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Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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Magnetic Binary Metal−Organic Framework As a Novel Affinity Probe for Highly Selective Capture of Endogenous Phosphopeptides Qianjing Liu,† Nianrong Sun,*,† Mingxia Gao,† and Chun-hui Deng*,‡ †

Department of Chemistry, Fudan University, 220 Handan Road, Yangpu District, Shanghai 200433, China Department of Chemistry, The Fifth People’s Hospital of Shanghai, Institutes of Biomedical Sciences, Collaborative Innovation Center of Genetics and Development, Fudan University, 220 Handan Road, Yangpu District, Shanghai 200433, China

‡

S Supporting Information *

ABSTRACT: Highly efficient detection of endogenous phosphopeptides from complex biosamples is essential in phosphopeptidomics analysis due to the severe disturbance caused by the chaotic biological environment. In this study, for highly selective capture of endogenous phosphopeptides, a magnetic binary metal−organic framework (MOF) with Zr−O and Ti−O centers (denoted as Fe3O4@PDA@Zr-Ti-MOF) was designed and synthesized by a facile postsynthetic method. Briefly, Zr-based MOF was first coated on the surface of magnetic Fe3O4 with polydopamine (PDA) as a linker, and then, the as-prepared Fe3O4@PDA@Zr-MOF was exposed to DMF solution containing TiCl4(THF)2, resulting in the successful synthesis of Fe3O4@PDA@Zr-Ti-MOF. This newly prepared Fe3O4@PDA@Zr-Ti-MOF owned the merits of large specific surface area, unique porous structure, and superparamagnetism as well as the enhanced dual affinities of Zr−O and Ti−O centers toward both endogenous mono-phospho-peptides and multiphospho-peptides, showing highly improved performance with better selectivity and sensitivity compared to single-metal centered MOFs (Fe3O4@PDA@Zr-MOF, Fe3O4@PDA@Ti-MOF). The Fe3O4@PDA@Zr-Ti-MOF was also successfully applied to extract endogenous phosphopeptides in biological sample of human saliva. As a result, 34 mono-phosphorylated peptides and 10 multi-phosphorylated peptides were detected from merely 1 μL of pristine human saliva, confirming its bright prospects in phosphopeptidomics analysis. KEYWORDS: Endogenous phosphopeptides, MOF, Metal oxide affinity chromatography, Affinity probe, Mass spectrometry

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INTRODUCTION

Up to now, various extraction methods have been introduced to separate phosphopeptides from complex samples, including immunoprecipitation,6 reverse-phase liquid chromatography (RPLC),7 ion exchange chromatography (IEC),8 immobilized metal ion affinity chromatography (IMAC),9 and metal oxide affinity chromatography (MOAC).10 Among these strategies, MOAC is one of the most frequently used tools due to the specific affinity between metal oxides (such as TiO2,10−12 ZrO2,13,14 Al2O3,15 SnO2,16 and ZnO17) and phosphate groups. Although many methods based on the MOAC have been developed and utilized for phosphopeptide enrichment, it still faces a great problem that different metal oxides exhibit different preference for monophosphopeptides or multiphosphopeptides, leading to the incomprehensive mapping of phosphopeptides.18,19 In order to overcome this problem, variations of binary metal oxides like rGR-TiO2-ZrO2, magG/ (Ti-Sn)O4, and magG/PD/(Zr-Ti)O4 were designed and prepared.19−21 Indeed, these binary metal oxides exhibited higher extraction efficiency toward phosphopeptides and

As one of the most significant post-translational protein modifications, protein phosphorylation participates in numerous biological and cellular activities, including the cell signaling, proliferation, differentiation, and apoptosis, which are closely associated with normal operation of the life cycle.1−3 Abnormity of protein phosphorylation can influence multiple properties of proteins, leading to a variety of diseases including cancer and so on.4 In general, enzymes in the body hydrolyze these abnormal phosphorylation into peptides that we called endogenous phosphopeptides. These endogenous phosphopeptides possessed higher clinic specificity than common ones. Thus, it is extremely necessary to have a better understanding of the process of phosphorylation and its relevant peptide sequence information, etc. Nevertheless, the complexity of organism, the poor ionization efficiency and low abundance of endogenous phosphopeptides as well as the interferences which come from non-phosphorylated peptides and proteins, etc., make it a great challenge to identify phosphopeptides directly by mass spectrometry.5 Thus, for phosphopeptidomics analysis, specific and selective extraction of phosphopeptides is an indispensable step. © XXXX American Chemical Society

Received: January 2, 2018 Revised: January 30, 2018 Published: February 12, 2018 A

DOI: 10.1021/acssuschemeng.8b00023 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering Scheme 1. Synthetic Procedure for Fe3O4@PDA@Zr-Ti-MOF

proteins from complex samples; superparamagnetism endowed materials with the ability to response to external magnetic field, thereby simplified the separation process. Amazingly, the asprepared materials maintained all the merits of the large specific surface area, unique microporous structure, and super-paramagnetism. More importantly, due to the dual affinities of Zr− O and Ti−O in MOFs toward phosphopeptides, it exhibited extraordinary performance in detecting phosphopeptides from tryptic digests of standard phosphorylated proteins as well as endogenous phosphopeptides from naturally obtained complex sample of human saliva.

achieved stronger specificity and better selectivity than single metal oxides. But these nonporous hybrids could not realize the enrichment of endogenous phosphopeptides with high selectivity and high efficiency due to the presence of highly abundant proteins in organism. Thus, to detect as many and complete endogenous phosphopeptides as possible, the exploitation of novel materials which not only containing binary or multimetal oxides but also having the size-exclusion ability may be a good solution. According to reports, metal−organic frameworks (MOFs) are prepared via the coordination of metal centers and organic ligands and feature, by the active metal sites, abundant available ligands as well as many properties including large surface areas, tunable high porosity, and the availability of further functionality.22−24 As a result, depending on the above merits, metal−organic frameworks have gained much attention in proteomics research over the past decades. For instance, Wang reported the MG@Zn-MOFs biocomposite for glycopeptide enrichment via the interaction between the targets and ligand groups;25 Zhao reported Fe3O4@PDA@Zr-MOF for phosphopeptide enrichment using the active metal sites.26 Recently, by virtue of their micropores for capturing small biological molecules and excluding large-size substances at the same time, various MOFs have been fabricated and widely used in peptidomics analysis.27 The special micropores of MOFs make the isolation of endogenous phosphopeptides much easier. However, among those reported materials of MOFs, including Fe-based MOFs of MIL-100, MIL-53, Zr-MOFs, and ErMOFs,26−28 their performances in phosphopeptide enrichment were not very satisfactory because of the incomplete removal of severe interferences. Therefore, the development of metal− organic frameworks with binary metal oxides and size-exclusion ability might overcome the shortcoming in endogenous phosphopeptide enrichment. Herein, for the first time, a magnetic binary metal−organic framework based on Zr−O clusters and Ti−O clusters (denoted as Fe3O4@PDA@Zr-Ti-MOF) was synthesized to improve the phosphopeptide enrichment efficiency. Simply, after grafting Zr-MOF on polydopamine (PDA) coated magnetic microspheres, a postsynthetic method was adopted to obtain magnetic Zr-Ti-MOF by replacing partial Zr atoms with Ti atoms. As is well-known, larger surface areas could offer more functionalized groups for improving the capture ability of materials; unique microporous structure could help to realize the peptides capture with simultaneous exclusion of large-size

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EXPERIMENTAL SECTION

Materials and Chemicals. Iron chloride hexahydrate (FeCl3· 6H2O), sodium acetate, ethanol, ethylene glycol, dopaminechloride, zirconiun tetrachloride (ZrCl4), titanium tetraisopropanolate, terephthalic acid (TPA), NaOH, dimethylformamide (DMF, 99%), methanol, ammonium bicarbonate (NH4HCO3), ammonium hydroxide (NH3·H2O), and phosphoric acid (H3PO4) were purchased from Shanghai Chemical Corp. Tetrachlorobis(tetrahydrofuran)titanium(IV) (TiCl4(THF)2) was purchased from Shanghai Titan Scientific Corp. β-Casein, bovine serum albumin (BSA), and trypsin from bovine pancreas were purchased from Sigma-Aldrich. 2,5-Dihydroxybenzoic acid (DHB), tris(hydroxymethyl) aminomethane (Tris) were purchased from J&K Scientific. ACN (HPLC-grade) and trifluoroacetic acid (TFA) were purchased from Merck (Darmstadt, Germany). Human serum was offered by Shanghai Zhongshan Hospital. All aqueous solutions were prepared using Milli-Q water from Milli-Q system (Millipore, Bedford, MA). Other chemicals were all of analytical grade. Synthesis of Fe3O4@PDA. First, 1.36 g (0.005 mol) FeCl3·6H2O and 3.6 g (0.044 mol) smashed sodium acetate were dissolved in 75 mL ethylene glycol. The mixture was stirred for 30 min and then transferred into a Teflon-lined stainless-steel autoclave. The autoclave was heated for 16 h at 200 °C. The obtained Fe3O4 was washed with deionized water five times and then dried in vacuum at 50 °C. Next, 0.32 g (1.7 mmol) dopamine chloride was dissolved in 80 mL Tris buffer containing 0.05 g (0.4 mmol) Tris. A 0.12 g portion of Fe3O4 was added and then stirred at room temperature for 12 h. The obtained Fe3O4@PDA was washed by deionized water for three times and then dried in vacuum at 50 °C. Synthesis of Fe3O4@PDA@Zr-MOF. A 0.12 g (0.7 mmol)portion of terephthalic acid and 0.16 g (0.7 mmol) ZrCl4 were dissolved in 60 mL DMF and then 0.1 g dried Fe3O4@PDA was added. The mixture was stirred for 1 h at 120 °C. The obtained Fe3O4@PDA@Zr-MOF was washed with DMF for three times and then dried in vacuum at 50 °C. Synthesis of Fe3O4@PDA@Ti-MOF. A 0.12 g (0.7 mmol)portion of terephthalic acid and 0.2 mL (0.67 mmol) titanium tetraisopropanB

DOI: 10.1021/acssuschemeng.8b00023 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering Scheme 2. Workflow of Phosphopeptide Enrichment from Biological Samples by Fe3O4@PDA@Zr-Ti-MOF

olate were dissolved in 60 mL DMF with 0.1 g dispersed Fe3O4@PDA. The suspension was stirred at 150 °C for 1 h. The obtained material was washed with DMF three times and then dried in vacuum at 50 °C. Synthesis of Fe3O4@PDA@Zr-Ti-MOF. The postsynthetic method was employed according to a previous report with a bit of alteration.29 Briefly, 0.04 g Fe3O4@PDA@Zr-MOF was dispersed in 30 mL DMF containing 0.1 g (0.3 mmol) TiCl4(THF)2. The suspension was then stirred at 85 °C for 24 h. The obtained product, denoted as Fe3O4@PDA@Zr-Ti-MOF, was washed with DMF three times and then soaked in methanol for 2 days with the solution replaced with fresh methanol every 12 h. At last, the washed material was dried in vacuum at 50 °C. Synthesis of Fe3O4@PDA@(Zr-Ti)O4. The synthetic procedure was employed according to a previous report with a little modification.21 In brief, 0.5 mL titanium isoproxide and 0.98g zirconium isopropoxide were dissolved in 50 mL ethanol. After ultrasonication for 30 min, 0.03 mg Fe3O4@PDA was added. In the meantime, 60 mL mixture solution containing 10 mL ethanol and 50 mL deionized water was added dropwise in above suspension under stirring. The mixture was then stirred for 8 h at room temperature. After that, the obtained composites were washed with ethanol and deionized water several times and dried in vacuum at 50 °C. Finally, the product was calcined in N2 at 400 °C for 2 h. Sample Preparation. A 1 mg (0.04 mmol) portion of β-casein was dissolved in 1 mL 25 mM NH4HCO3 buffer and denatured in boiling water for 10 min, respectively. The obtained solutions were then incubated with trypsin at 37 °C for 16 h with the mass ratio of trypsin to protein at 1:50 (w/w). The tryptic digests were diluted with loading buffer of 50%ACN/0.1%TFA (v/v) for enrichment experiments. A 25 mg (0.16 mmol) portion of DHB was dissolved by 1 mL buffer of 70%ACN/1% H3PO4 for further use. Fresh human saliva was taken from a healthy volunteer. Briefly, 2 mL saliva was diluted with 2 mL 0.2% TFA and centrifuged for 5 min at the speed of 8000 rpm. The supernatant was collected for further use. Enrichment of Phosphopeptides from Tryptic Digests of Standard or Complex Biosamples. The workflow of phosphopeptide enrichment is illustrated in Scheme 2. In detail, 200 μg of Fe3O4@ PDA@Zr-Ti-MOF was dispersed in 100 μL of mixture of peptides (diluted by 50%ACN/0.1%TFA) and the obtained solution was incubated in a vortex at 37 °C for 30 min. Then the material and solution were separated by a magnet. The supernatant was removed and the material was washed by loading buffer (50%ACN/0.1%TFA)

three times. After that, the enriched phosphopeptides were eluted with 10 μL 10% ammonium hydroxide (v/v) at 37 °C for 20 min. Finally, the eluent was collected and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS) or lyophilized for nano-LC-ESI-MS/MS analysis. Characterization and MS Analysis. Detailed information on instruments and characterization are listed in the Supporting Information

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RESULTS AND DISCUSSION Synthesis and Characterization of Fe3O4@PDA@Zr-TiMOF. The synthetic procedure for the Fe3O4@PDA@Zr-TiMOF was illustrated in Scheme 1. Briefly, Fe3O4 magnetic nanoparticles were prepared by a well-known solvothermal reaction, followed by the coating of polydopamine through a simple polymerization of dopamine. Then, Fe3O4@PDA@ZrMOF was obtained by the self-assembly of Zr-MOF on the surface of Fe3O4@PDA with the involvement of ZrCl4 and TPA. Finally, TiCl4(THF)2 was employed to react with Fe3O4@PDA@Zr-MOF and partial Zr atoms were replaced by Ti atoms,29 resulting in the successful synthesis of Fe3O4@ PDA@Zr-Ti-MOF. The morphology and microstructure of Fe3O4@PDA@ZrTi-MOF was characterized by various tools. As shown in TEM images (Figure 1), around 40 nm of PDA shell was grafted on magnetic microspheres (Figure 1a). By contrast, after the modification of Zr-Ti-MOF (Figure 1b), the surface of Fe3O4@ PDA particles was rough, indicating the successful synthesis of core−shell structured MOF composites. The coexistence of Zr and Ti element was proved by EDX analysis (Supporting Information Figure S1) with the weight proportion of 5.90% and 2.16% respectively, which was crucial for further enrichment. Moreover, the Fe3O4@PDA@Zr-Ti-MOF was characterized by XPS to explore the inner structure of Zr-Ti-MOF. The peaks of Zr and Ti could be observed directly from the wide scan spectrum (Figure 2a), which was consistent with the EDX result. Besides, in the high-resolution spectrum of Zr 3d5 (Figure 2b), two peaks could be distinguished with the binding energy at around 184.0 and 186.3 eV. Binding energy is a measure of electron density around atoms. Compared to C

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Fe3O4@PDA@Zr-Ti-MOF were tested using SQUID magnetometer. As shown in Figure 3, the magnetization values

Figure 1. TEM images of (a) Fe3O4@PDA and (b) Fe3O4@PDA@ZrTi-MOF.

original Zr-MOF whose peaks were located at 182.0 and 184.5 eV respectively,30 the shift of binding energies to larger values indicated an improved ability of Fe3O4@PDA@Zr-Ti-MOF to capture negatively charged groups like phosphoryl groups in phosphopeptides.31 Meanwhile, the peaks of Ti 2p3 were also investigated (Figure 2c). Similarly, the binding energy of these two peaks were centered at 459.8 and 465.9 eV respectively, higher than initial values of 458.1 and 463.8 eV.30 The increased binding energies of Zr and Ti may be attributed to the formation of Zr−Ti clusters on an atomic scale in the MOF. The structure of magnetic MOFs was further confirmed by wide-angle X-ray diffraction patterns (Supporting Information Figure S2). The diffraction peaks at 2θ = 7.5, 8.6, 25.8, 43.4, 50.4° were from the Zr-Ti-MOF and 2θ = 30.7, 35.8, 57.2° were from Fe3O4. Nitrogen adsorption−desorption isotherm was employed to investigate the surface areas and pore structure of Fe3O4@PDA@Zr-Ti-MOF, Fe3O4@PDA@ZrMOF, and Fe3O4@PDA@Ti-MOF (Figures S3 and S4 and Table S1 in the Supporting Information). The calculated surface area of Fe3O4@PDA@Zr-Ti-MOF was 745.2 m2/g, and the pore size is around 1.3 nm, which is similar to those of Fe3O4@PDA@Zr-MOF, Fe3O4@PDA@Ti-MOF, and ZrMOF’s in a former report.30 These results indicated that Ti clusters were introduced in MOF without destroying its micropores, and this meant that the Fe3O4@PDA@Zr-TiMOF would possess the ability to allow the small-size peptides into its porous channel while excluding large-size proteins. The merits of large surface area and suitable micropore structure of Fe3O4@PDA@Zr-Ti-MOF made it a potential probe to detect small-sized phosphopeptides in complex biosamples. The as-prepared material was endowed with magnetic responsiveness by introducing superparamagnetic Fe3 O4 cores. The magnetic properties of Fe3O4, Fe3O4@PDA, and

Figure 3. Magnetic hysteresis curves of Fe3O4, Fe3O4@PDA, and Fe3O4@PDA@Zr-Ti-MOF. The saturation magnetization values of Fe3O4, Fe3O4@PDA, and Fe3O4@PDA@Zr-Ti-MOF were 81.9, 64.1, and 39.6 emu·g−1, respectively.

decreased a bit with the coating of PDA and PDA@Zr-TiMOF shells but the magnetic responsiveness of Fe3O4@PDA@ Zr-Ti-MOF was still retained. Figure S5 displayed the rapid dispersion and separation of Fe3O4@PDA@Zr-Ti-MOF in water, which could avoid the loss of materials and make the enrichment procedure convenient and save time. Investigation of the Performance of Fe3O4@PDA@ZrTi-MOF for Phosphopeptide Enrichment. The enrichment procedure for phosphopeptides is displayed in Scheme 2. It mainly contains four steps: extraction, washing, elution, and MS analysis. The capability of Fe3O4@PDA@Zr-Ti-MOF in phosphopeptide enrichment was first investigated by tryptic digests of β-casein (with trace of α-casein), which is a standard phosphorylated protein. For comparison, the performances of Fe3O4@PDA@Zr-MOF and Fe3O4@PDA@Ti-MOF were also tested under the same conditions. In the direct MS analysis (Figure 4a), the spectrum was dominated by nonphosphopeptides and almost no phosphopeptides could be detected when the concentration of tryptic digests of β-casein was 10 ng/μL. But after treatment with Fe3O4@PDA@Zr-Ti-MOF, as shown in Figure 4b, seven mono-phosphorylated peptides, three multiphosphorylated peptides and four dephosphorylated fragments were detected with hardly any interference of non-phosphory-

Figure 2. XPS patterns of Fe3O4@PDA@Zr-Ti-MOF: (a) wide scan XPS spectrum, (b) high-resolution XPS spectrum of Zr 3d5, and (c) highresolution XPS spectrum of Ti 2p3. D

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Figure 4. MALDI-TOF mass spectra of tryptic digested β-casein (10 ng/μL): (a) before enrichment and (b−d) after treatment with (b) Fe3O4@ PDA@Zr-Ti-MOF, (c) Fe3O4@PDA@Zr-MOF, and (d) Fe3O4@PDA@Ti-MOF. Mass spectrometric peaks of mono-phosphorylated peptides were marked with red rhombuses, dephosphorylated fragment with triangles, multi-phosphorylated peptides with circles, and dephosphorylated fragment with stars.

Figure 5. MALDI-TOF mass spectra of tryptic digested β-casein (10 pg/μL): (a) before enrichment and (b−d) after treatment with (b) Fe3O4@ PDA@Zr-Ti-MOF, (c) Fe3O4@PDA@Zr-MOF, and (d) Fe3O4@PDA@Ti-MOF. Mass spectrometric peaks of phosphopeptides were marked with red rhombuses, dephosphorylated fragment, with triangles.

tryptic digests of β-casein was diluted by 1000-fold to 10 pg/μL (0.4 fmol/μL), no phosphopeptides could be detected in direct MS analysis (Figure 5a). After enriched by Fe3O4@PDA@ZrTi-MOF (Figure 5b), three phosphopeptides along with two dephosphorylated fragments could be detected with high intensities and signal-to-noise ratios (S/N). By contrast, only two peaks of phosphopeptides were observed with relatively lower intensities after enrichment by Fe3O4@PDA@Zr-MOF and Fe3O4@PDA@Ti-MOF, respectively (Figure 5c and d). What’s more, even when the concentration of tryptic digests of β-casein was as low as 1 pg/μL (0.04 fmol/μL), three phosphopeptides were still detected after treatment with Fe3O4@PDA@Zr-Ti-MOF (Figure S7). These results indicated the better enrichment efficiency of Fe3O4@PDA@Zr-Ti-MOF than that of single-metal centered Fe3O4@PDA@Zr-MOF and Fe3O4@PDA@Ti-MOF composites. Inspired by the above results, the enrichment performance of Fe3O4@PDA@Zr-Ti-MOF toward mono-phosphorylated peptide and multi-phosphorylated peptide was explored further.

lated peptides and the peak intensity of phosphopeptides was quite strong. Compared with the enrichment results of Fe3O4@ PDA@Zr-MOF and Fe3O4@PDA@Ti-MOF (Figure 4c and d), the number of mono-phosphorylated peptides and multiphosphorylated peptides captured by Fe3O4@PDA@Zr-TiMOF increased and the background of mass spectra was cleaner, demonstrating the extraordinary enrichment efficiency of this binary-metal centered MOF composite. The detailed information could be seen from Figure S6 and Table S2 of the Supporting Information. In comparison with former reported studies of MOF materials in which fewer phosphopeptides were detected27,28,31 or with lower peak intensities and severe noise,26 the result in this work was much better. The enhanced enrichment performance of Fe3O4@PDA@Zr-Ti-MOF may be attributed to the integration of Zr and Ti on an atomic scale in metal cluster of MOF. The competence of Fe3O4@PDA@Zr-Ti-MOF in phosphopeptide enrichment was further explored by tryptic digests of βcasein with lower concentrations. When the concentration of E

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Figure 6. MALDI-TOF mass spectra of a mixture of intact BSA and tryptic digests of β-casein: (a) before enrichment and (b) after treatment with Fe3O4@PDA@Zr-Ti-MOF. The mass ratios of BSA:β-casein were 1000:1. Mass spectrometric peaks of phosphopeptides were marked with red rhombuses, dephosphorylated fragments, with triangles.

Figure 7. MALDI mass spectra of endogenous phosphopeptides from pristine human saliva: (a) without enrichment and (b) after enriched by Fe3O4@PDA@Zr-Ti-MOF. Mass spectrometric peaks of mono-phosphorylated peptides were marked with red rhombuses, and those of multiphosphorylated peptides were marked with red circles.

toward both mono- and multi-phosphorylated peptides. Furthermore, different mole ratios of standard mono- versus multi-phosphorylated peptides were also employed for relative quantitative analysis (Figure S9). When the ratios of standard mono- versus multi-phosphorylated peptides were at 1:5, 1:10, 5:1, and 10:1, the signal intensities of phosphopeptides varied in direct proportion to the ratios of standard mono- versus multi-phosphorylated peptides, confirming that Fe3O4@PDA@

Standard mono-phosphorylated peptide (pSADGQHAGGLVK) at m/z 1219 and multi-phosphorylated peptide (TDHGAEIVYK [pS] PVVSGDT [pS] PRHL) at m/z 2624 were employed as standard samples. According to the results in Figure S8 of the Supporting Information, the detection limits of Fe3O4@PDA@Zr-Ti-MOF for mono-phosphorylated peptide and multi-phosphorylated peptide were 0.5 and 0.1 pg/μL, respectively, demonstrating its efficient affinity performance F

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total of 54 phosphorylated sites on 44 phosphopeptides including 34 mono- and 10 multi-phosphorylated peptides were detected from merely 1 μL of pristine human saliva (Table S3). The efficiency in detecting phosphopeptides from human saliva were greatly improved by Fe3O4@PDA@Zr-TiMOF compared to previous reports31,32 with only 17 and 14 phosphopeptides enriched, respectively.

Zr-Ti-MOF has no bias toward mono- and multi-phosphorylated peptides. Considering the microporous structure of Fe3O4@PDA@ZrTi-MOF, standard protein bovine serum albumin (BSA) was chosen to test its size-exclusion effect. In the mixture of BSA and tryptic digests of β-casein with mass ratio at 1000:1, BSA protein was obviously observed while phosphopeptides could hardly be detected before enrichment (Figure 6a). On the contrary, after extracted by Fe3O4@PDA@Zr-Ti-MOF, abundant phosphopeptides were identified with high intensities and no signal of BSA protein was observed (Figure 6b). The result confirmed that the employment of large proteins in the environment would not affect the affinity of Fe3O4@PDA@ZrTi-MOF composite toward small-size phosphopeptides, indicating its potential application in complex biosamples which contained severe interference from both non-phosphorylated peptides and large proteins. To further confirm the sizeexclusion effect of Fe3O4@PDA@Zr-Ti-MOF toward phosphopeptides, nonporous Zr/Ti based bimetallic compound of Fe3O4@PDA@(Zr-Ti)O4 was employed as the control group to extract phosphopeptides from complex mixture. As can be seen from the MS spectra in Figure S10, when tryptic digests of β-casein, phosphorylated protein α-casein and non-phosphorylated protein BSA were at the mass ratio of 1:5000:5000, the phosphopeptides extracted by Fe3O4@PDA@(Zr-Ti)O4 were limited and with low intensity, which could be blamed for the existence of high concentrations of large-size proteins that severely hindered the interaction between low concentrations of phosphopeptides and bimetallic composites. In the meantime, phosphorylated proteins adhered to the surface of materials. While for Fe3O4@PDA@Zr-Ti-MOF, the existence of abundant proteins almost had no influence on its interaction with small-size phosphopeptides and no phosphorylated proteins were detected. The results confirmed the sizeexclusion function of micropores of Fe3O4@PDA@Zr-TiMOF toward proteins including phosphorylated proteins and non-phosphorylated proteins. Furthermore, the reusability of Fe3O4@PDA@Zr-Ti-MOF was also tested by tryptic digests of β-casein. The composite was reused for five times. Before every enrichment cycle, the material was washed with eluant and loading buffer in order to remove the residues on its surface. As shown in Figure S11 (Supporting Information), after repeating the enriching process five times, the ability of Fe3O4@PDA@Zr-Ti-MOF for detecting phosphopeptides was almost the same as the initial material. Encouraged by all results above, Fe3O4@PDA@Zr-Ti-MOF composite was further applied to capture endogenous phosphopeptide in complicated biosample of human saliva. As a widely used clinical sample, human saliva is easy to get and contains many low-abundance endogenous phosphopeptides which could be potential biomarkers for disease diagnosis. The collected eluent was analyzed by MALDI MS and nano-LCESI-MS/MS simultaneously. As shown in Figure 7a, in the human saliva without enrichment, only one signal of phosphopeptide could be detected with extremely low intensity and the interferences were quite serious. However, after treatment with Fe3O4@PDA@Zr-Ti-MOF directly (Figure 7b), 25 peaks of phosphopeptides containing 16 mono- and 9 multi-phosphorylated peptides dominated the spectrum with scarcely any non-phosphorylated peptides. These peaks had been proved to be phosphopeptides by MALDI MS/MS (Figure S12). And after analyzed by nano-LC-ESI-MS/MS, a

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CONCLUSIONS In summary, a novel strategy was proposed for designing binary-metal centered magnetic metal−organic framework for phosphoproteome analysis. The as-prepared Fe3O4@PDA@ZrTi-MOF possessed the merits of large specific surface area and unique porous structure as well as the dual affinities of Zr−O and Ti−O. In comparison to similar materials with single metal centers (Fe3O4@PDA@Zr-MOF and Fe3O4@PDA@Ti-MOF), binary-metal centered Fe3O4@PDA@Zr-Ti-MOF could detect much more phosphopeptides including mono- and multiphosphorylated peptides and owned better selectivity and sensitivity. Furthermore, the performance of this composite in identifying endogenous phosphopeptides in human saliva was also excellent, exhibiting its great potential in phosphoproteome analysis and further disease diagnosis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.8b00023. [Fe3O4@PDA@Zr-Ti-MOF] (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (N.S.). *E-mail: [email protected] (C.-h.D.). ORCID

Chun-hui Deng: 0000-0002-8704-7543 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21425518, 21405022, and 21675034) and the National Basic Research Priorities Program of China (2013CB911201).

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REFERENCES

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DOI: 10.1021/acssuschemeng.8b00023 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acssuschemeng.8b00023 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX