mesoporous silica offer large surface area and the size-exclusion effect of the mesoporous structure also enhances the selectivity to the small-size target phosphopeptides. Second, the good magnetic response simplifies the enrichment process. Third, the introduction of ATP ligands offers excellent hydrophilicity, biocompatibility, and more active metal phosphonate sites toward phosphopeptides. With the unique advantages above, the obtained nanocomposite exhibited great enrichment capacity to phosphorylated peptides in the presence of interference proteins and nonphosphopeptides. Furthermore, MG@mSiO2-ATP-Ti4+ was successfully used to extract phosphorylated peptides from nonfat milk digest and human saliva and serum.
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EXPERIMENTAL SECTION
Chemicals. Graphene oxide (GO) was obtained from XFNAO Materials Tech Co. Ltd. (Nanjing, China). Tetraethyl orthosilicate (TEOS), 3-glycidoxypropyldimethoxymethylsilane (GLYMO), cetyltrimethylammonium bromide (CTAB), trifluoroacetic acid (TFA), Ti(SO4)2, dithiothreitol (DTT), iodoacetamide (IAA), and 2,5dihydroxybenzoic acid (DHB) of analytical grade were obtained from J&K (Beijing, China). HPLC grade acetonitrile (ACN) was obtained from Concord Technology Co. Ltd. (Tianjin, China). Adenosine-5′triphosphate disodium salt (ATP-Na2) and TiO2 beads (P25, 20 nm) were obtained from Macklin (Shanghai, China). Trypsin, bovine serum albumin (BSA), and β-casein were all obtained from SigmaAldrich (U.S.A). Nonfat milk was bought from a local supermarket. The healthy human serum was offered by Tianjin First Center Hospital. Deionized water was prepared using Milli-Q system (Millipore, Bedford, MA). All the other analytical grade chemicals were obtained from Tianjin Chemical Reagent Company (Tianjin, China). Characterization and Measurements. The morphology and structure of the series of products were investigated by transmission electron microscopy (TEM) on a JEM-2100 electron microscope (JEOL, Japan). Fourier transform infrared (FTIR) spectrum was collected on a TENSOR 37 (BRUKER, Germany) with KBr pellets. The crystalline structure of the resulting composites was characterized by a D/max-2500 X-ray diffractometer (RIGAKU, Japan) with Cu Kα radiation. The magnetism was measured with vibrating sample magnetometer (Quantum Design, SQUID VSM, U.S.A.). The X-ray photoelectron spectroscopy was performed with an ESCALAB 250Xi spectrometer. Nitrogen adsorption and desorption isotherms were collected using the Micromeritics 3Flex (U.S.A). Before measurement, the sample was degassed in a vacuum for 8 h at 250 °C. Preparation of MG@mSiO2 Nanocomposites. The Fe3O4-GO nanocomposites (denoted as MGNs) was first synthesized according to the previous strategy.45 Graphene oxide (60 mg), FeCl3·6H2O (400 mg), and NaAc (1.3 g) were dissolved in ethylene glycol (30 mL) under sonication to obtain a homogeneous suspension. Then, the above suspension was poured into an autoclave and reacted at 180 °C for 10 h. The resulting black solids were separated by using B
DOI: 10.1021/acssuschemeng.7b03607 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering Scheme 1. Illustration of the Synthesis of MG@mSiO2-ATP-Ti4+ Nanocomposites
Reusability Evaluation of MG@mSiO2-ATP-Ti4+. Typically, the nanocomposites were incubated with β-casein digest (100 fmol μL−1) for 30 min. After being collected by the magnetic field, the captured phosphopeptides were eluted by using 10% ammonia aqueous solution. Without additional regeneration step, the affinity particles were used to enrich phosphopeptides again under the same conditions. MALDI-TOF MS Analysis. The enrichment performance of MG@ mSiO2-ATP-Ti4+ toward phosphopeptides was characterized by MALDI-TOF MS with an AutoflexIII LRF 200-CID mass spectrometer (Bruker Daltonics, Germany). A 1 μL portion of the eluent was mixed with 1 uL the DHB (25 mg mL−1, containing 70% ACN and 1% H3PO4) completely and dropped upon the plate and analyzed by MALDI-TOF MS.
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Figure 1. TEM images of the (a) Fe3O4-GO, (b) MG@mSiO2, (c) MG@mSiO2-ATP-Ti4+ nanocomposites.
experiment. The typical IV isotherm defined by IUPAC can be observed in Figure 2, which proved existence of mesoporous
RESULTS AND DISCUSSION
Preparation and Characterization of MG@mSiO2ATP-Ti4+ Nanocomposites. The synthetic procedure of MG@mSiO2-ATP-Ti4+ was shown in Scheme 1. At first, Fe3O4-GO nanocomposites were prepared by the solvothermal method. Afterward, the surface of the magnetic composites were covered with a layer of mesoporous silica via a sol−gel reaction by taking advantage of TEOS and a surfactant CTAB. Next, ATP reacted with a kind of silane coupling agent (GLYMO) to form GLYMO-ATP compounds. And then the obtained compounds were grafted onto the inwalls of mesoporous channels. Finally, the Ti4+ were immobilized on the ATP moiety of the MG@mSiO2-ATP nanocomposites. The morphology of MG@mSiO2-ATP-Ti4+ nanocomposites was characterized by TEM. In Figure 1a, a plenty of scattered Fe3O4 nanoparticles with a mean size of about 200 nm appeared on the surface of GO sheet, indicating that Fe3O4GO nanocomposite was successfully synthesized. After coated silica, the evident silica layer and order mesoporous channels were observed in the TEM image of MG@mSiO2 (Figure 1b). Furthermore, the mesoporous structure of MG@mSiO2-ATPTi4+ still remained after modification of GLYMO-ATP compounds and immobilization of Ti4+ cations (Figure 1c). The pore structure of MG@mSiO2-ATP-Ti4+ nanocomposites was further investigated by N2 adsorption−desorption
Figure 2. N2 adsorption−desorption isotherm of MG@mSiO2-ATPTi4+ nanocomposites. (inset) Pore size distribution.
structure. The sudden rise of relative pressure from 0.4 to 0.8 demonstrates the uniform pore-size distribution of MG@ mSiO2-ATP-Ti4+ nanocomposites. The Brunauer−Emmett− Teller (BET) surface area and total pore volume of MG@ mSiO2-ATP-Ti4+ nanocomposites were calculated as 269 m2/g and 0.36 cm3/g, respectively. Furthermore, the average pore size could be estimated about 4.14 nm (the inset of Figure 2), which can exclude the large-size proteins in enrichment of phosphopeptides.48,49 In comparison with the MG@mSiO2 (Figure S1, Supporting Information), the pore size of MG@ C
DOI: 10.1021/acssuschemeng.7b03607 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering mSiO2-ATP-Ti4+ decreased by 1.48 nm, which suggests GLYMO-ATP compounds were modified on the inwall of mesoporous channels successfully. The FTIR spectrum was also used to illustrate the preparation of MG@mSiO2-ATP-Ti4+ nanocomposites. As can be seen from curve a in Figure 3, the peak at 580 cm−1 was
Figure 4. Hysteresis loops of (a) Fe3O4-GO and (b) MG@mSiO2ATP-Ti4+ nanocomposites. (inset) Dispersion of MG@mSiO2-ATPTi4+ composites (left); separation of MG@mSiO2-ATP-Ti4+ under an external magnet within 30 s (right).
the results were displayed in Figure 5. When using ACN/H2O (50/50, v/v, 0.1% TFA) as loading buffer, although the three characteristic phosphopeptide peaks (at m/z 2061, 2556, 3122) released from β-casein were detected (Figure 5a), a few nonphosphopeptides peaks were also observed. When the TFA content in loading buffer solution rose to 1% (Figure 5b), the number and intensity of the peaks for captured phosphopeptides were enhanced. The MS signals of nonphosphopeptides were eliminated effectively. In the third loading buffer (Figure 5c), the content of TFA was 2%, but no better enrichment performance was observed. In 2013, Zhou et al. developed a monodisperse microsphere-based Ti4+-IMAC material used in phosphoproteome enrichment and a high level of TFA (6%) was used for the sample loading.22 To further investigated enrichment performance of MG@mSiO2-ATP-Ti4+ in strong acidic conditions, the content of TFA in loading buffer was further increased to 5%. On the contrary, the MS intensity of phosphopeptides has decreased significantly (Figure 5d), which might be attributed to reduction of stability of the nanocomposite in highly acidic condition. According to above results, ACN/H2O (50/50, v/v, 1% TFA) was adopted as the optimal loading buffer in the following enrichment experiment. Application of MG@mSiO2-ATP-Ti4+ Nanocomposites in Phosphopeptides Enrichment from Standard Protein Digests. With the appropriate loading buffer, MG@mSiO2ATP-Ti4+ could extract phosphorylated peptides specifically from the digests of β-casein. Afterward, we evaluated the detection limit of MG@mSiO2-ATP-Ti4+ utilizing a series of concentration of β-casein digests. When the β-casein digest was diluted to 10 fmol μL−1 (Figure 6a), without any enrichment, only the nonphosphopeptides appeared in the spectrum. After treating with MG@mSiO2-ATP-Ti4+, six peaks of phosphorylated peptides were observed with clear background in Figure 6b. Furthermore, the MG@mSiO2-ATP-Ti4+ also showed high sensitivity to the low concentration of phosphopeptides. From Figure 6c, in the 50 amol μL−1 of βcasein digest, the synthesized nanomaterials could also extract phosphopeptides effectively. Even when the concentration of β-casein digest was decreased to 20 amol μL−1 (4 fmol), three phosphorylated peptides could still be detected (Figure 6d). These results indicated the MG@mSiO2-ATP-Ti4+ have high sensitivity to the low-abundance phosphopeptides, which could be ascribed to its large surface area and the strong affinity offered by the titanium ions immobilized on ATP moiety. In addition, the strong magnetic response of MG@mSiO2-ATP-
Figure 3. FTIR spectra of (a) Fe3O4-GO, (b) MG@mSiO2, (c) MG@mSiO2-ATP-Ti4+ nanocomposites.
attributed to the stretching vibration of Fe−O bond in Fe3O4, peaks at 1569 and 1643 cm−1 assigned to the stretching vibration of CC bond and the antisymmetrical vibration of COO− in GO. Besides, the peak of the O−H stretching was also observed at 3400 cm −1. After modification with mesoporous silica (curve b), the symmetric vibration and asymmetric stretching vibration of Si−O−Si were observed at 790 and 1084 cm−1. As shown from curve c, the new band at 1409 cm−1 proved the existence of CH2−N units and the new peak at 2940 cm−1 assigned to the CH2 stretching vibration, which indicated the successful modification of GLYMO-ATP on the surface of MG@mSiO2. Wide-angle X-ray diffraction pattern is shown in Figure S2 (Supporting Information). The six main characteristic diffraction peaks of (220), (311), (400), (422), (511), and (440) matched well with the typical cubic phase Fe3O4 (JCPDS 19-629) and remained unchanged after each step of modification (curve a−c). In addition, the elemental composition of MG@mSiO2-ATP-Ti4+ was also investigated by X-ray photoelectron spectrometry. As presented in Figure S3 (Supporting Information), the characteristic peaks of elements in MG@mSiO2-ATP-Ti4+ such as Fe 2p (710 eV), O 1s (532 eV), Ti 2p (458 eV), N 1s (400 eV), C 1s (284 eV), P 2p (143 eV), and Si 2p (103 eV) were all observed. Moreover, the weight percentage of the Ti4+ element was estimated at 6.9%. The results above further proved the successful preparation of MG@mSiO2-ATP-Ti4+. The magnetism of the obtained nanocomposites was evaluated by vibrating sample magnetometer. As shown in the magnetic hysteresis curves (Figure 4), compared with Fe3O4-GO, the magnetization saturation (Ms) values of MG@ mSiO2-ATP-Ti4+ reduced from 31.15 to 16.80 emu g−1 after multistep modification, but it still exhibited excellent magnetic response and can be separated from the suspension quickly (
