Dual-Functionalized Magnetic Metal–Organic Framework for Highly

Oct 17, 2017 - Herein, a novel dual-functionalized magnetic zirconium-based metal–organic framework (MOF) denoted as DFMMOF, with the purpose of com...
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Research Article pubs.acs.org/journal/ascecg

Dual-Functionalized Magnetic Metal−Organic Framework for Highly Specific Enrichment of Phosphopeptides Jianqiao Zhou,† Yulu Liang,† 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: The highly specific enrichment of phosphoproteins and phosphopeptides from intricate biological systems is the precondition of in-depth phosphoproteome research. Herein, a novel dual-functionalized magnetic zirconium-based metal−organic framework (MOF) denoted as DFMMOF, with the purpose of combining the affinity of immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) has been successfully synthesized. The inherent Zr−O cluster of DFMMOF particles acted as MOAC and the immobilized titanium(IV) ions served for IMAC. The obtained DFMMOF exhibited rapid magnetic separation (within 5 s), large surface area (237.9 m2 g−1), high binding capacity (100 mg g−1), and good postenrichment recovery (84.8%). Thanks to the strong affinity, low detection sensitivity (5 fmol) and high selectivity (β-casein/BSA with a molar ratio of 1:1000) for phosphopeptide enrichment were obtained using DFMMOF as absorbent. Moreover, the effective identification of phosphopeptides from real samples (human serum and nonfat milk) further confirmed the immense potential of DFMMOF as a promising candidate for the detection and extraction of trace amounts of phosphorylated peptides in complex biosamples. KEYWORDS: Phosphopeptide, Enrichment, Magnetic nanomaterials, Metal−organic framework, Immobilized metal ion affinity chromatography, Metal oxide affinity chromatography

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INTRODUCTION As one of the most prevalent and significant post-translational modifications,1 protein phosphorylation is a key regulator of a wide variety of biological processes involved in cell growth,2 metabolism,3 and some diseases like Alzheimer.4 As research continues, mass spectrometry (MS) has been recognized as a high-efficiency facility for phosphorylation determination based on its high sensitivity and resolution.5−7 Nevertheless, low ionization efficiency, incomplete phosphorylation at individual sites, and severe interference caused by nonphosphopeptides make the direct detection of phosphopeptides still a difficulty, especially for complex biological samples.8 Thus, the specific enrichment for low abundance phosphopeptides before MS analysis has become a prerequisite. To meet this demand, numerous materials and approaches including chemical derivatization,9,10 immunoprecipitation,11 ion exchange chromatography,12,13 inorganic salt affinity chromatography,14,15 immobilized metal affinity chromatography (IMAC),16−18 and metal oxide affinity chromatography (MOAC)19−22 have been developed. Among them, IMAC and MOAC have been regarded as the most frequently used © 2017 American Chemical Society

strategies. The enrichment mechanism of IMAC is the affinity between positively charged immobilized metal ions and negatively charged phosphate groups of phosphorylated proteins/peptides. As for MOAC, phosphorylated proteins/ peptides are trapped by reversible Lewis acid−base reaction provided by inherent metal oxide of the material. The strong affinity between metal centers and phosphate groups endows IMAC and MOAC based materials with a fast and efficient enrichment process. Despite many advantages of IMAC and MOAC, the overcoming of the loss of metal ions bounded on the surface of the matrix during the enrichment and washing procedure is still a great challenge. Besides, it has been reported that IMAC materials tend to capture multiply phosphorylated peptides while MOAC materials prefer monophosphorylated peptides.23,24 This may probably lead to the decrease of efficiency when used IMAC or MOAC alone. Thus, the joint Received: July 25, 2017 Revised: October 6, 2017 Published: October 17, 2017 11413

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ACS Sustainable Chemistry & Engineering Scheme 1. Schematic iIllustration of the Fabrication of DFMMOF

conditions and a characteristic structure containing many functional groups,40 the combination of PDA coating with magnetic materials has been applied to many research fields including phosphopeptide enrichment.41 Owing to the incredibly high surface area, abundant active metal sites, and convenience of modification, metal−organic frameworks (MOF) are emerging as a promising candidate for phosphoproteome analysis. Taking advantage of the interaction between the metal oxide cluster on the surface of MOFs and the phosphate groups of peptides, several MOFs such as FeMOF,42 Zr-MOF,43 and Er-MOF44 have been extensively employed in the enrichment of phosphopeptides. While other MOF materials suffering from a main weakness of low thermal and chemical stabilities, zirconium-based MOFs, along with their functionalized derivatives such as UiO-66-NO2 and UiO66-Br, stand out and draw much attention of the researchers. The high affinity between the zirconium atom and oxygen ligands results in unexceptional stable secondary building units (SBUs), leading to a strong resistance toward chemical treatments.45 This unprecedented stability endows Zr-based MOF with great potential in phosphoproteome research. Herein, a dual-functionalized magnetic metal−organic framework (DFMMOF) has been prepared via modifying UiO-66NH2 MOF onto PDA-coated magnetic spheres and subsequently immobilizing titanium(IV) ions on the surface of the obtained ATP grafted magnetic MOF (as shown in Scheme 1). The inherent Zr−O clusters of MOF play the role of MOAC, meanwhile the immobilized titanium(IV) ions on the surface of MOF act as IMAC, which endows the obtained material with high metal affinity toward both mono- or multi-phosphorylated peptides. Thus, this novel material combines the prominent advantages of rapid magnetic separation, large surface area, abundant functional sites of MOF, and highly specific enrichment capacity of both IMAC and MOAC. The high sensitivity, prominent selectivity, and rapid magnetic response exhibited during the enrichment procedure of different biosamples makes DFMMOF a promising candidate in the study of phosphoproteome.

use of IMAC and MOAC holds enormous potential for the phosphoproteome analysis. Massive effort has been devoted to the improvement of chelating ligand of IMAC materials since it determines the amount of the immobilized ions directly. Unfortunately, conventional chelators such as iminodiacetic acid (IDA),25 nitrilotriacetic acid (NTA),26,27 and polydopamine (PDA)28 are still suffering from the limitation of the quantity of the immobilized metal ions generally. In addition to this, the performance of IMAC materials is also constrained by nonspecific adsorption between nonphosphopeptides and hydrophobic residues of phosphate linkers via hydrophobic interaction. To overcome these adverse factors, great efforts have been devoted to the exploration of novel chelating ligands for IMAC materials. As the primary energy resource in a living organism, adenosine triphosphate (ATP) consists of three phosphate groups, which offers it a great advantage of immobilizing metal ions. Besides, purine base and pentose sugar groups of ATP may weaken the nonspecific adsorption by their great hydrophilicity.29,30 The unique superiority of ATP mentioned above makes it an excellent candidate for a novel chelator of IMAC materials. For traditional IMAC and MOAC materials, another factor that may cause a decrease in the efficiency of the enrichment process is the time-consuming separation procedure. Highspeed centrifugation is usually required during the enrichment and washing procedure, which leads to a troublesome loss of target peptides. The introduction of magnetic nanoparticles in phosphoproteome research greatly facilitates the separation of the trapped biomolecule from the sample solution, leading to a rapid and efficient enrichment procedure.31−35 However, most of the magnetic substrates suffer from relatively finite surface area as well as poor hydrophilicity, resulting in a low density of phosphonate ligands and a restricted ability in phosphopeptide enrichment. Consequently, core−shell magnetic nanomaterials with a magnetic core of good mangnetic responsiveness and a polymer shell providing plentiful functional sites and hydrophilic groups have been developed.36−39 One of the suitable polymers for magnetic core coating is polydopamine (PDA). With the easily spontaneous polymerization under mild 11414

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Preparation of Biosamples. β-Casein (1 mg) was added to the NH4HCO3 solution (1 mL, 100 mM) containing trypsin with a weight ratio of enzyme to protein at 1:50 and incubated at 37 °C for 18 h. BSA (1 mg) was first denatured in NH4HCO3 solution (100 μL, 50 mM) containing urea (8 M) at 56 °C for 15 min. After reducing by DTT (200 μL, 100 mM) at 56 °C for 1 h and alkylated using IAA (200 μL, 100 mM) at 37 °C for 30 min in the dark, the obtained solution was further diluted to 1 mL with NH4HCO3 and digested by trypsin with a weight ratio of enzyme to BSA at 1:50 at 37 °C for 16 h. Then 20 μL of human serum was first diluted by 120 μL of highly purified water and then denatured in boiling water. A total of 50 μL of nonfat milk was added in 1 mL of NH4HCO3 solution (25 mmol). The supernatant was separated by centrifugalization (16 000 rpm, 15 min), then denatured in boiled water, and finally digested with 50 μg of trypsin at 37 °C for 16 h. The obtained sample was stored at −20 °C for further use. Selective Enrichment of Phosphopeptide. Loading buffer of the ACN solution (50% with 2% TFA, v/v) and three washing buffers of (a) 50% (v/v) ACN solution containing 5% TFA and NaCl (200 mM), (b) 80% (v/v) ACN solution containing 5% TFA, and (c) 30% (v/v) ACN solution containing 0.1% TFA were prepared for the enrichment of phosphopeptides. A total of 20 μg of DFMMOF was first rinsed with the loading buffer and dispersed in the peptide mixture (200 μL). Then the mixture was incubated at room temperature for 30 min. The materials with captured peptides were collected by magnetic separation. After discarding the supernatant, 150 μL of three different washing buffers were used to wash the materials to reduce nonspecific binding. The target peptides were eluted with 5% ammonia aqueous solution (80 μL, w/w) by shaking for 15 min and sequentially analyzed by MALDI-TOF MS. Evaluation of Postenrichment Recovery. Two of the identical amounts of standard phosphopeptides (DSEQE(pS)DTLQK, m/z 1359.36) were first labeled with light or heavy isotopes utilizing the dimethyl labeling method.46 After that, the heavy isotope-labeled peptides were dissolved in loading buffer and enriched by DFMMOF. The resulting elute was blended with equal amounts of light isotopelabeled peptides and analyzed by MALDI-TOF MS. The recovery yield was determined by measuring the peak height ratio of heavy to light isotope-tagged standard phosphopeptides. MALDI-TOF MS Analysis. All MALDI-TOF MS experiments were performed on a Bruker Autoflex III MALDI-TOF mass spectrometer (Bruker Daltonics Bremen, Germany) in the positive-ion reflect mode with a smartbeam laser at 337 nm. The spectra were measured with an acceleration voltage of 19 kV. ACN-H2O−H3PO4 solution (70:29:1, v/v/v) containing DHB (25 mg) was used as a matrix solution. The eluting solution of phosphopeptides (0.5 μL) and the matrix solution (0.5 μL) were spotted on the MALDI plate for further analysis.

EXPERIMENTAL SECTION

Materials. Iron(III) chloride hexahydrate (FeCl3·6H2O), sodium acetate (NaAc), ethylene glycol (EG), N,N-dimethylformamide (DMF), acetonitrile (ACN), and formic acid (FA) were purchased from Tianjin Chemical Reagent Company (Tianjin, China). Dopamine hydrochloride, trihydroxy methyl aminomethane (Tris), zirconium chloride (ZrCl4), and 2-aminoterephthalic acid were purchased from Alfa Aesar (Shanghai, China). Titanium(IV) sulfate (Ti(SO4)2), ammonium bicarbonate (NH4HCO3), ammonium hydroxide (NH3·H2O), urea, and TiO2 beads (5 μm) were obtained from Macklin (Shanghai, China). Glutaraldehyde solution (50% in H2O, m/m) was from AMRESO (Solon, OH, U.S.A.). Dithiothreitol (DTT), iodoacetamide (IAA), trifluoroaceticacid (TFA), and 2,5dihydroxyl benzoic acid (DHB) were obtained from J&K Co., Ltd., (Beijing, China). Adenosine-5′-triphosphate disodium salt (ATP-Na2; ≥99.9%), trypsin (TPCK treated), β-casein, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Human serum was supplied by Tianjin First Center Hospital. Nonfat milk was bought from a local store. All aqueous solutions were prepared by Milli-Q water purification system (Millipore, Milford, MA). All other chemicals were at least of analytical grade and used without further purification. Characterization. Transmission electron microscopy (TEM) images were taken on a G2 F20 transmission electron microscope (FEI, Hillsboro, U.S.A.). Powder X-ray diffraction (XRD) patterns were evaluated on Rigaku D/max-2500 X-ray diffractometer with Cu Kα radiation. Fourier-transform infrared spectroscopy (FT-IR) was performed on a Nicolet AVATAR-360 Fourier transform infrared spectrometer (Nicolet, WI, U.S.A.) using KBr pellets. The X-ray photoelectron spectra (XPS) were performed by a Thermo Scientific ESCALAB 250Xi. A vibrating sample magnetometer (VSM) was used to analyzed the magnetic properties of the material (Quantum Design, San Diego, CA, U.S.A.). Nitrogen sorption isotherms were measured with a Micromeritcs Tristar 3000 analyzer (U.S.A.). Preparation of DFMMOF and Fe3O4@PDA@ATP-Ti4+. A total of 100 mg of Fe3O4 nanoparticles were first synthesized via hydrothermal reaction36 and then dispersed in Tris buffer (80 mL, 10 mM) containing dopamine hydrochloride (1 mg mL−1). After mechanical stirring for 12 h under ambient temperature, the resulting product (denoted as Fe3O4@PDA) was isolated magnetically and successively washed by highly purified water and ethanol. Next, 100 mg of Fe3O4@PDA was added to a DMF solution (80 mL) containing ZrCl4 (1 mmol) and 2-aminoterephthalic acid (1 mmol). After heating at 120 °C for 2 h, the resulting product was isolated magnetically and then redispersed in a fresh solution containing two MOF precursors in the concentrations mentioned above. After three assembly cycles, the product (denoted as Fe3O4@ PDA@UiO-66-NH2) was collected by magnetic separation, washed successively with DMF and ethanol, and dried under vacuum at 50 °C. The immobilization of titanium(IV) ions was according to the reported protocol but with some modifications.29 Briefly, amine groups on Fe3O4@PDA@UiO-66-NH2 (100 mg) were activated by glutaraldehyde solution (4 mL, 25%, w/w) in citrate buffer (100 mM, pH 5.0) under gentle vibration for 2 h. After being isolated magnetically and washed, the resulting product was dispersed in 4 mL of ATP solution (0.2 g mL−1, citrate buffer), gently vibrated for 2 h, and then washed thoroughly. Finally, the obtained product was added to 10 mL of Ti(SO4)2 (100 mM, 0.1% (v/v) FA) and vibrated for 2 h to immobilize titanium(IV) ions. The resulting product was isolated magnetically and washed by FA. Fe3O4@PDA@ATP-Ti4+ was prepared for comparison with DFMMOF. A total of 100 mg of the obtained Fe3O4@PDA was dispersed in 4 mL of ATP (0.2 g mL−1, 100 mM citrate buffer) and gently vibrated for 2 h. Then the resulting product was collected by magnetic separation and washed. After that 10 mL of Ti(SO4)2 (100 mM, 0.1% (v/v) FA) was added to immobilize titanium(IV) ions. The obtained Fe3O4@PDA@ATP-Ti4+ was gathered magnetically and washed by FA.

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RESULTS AND DISCUSSION The morphology and structure of MOF materials was investigated by transmission electronic microscopy (TEM). As shown in Figure 1a, the TEM image of Fe3O4@PDA nanoparticles exhibited clearly a core−shell structure containing a magnetic core and a layer of PDA. After coating with UiO-66NH2 on the surface of PDA, the MOF shell showed rougher morphology than PDA layer’s and could be distinguished from

Figure 1. TEM images of Fe3O4@PDA (a), Fe3O4@PDA@UiO-66NH2 (b), and DFMMOF (c). 11415

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ACS Sustainable Chemistry & Engineering the PDA shell because of its crystal structure (Figure 1b). The TEM image of DFMMOF (Figure 1c) also showed the typical core−shell structure and the MOF shell without much changes in morphology after the modification of ATP and the immobilization of titanium(IV) ions on the surface of Fe3O4@PDA@UiO-66-NH2. Furthermore, Fourier transform infrared (FT-IR) spectra were utilized to better understand the successful synthesis of PDA and MOF shell (Figure 2). The

Figure 3. XRD patterns of Fe3O4@PDA@UiO-66-NH2 (a) and DFMMOF (b).

materials are superparamagnetism. Despite the fact that after coating with the MOF layer and immobilization with the titanium(IV) ions the saturation magnetization (Ms) value of DFMMOF showed a slight decrease compared with the Ms value of Fe3O4@PDA, the DFMMOF could be isolated readily with the help of external magnet in only 5 s. High specific area is a significant property of MOFs, which involves the achievement of advanced enrichment performance for phosphopeptide. The N2 adsorption−desorption measurements were employed to evaluate the surface area of prepared materials (Figure S2). The N2 sorption isotherms showed typical IV type curves, and the as-synthesized material possessed a Brunauer−Emmett−Teller (BET) surface area of 237.9 cm2 g−1. On account of the presence of the Fe3O4 core, the BET surface area of DFMMOF is much lower than that for UiO-66-NH2. However, the value is satisfactory for enrichment purposes. The pore width was calculated to be 1.8 nm using the Barrett−Joyner−Halenda (BJH) method; thus, it could allow the phosphopeptides to enter in and meanwhile exclude proteins of high molecular weight. X-ray photoelectron spectroscopy (XPS) was employed for confirming the modification of phosphate groups from ATP and the immobilization of titanium(IV) ions. As shown in Figure S3, the C (284.8 eV), O (531.8 eV), N (399.7 eV), Fe (710.8 eV), Zr (182.9 eV), P (133.8 eV), and Ti (458.9 eV) element peaks were found, confirming the existence of phosphonate groups and titanium(IV) ions in DFMMOF. The atom percentages of zirconium and titanium are estimated to be 3.35% and 2.34%, respectively. For minimizing the interference of nonspecific binding and to achieve the optimal adsorption efficiency, the influence of different acidities of the loading buffer was tested. According to the results shown in Figure S4, a relative clear background and strong intensity of phosphopeptide peaks were obtained when the percentage of TFA was set as 2%. Thus, loading buffer containing 2% TFA was adopted for further enrichment process. To investigate the enrichment performance of DFMMOF, tryptic digests from β-casein were first incubated with the presence of DFMMOF in loading buffer. After isolating and washing procedure of the materials, the target phosphopeptides were eluted and analyzed. Figure 4a showed that the mass

Figure 2. FT-IR spectra of Fe3O4 (a), Fe3O4@PDA (b), Fe3O4@ PDA@UiO-66-NH2 (c), and DFMMOF (d).

absorption band at 590 cm−1 was ascribed to the Fe−O stretching vibration of Fe3O4. Compared with the FT-IR spectrum of Fe3O4, several new arising peaks were observed in the spectrum of Fe3O4@PDA. Peaks at 1574 and 1515 cm−1 originated from the aromatic CC stretching vibration, and peaks at 1342 and1291 cm−1 are assigned to the N−H bending vibration and C−O stretching vibration. After modifying with UiO-66-NH2, new characteristic adsorption peaks at 1566, 1430, and 1258 cm−1 corresponding to the carboxy group of the organic ligand in MOF showed a sharp increase. The strong adsorption band at 1045 cm−1 is attributed to the vibration of phosphate groups, implying the successful modification of ATP on the surface of UiO-66-NH2. Wide-angle X-ray diffraction (XRD) was used to investigate the crystalline structure and compositions of prepared materials. Both XRD patterns of Fe3O4@PDA@UiO-66-NH2 and DFMMOF presented intense diffraction peaks at 2θ = 30.1, 35.5, 43.2, 53.5, 57, and 62.8°, corresponding to the (220), (311), (400), (422), (511), and (440) planes of Fe3O4, respectively (Figure 3). Additionally, reflections at 2θ = 7.4 and 8.5° were typical of UiO-66-NH2,47 which confirmed the formation of crystalline UiO-66-NH2 MOF on the surface of Fe3O4 nanoparticles. Despite the fact that there might be a slight deconstruction of the framework caused by the interaction of zirconium centers with the decorated phosphonate groups of ATP, matching of the reflections at 2θ = 7.4 and 8.5° indicated the complete preservation of the MOF structure. The magnetic properties of Fe3O4, Fe3O4@PDA, and DFMMOF were characterized by a vibrating sample magnetometer (VSM). Figure S1 exhibits the magnetic hysteresis loop curves of these materials. As can be seen, there is no obvious remanence or coercivity, which indicates that all of the three 11416

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structure, along with the existence of Fe3O4 which decreases the loss of phosphopeptides efficiently. The high selectivity for phosphopeptide enrichment by DFMMOF was evaluated by a tryptic digests mixture of βcasein and BSA. As demonstrated in Figure 5, when the molar

Figure 5. MALDI-TOF mass spectra of a tryptic digests mixture of βcasein and BSA before (a) and after enrichment at molar ratios of 1:500 (b) and 1:1000 (c). The peaks of phosphopeptides are marked with asterisks, and the peaks of dephosphopeptides are marked with octothorpes.

Figure 4. MALDI-TOF mass spectra of tryptic digests of β-casein. Direct analysis (a) and after enrichment by DFMMOF with the amount of 2 pmol (b), 200 fmol (c), and 5 fmol (d). The peaks of phosphopeptides are marked with asterisks, and the peaks of dephosphopeptides are marked with octothorpes.

ratio of β-casein and BSA reached 1:500, MALDI mass spectra were dominated by nonphosphopeptides with high intensities before enrichment. However, after treatment with DFMMOF, two of the target phosphopeptides and the corresponding dephosphorylated peptide were well-distinguished. Even the molar ratio of BSA and β-casein was increased to 1000 times, and two expected phosphopeptides can still be identified. The result demonstrated that the prepared DFMMOF has a prominent selectivity for phosphopeptides enrichment, which could be mostly ascribed to the immobilization of a large amount of titanium(IV) ions acting as IMAC. The enrichment sensitivity and selectivity of DFMMOF toward β-casein digests is comparable to some novel IMAC or MOAC based affinity materials48,49 or even superior to some similar materials reported formerly on the basis of the joint use of IMAC and MOAC due to the simple one-step strategy and the rapid magnetic separation.50,51 The reproducibility of the enrichment procedure was assessed by performing three batches of enrichment processes under the same experiment and MALDI-TOF MS detecting conditions parallelly. As revealed in Figure S5, the resulting MS spectra showed no clear distinction of the enrichment potential between the three batches, indicating good batch to batch reproducibility.

spectrum of tryptic digests without enrichment was occupied by abundant nonphosphopeptides. The signal of phosphopeptides was suppressed severely due to its low-concentration in comparison to the nonphosphopeptides. Nevertheless, after enrichment by DFMMOF, three expected phosphopeptides with strong signal intensities showed up, along with their dephosphorylated peptides (Figure 4b). Detailed information on phosphopeptides trapped by DFMMOF from β-casein tryptic digestion is displayed in Table S1. Phosphopeptides are at low abundance as the composition existed in a real biological sample, which makes the sensitivity of phosphopeptide enrichment a crucial criterion to assess the performance of adsorbent on the phosphopeptide enrichment. Consequently, lower concentrations of β-casein tryptic digests were further enriched by DFMMOF. As revealed in Figure 4c, two phosphopeptides were clearly identified along with their dephosphorylated peptides. Even when the total amount of βcasein digests was decreased to an extremely low level of 5 fmol, the signal of one phosphopeptide could still be identified, which suggested that the as-synthesized DFMMOF has high enrichment sensitivity for phosphopeptides (Figure 4d). The low detection limit could be attributed to abundant Zr−O clusters and immobilized titanium(IV) ions of the framework 11417

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ACS Sustainable Chemistry & Engineering To further verify the robustness and reusability of DFMMOF, the same batch of material was recycled and reactivated by loading buffer before each enrichment procedure. As indicated in Figure S6, there were no notable differences between the results from three enrichment procedures despite a slight decline of the signal intensity due to the alkaline erosion of the material during the process of sample eluting. The result confirms the exceptional stability of Zr-based MOF toward chemical treatment as well as excellent reusability of DFMMOF for phosphopeptides enrichment. The adsorption capacity of DFMMOF was studied by incubating 1 μg of β-casein tryptic digests with a series of different dosages (1−50 μg) of the material, respectively. As shown in Figure S7, when the signal intensity of β1 (m/z = 2061.73) trapped by DFMMOF reached a maximum, the total amount of phosphopeptides in loading buffer was bound onto the material. The calculated adsorption capacity of DFMMOF was about 100 mg g−1, which is satisfactory for enrichment purposes owing to the large surface area of DFMMOF. The postenrichment recovery of phosphopeptides was evaluated by introducing a dimethyl labeling method. Briefly, two samples containing the same amount of standard phosphopeptide (DSEQE(pS)DTLQK) were treated with formaldehyde or deuterium formaldehyde, respectively. The first sample resulted in a 56 Da mass increase due to the introduction of four CH3 (two at the side chain of lysine and two at the N-terminal of the peptide). Similarly, the second sample produced a 64 Da mass increase with four hydrogen atoms of the peptide replaced by four CHD2. Then the second sample was applied in the above-mentioned trap and release process. The elute was fully blended with the first sample and then analyzed. As revealed in Figure 6, the postenrichment

Figure 7. MALDI-TOF mass spectra of human serum before (a) and after (b) enrichment by DFMMOF. The peaks of phosphopeptides are marked with asterisks and the peaks of dephosphopeptides are marked with octothorpes.

before enrichment. In contrast, four phosphopeptides and one dephosphopeptides with high peak intensity could be clearly identified after the treatment with DFMMOF. Detailed information is listed in Table S2. The outcome indicated a satisfactory selectivity toward phosphopeptide enrichment from complex biological samples as well as a great potential for further phosphoproteomics research using the novel DFMMOF material. To further confirm the practicability of DFMMOF in real samples and meanwhile demonstrate the advantage of combining IMAC and MOAC, tryptic digests of nonfat milk were used in the enrichment experiment as it had a large number of phosphopeptides with different phosphorylations. Fe3O4@PDA@ATP-Ti4+ (single IMAC method) and Fe3O4@ PDA@UiO-66-NH2 (single MOAC method) were synthesized as contrast. The enrichment ability of DFMMOF, Fe3O4@ PDA@ATP-Ti4+, and Fe3O4@PDA@UiO-66-NH2 toward phosphopeptides was investigated by three parallel enrichment processes. As revealed in Figure 8a, MS spectra of the digested nonfat milk without further treatment demonstrated that no phosphopeptide peak was detected owing to the severe interference by abundant nonphosphopeptides. After enrichment, Fe3O4@PDA@Ti4+ showed preference for multiphosphopeptides (Figure 8b), while Fe3O4@PDA@UiO-66-NH2 tended to capture more monophosphopeptides according the numbers and intensities of the peaks (Figure 8c). Nevertheless, both of them enriched fewer phosphopeptides than DFMMOF (10 monophosphopeptides and 5 multiphosphopeptides), indicating that the joint use of IMAC and MOAC endowed the designed DFMMOF with large improvement of the enrichment ability toward phosphopeptides. Detailed information is displayed in Table S3.

Figure 6. MALDI-TOF mass spectra of standard phosphopeptide DSEQE(pS)DTLQK (a mixture of unenriched fraction labeled by four CH3 and an equal amount of the enriched fraction labeled by four CHD2).

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recovery of DFMMOF toward phosphopeptides was calculated to be as high as 84.8%, indicating that DFMMOF could act as an effective adsorbent for phosphopeptides enrichment. Encouraged by the results of the enrichment experiments mentioned above, further research was introduced to investigate the practicability of DFMMOF on a complex biological sample using human serum. Figure 7a showed that only one peak of phosphopeptide could be observed along with abundant peaks of nonphosphopeptides dominating the spectra

CONCLUSIONS In summary, a novel dual-functionalized magnetic metal− organic framework has been successfully synthesized by introducing efficient and robust immobilization of titanium(IV) ions on UiO-66-NH2 shell coated Fe3O4@PDA nanoparticles for highly sensitive and selective enrichment of low-abundance phosphopeptides from complex samples. The UiO-66-NH2 MOF shell endows the nanomaterial with not only the 11418

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mass spectra of tryptic digests from β-casein after enrichment by DFMMOF using series loading buffer containing different concentrations of TFA; the reproducibility of enrichment procedure; reusability test of DFMMOF; binding capacity test and detailed information on the phosphopeptides captured by DFMMOF. (PDF)

AUTHOR INFORMATION

Corresponding Author

*Fax: (+86) 22-2350-2458. E-mail: [email protected]. ORCID

Langxing Chen: 0000-0002-8616-9207 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the National Natural Science Foundation of China (No. 21475067) and the Natural Science Foundation of Tianjin (No. 15JCYBJC20600).

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Figure 8. MALDI-TOF mass spectra of nonfat milk before (a) and after enrichment by (b) Fe3O4@PDA@Ti4+, (c) Fe3O4@PDA@UiO66-NH2, and (d) DFMMOF. The peaks of phosphopeptides are marked with asterisks, and the peaks of dephosphopeptides are marked with octothorpes.

improvement of hydrophilicity and biological compatibility but also abundant Zr−O clusters and functional sites to anchor chelating ligands for immobilizing a mass of titanium(IV) ions, resulting in an alliance for MOAC and IMAC to capture both mono- and multiple-phosphopeptides simultaneously. Additionally, DFMMOF possessed good mangnetic responsiveness, a higher recovery, and a large binding capacity, exhibiting great availability in the identification of low-abundance phosphopeptides from complex biosamples like human serum and nonfat milk. Further applications of this novel material are believed to provide a new approach to the efficient enrichment of phosphopeptides and a more comprehensive understanding of phosphoproteomics in modern life science.

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REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b02521. Several figures and tables are provided for further exhibition of the experiment data. Magnetic hysteresis curves of Fe3O4 (a), Fe3O4@PDA (b), and DFMMOF (c); the nitrogen adsorption−desorption isotherms of DFMMOF; XPS patterns of DFMMOF; MALDI-TOF 11419

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