Boronic Acid-Functionalized Magnetic MetalâOrganic Frameworks via

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Boronic acid-functionalized magnetic metal–organic frameworks via dual ligand strategy for highly efficient enrichment of phosphopeptides and glycopeptides Bin Luo, Qiang Chen, Jia He, Zhiyu Li, Lingzhu Yu, Fang Lan, and Yao Wu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06171 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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ACS Sustainable Chemistry & Engineering

Boronic acid-functionalized magnetic metal– organic frameworks via dual ligand strategy for highly efficient enrichment of phosphopeptides and glycopeptides Bin Luo, Qiang Chen, Jia He, Zhiyu Li, Lingzhu Yu, Fang Lan*, and Yao Wu* National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China (Corresponding author's email: [email protected]; [email protected])

KEYWORDS：Magnetic metal–organic frameworks, boronic acid, enrichment, phosphopeptides, glycopeptides

ABSTRACT To date, abundant strategies and affinity materials are proposed to enrich phosphopeptides and glycopeptides from biological systems. However, few affinity materials can meet the demand for enriching both phosphopeptides and glycopeptides with an integrated single material. In this work, a novel magnetic metal–organic framework nanocomposite with Zr4+ ions as metal units as well as terephthalic acid (PTA) and 1,4-phenylenebisboronic acid (denoted as PBA) as dual organic ligands, was designed and fabricated for

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highly efficient enrichment of both phosphopeptides and glycopeptides. It is worth mentioning that Zr4+ ions and PBA were well assembled into the MOF coating via the dual ligand strategy to achieve both phosphopeptide and glycopeptide enrichment by the Zr–O clusters based on metal oxide affinity chromatography (MOAC) and abundant boronic acid groups based on boronic acid affinity chromatography (BAAC) methods. The quick magnetic response performance and high surface area endowed the affinity material outstanding ability for phosphopeptide and glycopeptide enrichment in the model protein mixtures (β-casein, human IgG, and bovine serum albumin). Furthermore, the nanocomposites exhibited specific enrichment capacity in complex biological samples (rat brain and rat liver), revealing the great potential for the identification and analysis of trace phosphopeptides and glycopeptides in biological samples. INTRODUCTION Protein post-translational modifications (PTMs), endow additional functional groups (e.g., methyl, ubiquitin, phosphate or glycan) to proteins, partly increase protein diversity and complexity.1 PTMs have great importance in the regulation of protein functions and multiple cellular processes.2-4 Unfortunately, abnormal PTMs are always associated with various serious diseases.5-7 For example, the troublesome cancer is reported to be closely related to the abnormal phosphorylation and glycosylation which are considered as two most typical and extensive types of protein PTMs.8,9 Therefore, the identification with high coverage of phosphopeptides and glycopeptides plays


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crucial role in discovery of cancer biomarkers or other diseases. Considering the fast speed, high resolution, and high throughput, mass spectrometry (MS) is widely employed in the proteomics research. However, the severe obstacles, originate from diversity of protein samples, low abundance of PTM proteins and strong signal suppression from the abundant background proteins, make it still a challenge.10,11 Thus, the sample pretreatment and enrichment process are prerequisites for identification and analysis of trace phosphopeptides and glycopeptides in biological samples. To date, various strategies have been developed for phosphopeptide enrichment, including metal oxide (zirconium dioxide (ZrO2) and titanium dioxide (TiO2)) affinity chromatography (MOAC),12,13 immobilized metal ion (Zr4+, Ti4+, Fe3+, and Zn2+) affinity chromatography (IMAC),14,15 and strong ion exchange chromatography (SAX).16 Among these methods, the affinity materials based on MOAC and IMAC techniques are most commonly used, while the MOAC method is more effective in the enrichment of phosphopetides because of the bidentate interactions between the metal oxides and phosphate groups as compared with IMAC method.17 As for glycopeptide enrichment, hydrazine chemistry,18 lectin affinity chromatography,19 boronic acid affinity

chromatography

(BAAC),20,21

and

hydrophilic

interaction

liquid

chromatography (HILIC)22,23 are the common approaches. However, high cost of hydrazine chemistry, excessive specificity of lectin affinity chromatography, and nonspecific adsorption of HILIC method have limited their application to some extent.24 Due to the high specificity towards glycopeptides and convenient capture and elution processes via environmental stimuli response, BAAC method holds huge



potential for glycopeptide enrichment. Therefore, combining with the MOAC and BAAC techniques in an integrated single material would be an advisable strategy for both phosphopeptide and glycopeptide enrichment. In recent years, metal-organic frameworks (MOFs), consisted of metal-containing units and organic ligands, have become a popular class of porous material and been widely applied in gas storage, catalysis, drug delivery, and separation fields due to their high porosity, high surface area, and tunable properties.25-28 Magnetic MOFs have attracted increasing attention in proteomics application, particularly enzyme immobilization, phosphopeptide enrichment, and glycopeptide enrichment.29,30 In addition, the post-synhtetic modification strategy is usually applied to develop new functional MOFs, leading to their broader application.31,32 However, few studies have reported that metal ions and organic ligands simultaneously could serve as affinity sites for both phosphopeptide and glycopeptide enrichment. It would be a meaningful and challenging work to design and prepare a kind of functionalized magnetic MOF material for both phosphopeptide and glycopeptide enrichment. Herein, we designed and synthesized a kind of novel magnetic MOF nanocomposite as a platform for both phosphopeptide and glycopeptide enrichment via the metal oxide clusters and boronic acid groups on the organic ligands (Scheme 1A). The design route has the following attractive features. First, the SPIOs (superparamagnetic iron oxide nanoparticles) core, prepared by typical solvothermal reaction, endowed the nanocomposites excellent magnetic responsiveness which would be beneficial for simplification of the enrichment process and rapid recovery of the affinity material.


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Second, thanks to the excellent chelation between metal ions and abundant hydroxyls of the SiO2 middle layer, the epitaxial growth of MOFs on the surface of SPIOs@SiO2 nanospheres could be easily achieved. Third, the functionalization of MOFs was usually realized by post-synthetic modification. However, the complicated and tedious modification processes sharply decreased efficiency of PSM strategy. In this work, the dual organic ligands strategy was employed to prepare the boronic acid functionalized magnetic Zr-MOF nanocomposites (denoted as SPIOs@SiO2@MOF) by one step for the first time. In order to introduce boronic acid groups, both terephthalic acid (PTA) and 1,4-phenylenebisboronic acid (PBA) served as dual ligands together with Zr4+ metal ions to construct the Zr-MOF structure. As expected, successful introduction of a large number of boronic acid groups was beneficial to enrich glycopeptides. On the other hand, the SPIOs@SiO2@MOF nanocomposites could also be applied to enrich phosphopeptides based on strong binding between Zr-O clusters and phosphopeptides. To our knowledge, this was the first magnetic MOF affinity material that not only metal ions but also functional organic ligands served as dual affinity sites for phosphopeptide and

glycopeptide

enrichment

respectively.

Overall,

the

as-prepared

SPIOs@SiO2@MOF nanocomposites possessed excellent magnetic responsiveness and high surface area, exhibiting great performance in phosphopeptide and glycopeptide enrichment. More importantly, our work would provide a promising design platform of multifunctional magnetic MOF affinity materials for multiple peptide enrichment, throwing light on comprehensive peptidomes analysis.



Scheme 1. Schematic representation of the (A) synthetic route and (B) workflow of phosphopeptide or glycopeptide enrichment by SPIOs@SiO2@MOF nanocomposites.

EXPERIMENTAL SECTION Materials Iron(III) chloride hexahydrate (FeCl3·6H2O), zircomiun tetrachloride (ZrCl4), ammonium acetate (NH4Ac), bovine serum albumin (BSA), tetraethoxysilane (TEOS), 2,5-dihydroxyl benzoic acid (DHB), β-casein (from bovine milk), dithiothreitol (DTT), iodoacetamide (IAA), PNGase F, trypsin (TPCK treated), and human serum immunoglobulin G (human IgG) were purchased from Sigma Aldrich. Commercial TiO2 microspheres, polyetherimide (PEI, Mw 25,000), terephthalic acid (PTA), 1,4phenylenebisboronic acid (PBA), and trifluoroacetic acid (TFA) were purchased from Aladdin (Shanghai, China). Ethylene glycol (EG), trisodium citrate dihydrate (Na3CT),


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anhydrous ethanol, acetic acid (HAC), ammonium hydroxide (NH3·H2O, 28 wt%), dimethyl formamide (DMF), ammonium bicarbonate (NH4HCO3), urea, and acetonitrile (ACN) were purchased from Forest Science and Technology Development Co. Ltd. (Chengdu, China). Characterizations Scanning electron microscopy (SEM) images and Energy dispersive X-ray (EDX) data were collected on a Hitachi S-4800 scanning electron microscope (Japan). Transmission electron microscopy (TEM) images were obtained by a JEOL JEM100CX transmission electron microscope (Japan). Crystal structure of samples with angles ranging from 10° to 80° performed on the powder X-ray diffraction (XRD, X' Pert Pro MPD, Philips, Netherlands). Spectrometer (FT-IR, PE spectrometer) was employed to obtain fourier transform infrared spectra with wave number range 5004000 cm-1. Thermogravimetric analysis (TGA) was performed under a nitrogen atmosphere with a heating rate of 10 K/min from room 35 to 900 °C (STA449 C Jupiter, NETZSCH). The magnetization of the sample with field from 0 to 18000 Oe was analyzed at 300K by vibrating sample magnetometer (VSM, model BHV-525, Riken Japanese Electronics Company). Surface area and pore size analyzer (QuadraSorb SI, America) was employed to study the surface and BJH pore size distribution at 77K. The all MALDI mass spectra were obtained by AB Sciex 5800 MALDI-TOF/TOF mass spectrometer (AB SCIEX, CA). Synthesis of the Fe3O4 nanoparticles (SPIOs)



Fe3O4 nanoparticles were prepared via a typical solvothermal reaction. For the synthesis, 1.157 g of FeCl3·6H2O was dissolved in 60 mL of EG, and 0.4 g of Na3CT was added with stirring. Thereafter, 3.303 g of NH4Ac was added with magnetic stirring for one hour. The mixture was breathed into stainless steel autoclave and maintained at 200 °C for 16 h. Finally, the sediment was collected by magnet separation and washed thoroughly with ethanol and deionized water for several times. The Fe3O4 nanoparticles were re-dispersed in deionized water (15 mL) for subsequent use. Synthesis of the SPIOs@SiO2 nanospheres Firstly, 5 mL the above SPIOs suspension was added to 20 mL HCl (0.1 M) and sonicated for 30 min. Then, the SPIOs nanoparticles were washed with deionized water for three times, and added into the mixed solvent containing deionized water (8 mL) and ethanol (32 mL). Followed by a sequential addition of ammonia(28%, 0.5 mL) and TEOS (0.5 mL), the resulting mixture was sonicated for another 10 min and stirred dramatically for 4 h at room temperature. The obtained SPIOs@SiO2 nanospheres were rinsed with ethanol, deionized water, and DMF for several times, respectively. Finally, the SPIOs@SiO2 nanospheres were re-dispersed in 3 mL DMF for subsequent use. Synthesis of SPIOs@SiO2@MOF nanocomposites One mL of the above SPIOs@SiO2 nanospheres and 0.232 g of zircomiun tetrachloride (ZrCl4) were added into 30 mL DMF with ultrasonic vibration for 30 min. 0.083 g of terephthalic acid (PTA) and 0.082 g of 1,4-phenylenebisboronic acid (PBA) were absolutely dissolved in 10 mL DMF. Then, the two solutions were mixed and heated at 120℃ under vigorous stirring for 4 h. The obtained SPIOs@SiO2@MOF


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nanocomposites were washed with DMF and acetone, and then dried overnight under vacuum. Synthesis of SPIOs@PEI@PBA nanospheres Firstly, 5 mL the above SPIOs suspension and 0.4 g polyetherimide (PEI) were added in 20 mL deionized water and continuously stirred for 24 h at room temperature. Then, the products were collected and washed with deionized water for three times. The obtained SPIOs@PEI nanospheres were dispersed in 20 mL deionized water containing 1.0 g 1,4-phenylenebisboronic acid (PBA). The mixture was stirred for 12 h at room temperature. Finally, the SPIOs@PEI@PBA nanospheres were washed with deionized water and re-dispersed in 1mL water for subsequent use. Sample preparation For the tryptic digests of standard proteins：β-casein (1 mg) was dissolved in 1 mL NH4HCO3 solution (50 mM pH 8.2) and digested with trypsin (40:1, w/w) at 37 °C for 16 h. Human serum immunoglobulin G (2 mg) and bovine serum albumin (4 mg) were respectively dissolved in 1 mL of buffer containing 50 mmol L-1 NH4HCO3 and 8 mol L-1 urea. After the addition of 100 μL DTT (1 mol L-1) and allowing to stand at 60 °C for 1 h, the protein was alkylated by 14.8 mg IAA at room temperature in the dark for 45 min. Finally, the mixture was diluted with NH4HCO3 (50 mM pH 8.2) and incubated at 37 °C for 16 h with trypsin at the mass ratio of enzyme to protein of 1:40 (w/w). The obtained tryptic digests were diluted to different concentrations with loading buffer (100 mM NH4HCO3 for IgG, 50%ACN/0.25% TFA for β-casein). The tryptic digest of BSA was lyophilized and stored at -20 °C for further use.



For the tryptic digests of proteins extracted from rat brain and rat liver: A Sprague Dawley male rat was sacrificed. First, the brain and liver were promptly taken out, then cut into bits, and washed with saline. Then, the brain and liver tissues were ground to powder in a mortar using liquid nitrogen. The sample was added into a new 1.5 mL EP tube, and each tube was added 400 uL of SDT lysis buffer. After that, the sample was blended and heated at 100℃ for 10 min. The sample was then cooled to room temperature with ice bath ultrasonic and loaded onto a 0.22 μm membrane tube followed by centrifugation at 14000 × g for 20 min. The FASP enzymatic hydrolysis was performed as follow. First, 100 μL of 50 mM iodoacetamide in UA buffer was added to the filter, and the sample was then incubated for 30 min at room temperature keeping in dark place. Then, 100 μL of 25 mM ABC (Applied Biosystems, Foster City, CA, USA) was added to each filter followed by centrifugation at14000 × g for 30 min. The protein suspensions were then digested with 40 μL of trypsin buffer at 37℃ for 18 h. Finally, The filtrate was collected by centrifugation at 14000g at room temperature for 30min, and the collected tube was added 40μL of 25 mM/L ABC buffer and the the filtrate was collected by centrifugation at 14000g at room temperature for 30min. The all collected filtrate was lyophilized for further use. Enrichment of phosphopeptides or glycopeptides from standard tryptic digests The SPIOs@SiO2@MOF nanocomposites (10 mg) were dispersed in 1 mL loading buffer (50%ACN/0.25%TFA for phosphopeptides, 100 mM NH4HCO3 for glycopeptides) for further use. For phosphopeptide enrichment: 20 μL of the above material suspension was added


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into 200 μL of tryptic digests (from β-casein or β-casein and BSA mixture) and the obtained mixture was vibrated in a shaker at room temperature for 45 min. After removal of the supernatant with the help of magnetic separation, the SPIOs@SiO2@MOF nanocomposites were washed with buffer (50%ACN/0.25%TFA) for three times to remove the nonspecifically adsorbed peptides. After that, 10 μL NH3·H2O (10 wt%) was added and sharply vibrated in the vortex to elute the phosphopeptides for 30 min. The eluate was analyzed by MALDI-TOF MS. For glycopeptide enrichment: 20 μL of the above material suspension was added into 200 μL of tryptic digests (from IgG or IgG and BSA mixture) and the obtained mixture was vibrated in a shaker at room temperature for 45 min. After removal of the supernatant with the help of magnetic separation, the SPIOs@SiO2@MOF nanocomposites were washed with buffer (100 mM NH4HCO3) for three times to remove the non-glycopeptides. After that, 10 μL of 50%ACN/1%TFA buffer was added and sharply vibrated in the vortex to elute the glycopeptides for 30 min. The eluate was analyzed by MALDI-TOF MS. Enrichment of phosphopeptides or glycopeptides from tryptic digests of proteins extracted from rat brain and rat liver For phosphopeptide enrichment: 0.5 mg tryptic digests of proteins extracted from rat brain was diluted with 500 mL loading buffer (50%ACN/0.25%TFA), and the the enrichment and elution processes were same as that in the peptide mixture enrichment. The elution was then lyophilized to dryness and analyzed by the Elite-LC-MS/MS (Thermo Orbitrap-Elite). The data were searched against the Uniprot rat reference



proteome database. For glycopeptide enrichment: 0.5 mg tryptic digests of proteins extracted from rat liver was diluted with 500 mL loading buffer (100 mM NH4HCO3), and the the enrichment and elution processes were same as above. The elution was then lyophilized to dryness and then redissolved in 100 μL 25 mM ABC buffer (H2O18 preparation). Then, 50 units of PNGase F were added to the solution and the mixture was incubated at 37 °C for 3 h. Finally, the mixture was lyophilized to dryness and analyzed by the Elite-LC-MS/MS (Thermo Orbitrap-Elite). The data were searched against the Uniprot rat reference proteome database. RESULTS AND DISCUSSION Characterization of SPIOs@SiO2@MOF nanocomposites The morphology and microstructure of all above samples were characterized by Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) (Fig. 1(A–F)). SEM images indicated that SPIOs, SPIOs@SiO2 nanospheres, and SPIOs@SiO2@MOF nanocomposites all had a uniform spherical morphology and good dispersion (Fig. 1(A–C)). As for TEM images, SPIOs@SiO2 nanospheres exhibited a thin and smooth SiO2 shell around the spherical SPIOs (Fig. 1E). After modification with MOFs, the surface of as-prepared SPIOs@SiO2@MOF nanocomposites was rather rough and distinct from the smooth surface of SPIOs@SiO2 nanospheres (Fig. 1F).


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Figure 1.

SEM micrographs and TEM micrographs of the naked SPIOs (A, D),

SPIOs@SiO2 nanospheres (B, E), and the SPIOs@SiO2@MOF nanocomposites (C, F). To further prove the successful modification with the SiO2 shell and MOF coating, Energy dispersive X-ray (EDX) (Fig. 2A) and Fourier transform infrared (FT-IR) spectroscopy (Fig. 2B) were employed to analyze the SPIOs, SPIOs@SiO2 nanospheres, and SPIOs@SiO2@MOF nanocomposites. As shown in Fig. 2A, EDX analysis of SPIOs@SiO2@MOF nanocomposites confirmed the existence of Zr element with atomic ratio of 4.60%, indicating that Zr-MOF shell had been successfully grafted. What’s more, the B element with atomic ratio of 2.89% exhibited that the organic ligand PBA was successfully introduced. The high content of B element would promote glycopeptide enrichment. Compared with the FT-IR spectrum of SPIOs (Fig. 2B (a)), a new characteristic absorption peak (1091 cm-1, νSi–O–Si ) in the spectrum of SPIOs@SiO2 nanospheres demonstrated the successful coating of SiO2 shell (Fig. 2B(b)). There were some new peaks in 1500–1600 cm−1 in FT-IR spectra of SPIOs@SiO2@MOF nanocomposites, corresponding to the groups of aromatic rings (Fig. 2B(c)).



Figure 2. (A) Energy dispersive X-ray (EDX) spectrum data of SPIOs@SiO2@MOF nanocomposites. (B) FTIR spectra of (a) SPIOs, (b) SPIOs@SiO2 nanospheres, and (c) SPIOs@SiO2@MOF nanocomposites. The nitrogen adsorption–desorption analysis (Fig. 3A) and X-ray diffraction (XRD) (Fig. 3B) were used to further analyze the property of MOF shell. Fig. 3A showed that the SPIOs@SiO2@MOF nanocomposites had a typical IV isotherm with a BET surface area of 249.75 m2 g−1 calculated by the Langmuir method.33 And the tiny pore of the MOF shell was estimated to be 6.8 angstroms based on density functional theory method. The property of porous nature confirmed the presence of the framework structure of MOFs. As shown in Fig. 3B, XRD patterns of the SPIOs, SPIOs@SiO2 nanospheres, and SPIOs@SiO2@MOF nanocomposites had typical diffraction peaks at 30.3° (220), 35.2° (311), 43.5° (400), 53.4° (422), 57.1° (511), and 62.8° (440), which were in accordance with the standard XRD data cards of Fe3O4 (JCPDS no.19-06290).34 The results indicated that the SiO2 coating and MOF shell had no influence on the magnetic crystalline phase. The additional three new diffraction peaks at around 7.4°, 8.4°, and 25.8° could be indexed to the crystalline structure of the MOFs formed from


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zircomiun tetrachloride, PTA, and PBA, indicating that the MOF shell was successfully formed (Fig. 3B(c)).

Figure 3. (A) N2 adsorption−desorption isotherm and pore size distribution (inset) of the SPIOs@SiO2@MOF nanocomposites. (B) XRD patterns of (a) SPIOs, (b) SPIOs@SiO2 nanospheres, and (c) SPIOs@SiO2@MOF nanocomposites. The standard XRD pattern of SPIOs (JCPDS no. 19-06290) is shown by solid bars. The magnetic properties of the SPIOs@SiO2@MOF nanocomposites were characterized using thermogravimetric analysis (TGA) (Fig. 4A) and vibrating sample magnetometer (VSM) (Fig. 4B). The TGA curves showed that the mass loss of around 18% of SPIOs was attributed to the physically adsorbed water (Fig. 6a). The mass loss of SPIOs@SiO2 nanospheres increased to about 20% because of the extra coated SiO2 layer (Fig. 4A(b)). After coating the MOF shell, it could be calculated that the weight loss of SPIOs@SiO2@MOF nanocomposites was about 39%, indicating the presence of a high amount of MOF on the nanocomposites (Fig. 4A(c)). Accordingly, the magnetic content of SPIOs@SiO2@MOF nanocomposites was as high as around 61%. The magnetic hysteresis curves showed that all samples had no obvious coercivity or remanence,35 demonstrating that the SPIOs@SiO2@MOF nanocomposites exhibited



superparamagnetism. The superparamagnetism derived from the small clusters in the SPIOs, which behaved as superparamagnets with a saturation magnetization value of 58 emu g−1 (Fig. 4B(a)). After coating the SiO2 layer and MOF shell, the saturation magnetization of the SPIOs@SiO2@MOF nanocomposites was about 36 emug-1 (Fig. 4B(c)). Meanwhile, the SPIOs@SiO2@MOF nanocomposites exhibited excellent magnetic response, could be separated from the solution in less than 10 s using an adscititious magnetic field. Overall, the great magnetic properties endowed the SPIOs@SiO2@MOF nanocomposites tremendous advantages for magnetic separation in enrichment application.

Figure 4. (A) TGA curves of the (a) SPIOs, (b) SPIOs@SiO2 nanospheres, and (c) SPIOs@SiO2@MOF nanocomposites. (B) VSM curves of the (a) SPIOs, (b) SPIOs@SiO2 nanospheres, and (c) SPIOs@SiO2@MOF nanocomposites. Application of the SPIOs@SiO2@MOF composites in phosphopeptide and glycopeptide enrichment from the tryptic digest of standard proteins To investigate the phosphopeptide and glycopeptide enrichment capacity of the SPIOs@SiO2@MOF nanocomposites, tryptic digest of the standard phosphoprotein (βcasein) and glycoprotein (IgG) were respectively employed as the testing samples. The


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workflow of phosphopeptide or glycopeptide enrichment from complex samples was displayed in Scheme 1B. Generally, the standard peptide mixture was mixed with SPIOs@SiO2@MOF nanocomposites in loading buffer and incubated at room temperature. Subsequently, the washing buffer was applied to remove the nonspecifically adsorbed peptides for several times. Finally, the captured target peptides were eluted and analyzed by MALDI-TOF MS. For phosphopeptide enrichment, the SPIOs@SiO2@MOF nanocomposites specifically captured and enriched phosphopeptides based on MOAC method. As shown in Fig. 5a, only one phosphopeptide with very weak signal intensity was detected because of the low abundance of phosphopeptides and serious signal suppression by the vast nonphosphorylated peptides. After enrichment with SPIOs@SiO2@MOF Figure 5. MALDI-TOF mass spectra of (a) β-casein digest (10-6 M) and (b) β-casein

digest (10-6 M) after enrichment with the SPIOs@SiO2@MOF nanocomposites. (s, monophosphopeptide; m, multiphosphopeptide; #, dephosphorylated peptide).



nanocomposites, the signals of non-phosphorylated peptides disappeared and the peaks of phosphopeptides dominated the spectrum with a clear background. Three phosphopeptides (β1s, β2s, and β3m) along with their dephosphorylated counterparts were detected with strong signals (Fig. 5b). The detailed information of the enriched phosphopeptides from tryptic digest of β-casein was listed in Table S1 (SI). As for glycopeptide enrichment, the BAAC method was employed to selectively enrich glycopeptides. Before enrichment, the signals of glycopeptides were severely suppressed by the abundant subsistent non-glycosylated peptides (Fig. 6a). However, after being treated by SPIOs@SiO2@MOF nanocomposites, twenty-five glycopeptides in total were identified with high intensity. And the peaks of glycopeptides dominated the spectrum with a clear background, indicating the excellent selectivity of SPIOs@SiO2@MOF nanocomposites towards glycopeptide enrichment (Fig. 6b). The detailed information of the enriched glycopeptides from

Figure 6. MALDI-TOF MS spectra of (a) direct analysis of human IgG digest (10-7 M) and (b) after enrichment by SPIOs@SiO2@MOF nanocomposites.


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tryptic digest of IgG was listed in Table S2 (SI). As a result, the SPIOs@SiO2@MOF nanocomposites combining with IMAC and BAAC methods could be applied to specifically enrich not only phosphopeptides but also glycopeptides. Figure 7. MALDI-TOF mass spectra of the tryptic digests of β-casein (10-8 M): (a)

direct analysis, (b) analysis after enrichment using the commercial TiO2 microspheres, and (c) analysis after enrichment by the SPIOs@SiO2@MOF nanocomposites. (s, monophosphopeptide; m, multiphosphopeptide; #, dephosphorylated peptide). Furthermore, to verify the high efficiency of SPIOs@SiO2@MOF nanocomposites for phosphopeptide or glycopeptide enrichment, we employed commercial TiO2 microspheres (for phosphopeptide enrichment) and as-prepared SPIOs@PEI@PBA nanospheres (for glycopeptide enrichment) as controls. The tryptic digest mixture of βcasein (10-8 M) was used to evaluate the phosphopeptide enrichment performance between the SPIOs@SiO2@MOF nanocomposites and commercial TiO2 microspheres. As for SPIOs@SiO2@MOF nanocomposites, the phosphopeptides were easily detected



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with a clear background after enrichment (Fig. 7b). However, there were many nonphosphorylated peaks in the spectrum after enrichment with the commercial TiO2 microspheres (Fig. 7c). And the MS signal intensity of enriched phosphopeptides was much lower than that of SPIOs@SiO2@MOF nanocomposites. Figure 8. MALDI-TOF MS spectra of (a) direct analysis of human IgG digest (10-8 M),

(b) after enrichment by SPIOs@PEI@PBA nanospheres, and (c) enrichment by SPIOs@SiO2@MOF nanocomposites. On the other hand, the tryptic digest mixture of IgG (10-8 M) was used to evaluate the glycopeptide

enrichment

performance

between

the

SPIOs@SiO2@MOF

nanocomposites and SPIOs@PEI@PBA nanospheres. As shown in Fig. 8c, only six glycopeptides could be found with abundant peaks of non-glycosylated peptides, and that the MS signal intensity of enriched glycopeptides was relative low after enrichment by SPIOs@PEI@PBA nanospheres. These results indicated that the enrichment


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efficiency of SPIOs@SiO2@MOF nanocomposites was evidently superior to those contrast materials, which might be ascribed to unique boronic acid functionalized ZrMOF structure and excellent magnetic response property.

Figure 9. (A) MALDI-TOF mass spectra of the tryptic digest of β-casein with different concentrations after enrichment with SPIOs@SiO2@MOF nanocomposites: (a) 10-9 M and

(b)

10-10

M.

(s,

monophosphopeptide;

m,

multiphosphopeptide;

#,

dephosphorylated peptide). (B) For the glycopeptide enrichment from IgG tryptic digest: (a) 10-9 M, (b) 10-10 M, and (c) 10-11 M. In consideration of the low concentration of phosphopeptides or glycopeptides in the complex biological samples, the detection sensitivity is regarded as the significant performance. To confirm the sensitivity of the SPIOs@SiO2@MOF nanocomposites, different concentrations of β-Casein tryptic digests and IgG tryptic digests were used as the test samples. As shown in Fig. 9A, all three phosphopeptides could be still detected with relatively high signal even the β-casein digest concentration was as low as 10-10 M. As for glycopeptides, tryptic IgG digests with different concentrations (10−9 M, 10−10



M, and 10−11 M) were enriched by SPIOs@SiO2@MOF nanocomposites, and that 11, 9 and 6 glycopeptides could be still clearly detected respectively (Fig. 9B). As a result, the SPIOs@SiO2@MOF nanocomposites possessed prominent sensitivity toward phosphopeptide and glycopeptide enrichment.

Figure 10. (A) MALDI-TOF mass spectra for phosphopeptide enrichment from a tryptic digest mixture of β-casein and BSA at a molar ratio of 1:100: (a) before enrichment, (b) afer enrichment; 1:400: (c) afer enrichment. (s, monophosphopeptide; m, multiphosphopeptide; #, dephosphorylated peptide). (B) MALDI-TOF mass spectra for glycopeptide enrichment from a tryptic digest mixture of IgG and BSA at a molar ratio of 1:100: (a) before enrichment, (b) afer enrichment; 1:500: (c) afer enrichment. The detection selectivity of affinity materials is also regarded as the significant index because of the abundant interferential peptides in the complex biological samples. Therefore, the mixture of β-Casein or IgG and BSA with different molar ratios were employed to evaluate selectivity of the SPIOs@SiO2@MOF nanocomposites. When the molar ratio of β-casein and BSA was 1:100, the MS signals of phosphopeptides


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were badly overwhelmed by the non-phosphopeptides in direct MS analysis (Fig. 10A(a)). After selective enrichment, all three phosphopeptides dominated the spectrum with a clear background (Fig. 10A(b)). With an increasing ratio as 1:400, three phosphopeptides could be easily detected with high signal intensity after enrichment processes (Fig. 10A(c)). For glycopeptides, with a molar ratio of 1:100 (IgG: BSA), all MS signals of glycopeptides appeared in mass spectrum without any interferential peptides (Fig. 10B(b)). Furthermore, 9 glycopeptides could still be identified when the molar ratio of IgG and BSA was as low as 1:500 (Fig. 10B(c)). All the results indicated that the SPIOs@SiO2@MOF nanocomposites were endowed with excellent selectivity and specificity by the high surface area and excellent performance of MOFs. In addition, the recyclability of the SPIOs@SiO2@MOF nanocomposites was evaluated (Fig. S1 in the SI). We employed β-casein digest as an analyte in the enrichment of each test. The phosphopeptides were clearly detected in the β-casein digest after enrichment by SPIOs@SiO2@MOF nanocomposites. Whether using the signal intensity or the identified phosphopeptides, the stabilizing effect can be obtained for the first cycle and the fifth cycle, suggesting that the nanocomposites owned prominent recyclability. Application of the SPIOs@SiO2@MOF composites in phosphopeptide and glycopeptide enrichment from the complex biological samples Inspired by the above excellent enrichment performance from the tryptic digest of standard proteins, SPIOs@SiO2@MOF nanocomposites were further applied to enrich phosphopeptides and glycopeptides from the real complex biological samples (tryptic



digest of proteins extracted from the mouse brain and mouse liver). For phosphopeptide enrichment, the tryptic digest of proteins extracted from the mouse brain was incubated with the SPIOs@SiO2@MOF nanocomposites, and the eluent was analyzed by EliteLC-MS/MS. The result was analyzed by database searching with the high confident false discovery rate (FDR≤0.01) for phosphopeptide identification. As shown in Fig. S2, a total of 174 unique phosphopeptides were identified after enrichment with the SPIOs@SiO2@MOF nanocomposites. The explicit data of about the identified phosphopeptides was shown in Table S3 in the SI. Interestingly, the multiphosphopeptides occupied about 39.1% of the total identified phosphopeptides, which was larger than commercial TiO2,36 implying that the SPIOs@SiO2@MOF nanocomposites tend to enrich multiphosphopeptides. To date, it is still a challenge on the enrichment and analysis of multiphosphopeptides due to the poor ionization efficiency of multiphosphopeptides and severe suppression by the coexistence of nonphosphorylated peptides and monophosphopeptides.37 The emergence of the SPIOs@SiO2@MOF nanocomposites would provide a promising platform to enrich multiphosphopeptides in real biological samples. On the other hand, the SPIOs@SiO2@MOF nanocomposites could identified 152 unique glycopeptides from the tryptic digest of proteins extracted from the mouse liver. The detailed data of about the identified glycopeptides was shown in Table S4 in the SI. Overall, the above results revealed that SPIOs@SiO2@MOF nanocomposites could selectively enrich the lowabundance phosphopeptides and glycopeptides from the real complex biological samples, holding enormous potential for peptidomics research.


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CONCLUSIONS A novel dual-functional magnetic MOF nanocomposite was ingeniously designed and prepared by a facile synthetic route for efficient enrichment of phosphopeptides and glycopeptides. The as-prepared SPIOs@SiO2@MOF nanocomposites possessed the excellent properties of superior magnetic response, large surface area, and porous performance, exhibiting great convenience as well as high selectivity and detection sensitivity towards phosphopeptide and glycopeptide enrichment in tryptic digest of standard proteins. Furthermore, the results of the selective enrichment toward phosphopeptides and glycopeptides from real biological samples (rat brain and rat liver lysate) further demonstrated the great practicability of this affinity material in identifying low abundance phosphopeptides and glycopeptides from complex biological samples. Regrettably, the SPIOs@SiO2@MOF nanocomposites could not enrich phosphopeptides and glycopeptides synchronously in one enrichment process because the enrichment environment is different for phosphopeptide and glycopeptide enrichment. This will be the direction of our future work. In conclusion, we offered a general strategy to guide the design of multifunctional magnetic MOF affinity materials for multiple peptide enrichment, providing a huge potential for comprehensive peptidomes analysis. ASSOCIATED CONTENT Supporting Information The figure of identified phosphopeptides from tryptic digests of rat brain lysate after enrichment with the SPIOs@SiO2@MOF nanocomposites; Detailed information on the



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phosphopeptides and glycopeptides enriched by SPIOs@SiO2@MOF nanocomposites from tryptic digests of β-casein, IgG, rat brain lysate, and rat liver lysate. AUTHOR INFORMATION Corresponding Author Yao Wu ([email protected]); Fang Lan ([email protected]). Notes All authors declare no competing financial interests. ACKNOWLEDGMENT Financial support is gratefully acknowledged from National Key Research and Development Program of China (No. 2016YFC1100403), National Natural Science Foundation of China (No.31171037, 51673124), the Research Program of Sichuan Province (No. 2018GZ0324), and Innovative Spark Program of Sichuan University (No.

2018SCUH0044).

We

gratefully

acknowledge

Shanghai

Omicsspace

Biotechnology Co. Ltd. for technical assistance with the peptide/protein identification. REFERENCES 1. Mann, M.; Jensen, O. N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 2003, 21, 255-261, DOI 10.1038/nbt0303-255. 2. Gruber, W.; Scheidt, T.; Aberger, F.; Huber, C. G. Understanding cell signaling in cancer stem cells for targeted therapy-can phosphoproteomics help to reveal the secrets? Cell Communication and Signaling 2017, 15, 12-27, DOI 10.1186/s12964-017-0166-1.


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TOC

A novel magnetic metal-organic framework nanocomposite (SPIOs@SiO2@MOF) was designed and fabricated for highly efficient enrichment of both phosphopeptides and glycopeptides.


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