Dendritic Mesoporous Silica Nanoparticles with Abundant Ti4+ forPhosphopeptide Enrichment from Cancer Cells with 96% SpecificityYayun Hong,† Yating Yao,‡ Hongli Zhao,*,† Qianying Sheng,† Mingliang Ye,‡ Chengzhong Yu,*,§

and Minbo Lan*,†

†Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China Universityof Science and Technology, Shanghai 200237, People’s Republic of China‡Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute ofChemical Physics, Chinese Academy of Sciences (CAS), Dalian, China§Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia

*S Supporting Information

ABSTRACT: Selective enrichment and sensitive detection ofphosphopeptides are of great significance in many bioapplications.In this work, dendritic mesoporous silica nanoparticles modified withpolydopamine and chelated Ti4+ (denoted DMSNs@PDA-Ti4+)were developed to improve the enrichment selectivity ofphosphopeptides. The unique central-radial pore structures endowedDMSNs@PDA-Ti4+ with a high surface area (362 m2 g−1), a largepore volume (1.37 cm3 g−1), and a high amount of chelated Ti4+ (75μg mg−1). Compared with conventional mesoporous silica-basedmaterials with the same functionalization (denoted mSiO2@PDA-Ti4+) and commercial TiO2, DMSNs@PDA-Ti4+ showed betterselectivity and a lower detection limit (0.2 fmol/μL). Moreover,2422 unique phosphopeptides were identified from HeLa cellextracts with a high specificity (>95%) enabled by DMSNs@PDA-Ti4+, better than those in previous reports.

Protein phosphorylation, a widely found protein post-translational modification, is involved in many important

cellular activities including signaling transduction, molecularrecognition, and metabolic process.1−5 Nearly one-third of allproteins in eukaryotes are phosphoproteins which may containpotential biomarkers with clinical significance.6−8 Investigationof protein phosphorylation under different physiologicalconditions is important in understanding the signalingpathways, the mechanism of disease generation, anddiagnosis,9,10 where sensitive detection of phosphorylatedproteins in biosamples is the key step.11−13 Currently, massspectrometry (MS)-based technologies are the primaryanalytical tools for accurately identifying and locatingphosphorylation sites due to their high sensitivity and strongsequence analysis capability.14,15 However, the low abundanceand low ionization efficiency of phosphopeptides make themdifficult to be identified by direct MS analysis.16−19 Therefore,separation and enrichment of phosphopeptides from compli-cated samples prior to MS analysis is necessary.To date, many materials and techniques have been developed

for phosphopeptide enrichment. Immobilized metal ion affinitychromatography (IMAC) is one of the most widely usedenrichment techniques in phosphoproteomics analysis. Never-theless, the major limitation of this method is the high level ofnonspecific binding of acidic peptides.20−23 Although much

effort has been devoted to improving the enrichment selectivityof IMAC materials, the results are still unsatisfactory. Theenrichment selectivity of IMAC materials mainly depends onthe hydrophilicity of materials and the amount of theimmobilized metal ions. On one hand, phosphopeptides showhigher hydrophilicity than other peptides;24−27 thus, increasingthe hydrophilicity of the materials can improve the enrichmentselectivity of phosphopeptides. On the other hand, IMACmaterials primarily rely on the electrostatic interactionsbetween phosphate groups and metal ions (e.g., Ti4+, Fe3+,Zn2+, Ga3+, and Zr4+);28−30 thus, the more the immobilizedmetal ions, the better the enrichment selectivity. Therefore, anIMAC material with the above properties is anticipated to haveexcellent performance for phosphopeptide enrichment.In order to increase the amount of the immobilized metal

ions and subsequent phosphopeptide entrapment, high surfacearea and suitable pore structures are both important forcandidate materials. Although mesoporous silica materials withhigh surface areas have been widely used in the synthesis ofIMAC materials, most of them have relatively small pore sizes

Received: March 27, 2018Accepted: May 25, 2018Published: May 25, 2018

Article

pubs.acs.org/acCite This: Anal. Chem. 2018, 90, 7617−7625

© 2018 American Chemical Society 7617 DOI: 10.1021/acs.analchem.8b01369Anal. Chem. 2018, 90, 7617−7625

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(<3 nm).31,32 Such small mesopores could be easily blocked inthe following modification process. In addition, for small poreswith long pore length, the so-called “shadow effect” wouldhamper the diffusion and release of target phosphopep-tides.33−35 In this regard, dendritic mesoporous silica nano-particles (DMSNs),36−39 a new type of mesoporous silicamaterial, are considered as an optimal alternative to conven-tional mesoporous silica. DMSNs have a unique central-radialpore structure, large pore sizes, and highly accessible surfaceareas; thus, polymers can be easily modified onto the inner wallof the pores without significant pore blocking, therebyincreasing the binding amount of chelating ligands. Simulta-neously, the large pore size and short pore length can preventthe “shadow effect” and provide efficient mass transporta-tion.40,41 Polydopamine (PDA) with favorable hydrophilicityand biocompatibility possesses high cross-linking ability tometal ions.42−46 The modification of PDA on DMSNs isexpected to provide a new type of IMAC material forphosphopeptide enrichment with high performance.Herein, a novel IMAC material, dendritic mesoporous silica

nanoparticles modified with PDA and chelated Ti4+ (denoted asDMSNs@PDA-Ti4+), has been synthesized for phosphopeptideenrichment. DMSNs@PDA-Ti4+ exhibit a large pore size of18.8 nm, a surface area of 362 m2 g−1, a pore volume of 1.37cm3 g−1, and a high chelated Ti4+ amount of 75 μg mg−1. Theefficacy of DMSNs@PDA-Ti4+ has been compared withconventional mesoporous silica-based materials (namedmSiO2@PDA-Ti4+) and commercial TiO2 for phosphopeptideenrichment in various biological samples, including standardphosphoprotein, nonfat milk, human serum, and HeLa cellextracts. DMSNs@PDA-Ti4+ demonstrate a low phosphopep-tide detection limit of 0.2 fmol/μL and an extremely highspecificity (>95%) of phosphopeptides identified from HeLacell extracts, showing great promise for phosphoproteomestudies.

■ EXPERIMENTAL SECTIONMaterials and Chemicals. Cetyltrimethylammonium bro-

mide (CTAB), tetraethyl orthosilicate (TEOS), triethanol-amine (TEA), sodium salicylate (NaSal), dopamine hydro-chloride (DA·HCl), and Ti(SO4)2 were purchased fromAladdin. Ammonium bicarbonate (NH4HCO3), urea, proteaseinhibitor cocktail (components: AEBSF, Aprotinin, Bestatin, E-64, Leupeptin, Pepstatin A), Triton X-100, β-glycerophosphatedisodium salt hydrate (C3H7Na2PO6), sodium pyrophosphatetetrabasic (Na4P2O7), sodium fluoride (NaF), sodiumorthovanadate (Na3VO4), α-casein (from bovine milk), 2,5-dihydroxybenzoic acid (DHB), bovine serum albumin (BSA),and trypsin were obtained from Sigma-Aldrich. Acetonitrile(ACN) and trifluoroacetic acid (TFA) were chromatographicgrade. Commercial TiO2 (5 μm) was produced by Shimadzu.Nonfat milk was bought from a local shop. Water used inexperiments was purified using a Milli-Q system (Millipore,Bedford, MA). All other reagents used in this research were atleast of analytical grade without further purification.Preparation of DMSNs@PDA-Ti4+ and mSiO2@PDA-

Ti4+. DMSNs were synthesized according to a literaturemethod.37 Typically, 0.136 g of TEA was added to 50 mL ofdeionized water. The solution was stirred at 80 °C in an oilbath for 0.5 h. Afterward, 0.76 g of CTAB and 0.336 g of NaSalwere added and kept under stirring for another 1 h. Then, 8 mLof TEOS was added to the water−CTAB NaSal−TEA solutionunder a gentle stirring rate of 200 rpm for 2 h. The products

were collected by centrifugation and washed with ethanol threetimes. Finally, the obtained products were extracted with HCland methanol solution at 65 °C three times to remove thetemplate and dried in vacuum at room temperature overnight.The synthesis of mSiO2 was similar to that of DMSNs, exceptNaSal was not added during the reaction.The dried DMSNs and mSiO2 were modified with PDA by

oxidative self-polymerization of DA in a mild condition. Briefly,0.4 g of DA·HCl was dissolved in 200 mL of Tris buffer (10mM, pH 8.5) by 5 min ultrasonication, and then 0.2 g ofDMSNs or mSiO2 was added and sonicated for another 5 min.The above solution was transferred into a 500 mL flask andstirred for 12 h at 30 °C. The products were collected bycentrifugation and washed with water several times.Finally, the DMSNs@PDA or mSiO2@PDA were incubated

in Ti(SO4)2 solution (100 mM) for 2 h to immobilize Ti4+

cations. The products were washed with deionized waterseveral times and then lyophilized to dryness.

Characterization. Transmission electron microscopy(TEM) images were carried out by JEOL JEM-1400 trans-mission electron microscope (JEOL, Japan). Field emissionscanning electron microscope (FE-SEM) images were obtainedby Nova NanoSEM 450 field emission scanning electronmicroscope (FEI, USA). The elemental mapping patterns wereobtained using a XM-2 EDS (EDAX, USA) equipped with aTecnai G2 F30 S-TWIN field emission transmission electronmicroscope (FEI, USA). Fourier transform infrared (FT-IR)spectra were performed on a Nicolet 6700 using KBr pellets(Thermo Fisher Scientific, USA). Thermogravimetric analysis(TGA, Pyris 1 TGA, Perkinelmer, USA) was performed fromRT to 800 °C at a heating rate of 20 °C/min underatmosphere. Inductively coupled plasma-atomic emissionspectrometry (ICP-AES) was carried out with a Varian ICP-710ES instrument (Varian, USA). Nitrogen adsorption−desorption isotherms were measured on an ASAP 2020apparatus (Micrometritics, USA). The specific surface areaand pore volume were determined by the Brunauer−Emmett−Teller (BET) method and the Barrett−Joyner−Halenda (BJH)model, respectively.

Preparation of Protein Digestion. α-Casein (1 mg) wasdissolved in 1.0 mL of ammonium bicarbonate buffer (50 mM,pH 8.2) and digested by trypsin for 17 h at 37 °C with anenzyme/substrate ratio of 1:25 (w/w).BSA (1 mg) was dissolved in 1 mL of denaturing buffer (8 M

urea, 50 mM NH4HCO3, pH 8.2). Afterward, the proteinsolution was incubated with 5 μL of dithiothreitol (200 mM) at56 °C for 45 min. Then, 20 μL of iodoacetamide (200 mM)was added and incubated for another 30 min in the dark. Theprotein mixture was digested by trypsin at an enzyme/substrateratio of 1:25 (w/w) for 17 h at 37 °C. The protein digests werelyophilized to dryness and stored in a cryogenic refrigerator at−80 °C before use.Human serum samples were collected from healthy people

and obtained from the Sixth People’s Hospital affiliated withShanghai Jiaotong University according to the standard clinicalprocedures. The serum sample was stored at −80 °C and usedfor endogenous phosphopeptide enrichment without furthertreatment.Nonfat milk (30 μL) was diluted with 1 mL of NH4HCO3

(25 mM, pH = 8.0), and then the mixtures were centrifuged at16 000 g for 15 min. The supernatant was collected anddenatured at 100 °C for 10 min. Subsequently, the mixtureswere treated with 40 μg of trypsin for 17 h at 37 °C. The

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obtained proteolytic digests were stored at −80 °C for furtheruse.HeLa cells were cultured to 90% confluence in 15 cm

diameter dishes. After being washed twice with ice-cold PBSbuffer (pH 7.4), the cells were resuspended in lysis buffer (8 Murea, 50 mM Tris-HCl, pH 7.4) consisting of 2% proteaseinhibitor cocktail, 1% Triton X-100, 1 mM C3H7Na2PO6, 1 mMNa4P2O7, 1 mM NaF, and 1 mM Na3VO4. The suspension wasthen sonicated for 180 s (3 s sonication was followed with 3 sinterval) in ice. The cell debris was removed by centrifugationat 15 000 g at 4 °C for 20 min. The supernatant was collected,and five volumes of precipitation solution (acetone/ethanol/acetic acid, 50/49.9/0.1, v/v/v) was added to precipitate theprotein at −20 °C for at least 2 h and then centrifuged at 15000 g at 4 °C for 20 min to collect the protein precipitate.Subsequently, the obtained protein pallet was resuspended in50 mM NH4HCO3 buffer, and the concentration of the proteinwas measured by Bradford assay. The procedure of the proteindigestion was the same as that of BSA.Selective Enrichment of Phosphopeptides from

Protein Digests and Tryptic Digests of HeLa CellExtracts. Twenty micrograms of DMSNs@PDA-Ti4+ wasadded into 200 μL of loading buffer (ACN/H2O/TFA,50:49.5:0.5, v/v/v) containing α-casein tryptic digest (2 μL),human serum (2 μL), or proteins extracted from nonfat milk (2μL), and the mixture was incubated at room temperature for 30min. Subsequently, the materials were collected by centrifuga-tion and rinsed with 100 μL of washing buffer (ACN/H2O/TFA, 50/49.9/0.1, v/v/v) three times. Then the phosphopep-tides were eluted from the materials with 10 μL of NH4OHsolution (0.4 M) for 20 min incubation. The eluent wascollected by centrifugation and analyzed by MALDI-TOF MS.For HeLa cell extracts enrichment, 200 μg HeLa cell extracts

were diluted with 500 μL of loading buffer (DMSNs@PDA-Ti4+: 1% TFA in 1:1 ACN-H2O; commercial TiO2: 0.1% TFA,2 M lactic acid solution in 1:1 ACN-H2O), and then 2 mg ofDMSNs@PDA-Ti4+ or commercial TiO2 was respectivelyadded for phosphopeptide enrichment. The following enrich-ment procedure was the same as above. The eluent wascollected and lyophilized for nano-LC-MS/MS analysis.MALDI-TOF MS Analysis. One microliter analytes were

dropped on the plate, and then 1 μL of matrix (DHB, 25 mg/mL, ACN/H2O/H3PO4, 70:29:1, v/v/v) was dropped andanalyzed by MALDI-TOF MS. All the MALDI-TOF MSanalyses were performed on a 4800 plus MALDI-TOF MS (ABSciex, USA) with a Nd:YAG laser at 355 nm, a laser pulsefrequency of 200 Hz, and an acceleration voltage of 20 kV inreflect positive ion mode. The scan range was 1000−3500 m/z.Nano LC-MS/MS Analysis. The nano LC-MS/MS analyses

were performed on a Dionex UltiMate 3000 RSLCnano system(Thermo Scientific, USA) with a Q-Exactive mass spectrometer(Thermo Scientific, USA). The lyophilized peptides wereresuspended in 1% FA/H2O and automatically loaded onto a 3cm C18 trap column (200 μm i.d.) at a flow rate of 5 μL/min.Subsequently, the peptides were separated on a reverse C18capillary analytical column (75 μm × 15 cm) under a lineargradient elution condition. For the capillary analytical column(75 μm i.d.), one end of the fused-silica capillary was firstmanually pulled to a fine point with a spray tip and then packedin-house with C18 AQ particles (5 μm, 120 Å). For RPLCseparation, mobile phase A (0.1% FA in H2O) and mobilephase B (0.1% FA in 80% ACN) were used to establish thelinear gradient elution method which was carried out as follows:

0−4% mobile phase B for 2 min; 4−35% B for 90 min; 35−45% B for 10 min; 45−90% B for 5 min; 90% B for 5 min; andfinally equilibration with mobile phase A for 15 min. The flowrate was adjusted to ∼300 nL/min.The Q-Exactive mass spectrometer was operated in data-

dependent MS/MS acquisition mode. The electrospray voltagewas set to 2.0 kV, and the temperature of the ion transfercapillary was set as 250 °C. The full MS scan acquired in theOrbitrap mass analyzer was from m/z 400 to 2000 with a massresolution of 70 000 (m/z 200). The 12 most intense parentions with charge states ≥2 from the full scan were fragmentedby higher-energy collisional dissociation (HCD) with anormalized collision energy (NCE) of 27%. The MS/MSacquisitions were performed in Orbitrap with a resolution of 35000 (m/z 200), and the automatic gain control (AGC) targetwas set to 1 × 105 with a max injection time of 120 ms.Dynamic exclusion was set as 30 s. System control and datacollection were carried out by Xcalibur software.

Database Retrieval and Data Analysis. The MS raw datafiles were exported using the Proteome Discoverer software(Thermo Fisher Scientific, version 1.4.0.288), and the MS/MSresults were searched by Mascot (Matrix Science, London,U.K.; version 2.3) against a Uniprot-SwissProt database(taxonomy: human, 20201 entries). The data retrievalparameters were as follows: precursor-ion mass tolerance, 10ppm; fragment-ion mass tolerance, 0.05 Da. Two missedcleavages were allowed by trypsin digestion. Carbamidomethylof cysteine (C) was set as fixed modification. Oxidation ofmethionine (M) and phosphorylation of serine/threonine/tyrosine (S/T/Y) were set as the variable modifications.Peptide level false discovery rates (FDR) were controlledlower than 1% by the percolator algorithm. Finally, thePhosphoRS 3.0 was used to determine the probability ofphosphorylation sites.

■ RESULTS AND DISCUSSIONSynthesis and Characterization of DMSNs@PDA-Ti4+.

The synthesis procedure for DMSNs@PDA-Ti4+ is illustratedin Scheme 1a. DMSNs with a central-radial pore structure weresynthesized by a sodium salicylate assisted method.37 The poresurface with Si−OH groups was modified with PDA byoxidative self-polymerization of DA in a mild condition.Subsequently, Ti4+ was immobilized on the PDA-modifiednanoparticles (DMSNs@PDA) by the chelation of PDA withTi4+ to obtain DMSNs@PDA-Ti4+.The morphology and size of the products were observed by

TEM and SEM. TEM images (Figure 1a) reveal that DMSNsare monodispersed with a uniform diameter of ∼150 nm andobvious central-radial dendritic mesopores. After PDA coating,both DMSNs@PDA and DMSNs@PDA-Ti4+ have no obviouschange in particle sizes (Figure 1b, c). SEM images (Figure1d−f) show that the all three samples possess large poreopenings, indicating that the PDA coating and Ti4+ chelatingprocedures did not block the large mesopores. Scanning TEM(STEM) image (Figure 1g) of DMSNs@PDA-Ti4+ combinedwith EDS elemental mapping of a single particle (Figure 1h−m) clearly reveal that Si, C, N, O, and Ti elements arehomogeneously distributed in DMSNs@PDA-Ti4+. In addition,TEM images of mSiO2 synthesized without NaSal, mSiO2@PDA, and mSiO2@PDA-Ti4+ show typical small mesopores(Figure S1), in accordance with the literature report.47,48

FTIR spectra of DMSNs, DMSNs@PDA, and DMSNs@PDA-Ti4+ were recorded to provide qualitative information for

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PDA coating and Ti4+ chelating (Figure 2a). Compared toDMSNs, DMSNs@PDA display new characteristic adsorption

peaks: the band at 1619 cm−1 is attributed to the N−Hstretching, and the peaks at 1503 and 1447 cm−1 are assigned tobenzene ring C−C vibration.42 After chelating Ti4+, a newadsorption peak appearing at 606 cm−1 is attributed to Ti−Ostretching.49

TGA was further utilized to confirm the large capacity ofDMSNs for PDA coating. As shown in Figure 2b, the weightratio of PDA in DMSNs@PDA is 37.04%, while in mSiO2@PDA only 25.49%, indicating that the large and open dendriticmesopores of DMSNs can provide larger capacity for PDAcoating than mSiO2. In addition, the mass ratio of DMSN toDA was optimized during the PDA modification process (seeSupporting Information Figure S2), reaching a saturated PDAloading at a mass ratio (DMSN to DA) of 1:2.In theory, the amount of immobilized Ti4+ is proportional to

the amount of coated PDA. Thus, ICP-AES was used toquantify the amount of immobilized Ti4+. For DMSNs@PDA-Ti4+, the amount of Ti4+ was as high as 75 μg mg−1, which ishigher than that of mSiO2@PDA-Ti4+ (48 μg mg−1) and manyprevious IMAC nanomaterials, such as Fe3O4@SiO2@PEG-Ti4+ (32.98 μg mg−1),50 MCNC@PMAA@PEGMP-Ti4+ (41μg mg−1),51 and Fe3O4@SiO2@(HA/CS)10-Ti

4+ (44.38 μgmg−1).52 These results suggest that the DMSNs@PDA possessstrong ability for Ti4+ chelating due to the unique structure ofDMSNs.Furthermore, N2 adsorption−desorption was performed to

study the texture properties. The surface area of DMSNs wascalculated to be 679 m2 g−1, and the pore volume was 2.91 cm3

g−1, indicating a highly accessible surface area for PDA and Ti4+

loading. The pore size of DMSNs was calculated to be 23.2 nm(Figure 2c inset), which could provide enough space to loadPDA without significant pore blocking. After modification withPDA and Ti4+ (Figure 2d), the DMSNs@PDA-Ti4+ still have ahigh surface area (361 m2 g−1) and large pore volume (1.37 cm3

g−1) that could provide a large number of affinity sites forphosphopeptide enrichment. In addition, the large pore size ofDMSNs@PDA-Ti4+ (18.8 nm) could effectively prevent the“shadow effect” from the small and deep pores. For

Scheme 1. (a) Synthesis Flow Chart of DMSNs@PDA-Ti4+.(b) Processing of Phosphopeptide Enrichment from ProteinDigestion Using DMSNs@PDA-Ti4+

Figure 1. TEM and SEM images of DMSNs (a, d), DMSNs@PDA (b,e), DMSNs@PDA-Ti4+ (c, f); STEM image of DMSNs@PDA-Ti4+

(g); EDX elemental mapping images of Si (h), C (i), N (j), O (k), andTi (l, m) of the nanoparticle highlight in orange square in (g).

Figure 2. (a) FT-IR spectra of (i) DMSNs, (ii) DMSNs@PDA, and(iii) DMSNs@PDA-Ti4+; (b) TGA curves of (i) DMSNs, (ii)mSiO2@PDA, and (iii) DMSNs@PDA; N2 adsorption−desorptionisotherms and pore size distributions (inset) of (c) DMSNs and (d)DMSNs@PDA-Ti4+.

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comparison, the surface area and pore properties of mSiO2 andmSiO2@PDA-Ti4+ were also investigated (Figure S3). Thesurface area of mSiO2 is 436 m2 g−1 , and the pore volume is0.89 cm3 g−1. After PDA and Ti4+ coating, the surface area andpore volume decreased to 174 m2 g−1 and 0.36 cm3 g−1,respectively, which are lower than that of DMSNs@PDA-Ti4+.Selective Enrichment of Phosphopeptides from α-

Casein Tryptic Digests. To demonstrate the phosphopeptideenrichment efficacy of DMSNs@PDA-Ti4+, α-casein trypticdigests were selected as the model sample. The procedure forthe enrichment of phosphopeptides from protein digestion isshown in Scheme 1b. For comparison, mSiO2@PDA-Ti4+ andcommercial TiO2 were also used to enrich phosphopeptidesfrom α-casein tryptic digests. As shown in Figure 3a, for direct

analysis of α-casein tryptic digests at a concentration of 4 ×10−7 M, merely eight phosphopetides were detected with weakMS intensity due to the severe interference of the abundantnonphosphopeptides. However, after enrichment by DMSNs@PDA-Ti4+ (Figure 3b), almost all of the nonphosphopeptideswere removed, and 36 phosphopetides (the detailedinformation on the identified phosphopeptides is given inTable S1) were identified with greatly improved signal-to-noise(S/N) ratio. Simultaneously, a majority of the correspondingdephosphorylated peptides with mass loss of 98 Da were alsoobserved. For mSiO2@PDA-Ti4+ and commercial TiO2, thenumber of identified phosphopeptides was reduced to 24 and18 phosphopeptides, respectively (Figure S4). The numbers ofphosphopeptides enriched by the three materials from α-caseintryptic digests are directly compared in Figure 4a. Theenrichment capacity of the three materials was also investigatedusing different amounts of nanoparticles to enrich phospho-peptides from a fixed amount of α-casein tryptic digests (1 μg).As shown in Figure 4b, the phosphopeptide loading capacitiesof DMSNs@PDA-Ti4+, mSiO2@PDA-Ti4+, and commercialTiO2 were calculated to be about 100, 67, and 40 mg g−1,respectively. These results demonstrate that the DMSNs@PDA-Ti4+ have an outstanding phosphopeptide enrichmentperformance and large phosphopeptide loading capacity due tothe excellent hydrophilicity, highly accessible surface area, andlarge amount of immobilized Ti4+.

The selectivity of the three materials for phosphopeptideenrichment was also compared by MALDI-TOF MS. As shownin Figure 5, when the molar ratio of α-casein and BSA was

1:5000 (loading buffer: 50% ACN, 6% TFA), 20 phosphopep-tides were identified with a clean background after enrichmentwith DMSNs@PDA-Ti4+ (Figure 5a). However, for mSiO2@PDA-Ti4+ (Figure 5b), only eight phosphopeptides with agreatly reduced signal intensity were observed together withsome nonphosphopeptides. For commercial TiO2, the spec-trum was dominated by nonphosphopeptides, and only fourphosphopeptides could be identified (Figure 5c). The numberand signal intensity of enriched phosphopeptides by threematerials are plotted in Figure 6 for direct comparison, whichclearly show that DMSNs@PDA-Ti4+ have significantly higherphosphopeptides enrichment selectivity and efficiency than

Figure 3. MALDI-TOF mass spectra of the tryptic digest of α-casein(4 × 10−7 M, 200 μL) (a) before and (b) after enrichment byDMSNs@PDA-Ti4+. # indicates dephosphorylated peptides.

Figure 4. (a) Comparison of the number of identified phosphopep-tides enriched from α-casein and nonfat milk by DMSNs@PDA-Ti4+,mSiO2@PDA-Ti4+, and commercial TiO2; (b) Comparison ofphosphopeptide loading capacity among DMSNs@PDA-Ti4+,mSiO2@PDA-Ti4+, and commercial TiO2.

Figure 5. MALDI-TOF mass spectra of the tryptic digest mixtures ofα-casein and BSA after enrichment by (a) DMSNs@PDA-Ti4+, (b)mSiO2@PDA-Ti4+, and (c) commercial TiO2 at a molar ratio of1:5000 (loading buffer: 50% ACN, 6% TFA).

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mSiO2@PDA-Ti4+ and commercial TiO2, in terms of both thenumber and signal intensity of captured phosphopeptides. Thistrend is further confirmed when the acidity of loading bufferwas decreased from 6% to 3% TFA (see SupportingInformation Figure S5).For sensitivity measurement, different concentrations of α-

casein tryptic digests were used to determine the detectionlimit. As shown in Figure 7, 12 phosphopeptides could becaptured by DMSNs@PDA-Ti4+ in 1 fmol/μL α-casein trypticdigest, and the minimum detection limit of DMSNs@PDA-Ti4+

for phosphopeptide enrichment was as low as 0.2 fmol/μL.However, the minimum detection limits of mSiO2@PDA-Ti4+

and commercial TiO2 were 1 and 2 fmol/μL, respectively(Figure S6). Obviously, the DMSNs@PDA-Ti4+ have a better

selectivity and lower detection limit for phosphopeptideenrichment than the other two materials, which is attributedto the excellent hydrophilicity and high density of immobilizedTi4+ as well as the large and accessible mesopores.

Selective Enrichment of Phosphopeptides from RealBiological Samples. DMSNs@PDA-Ti4+ with outstandingenrichment efficiency were further used to capture phospho-peptides from complex biological samples. Nonfat milk, humanserum, and HeLa cell extracts were selected as real biologicalsamples. For tryptic digests of nonfat milk, 30 phosphopetideswere observed with significantly enhanced S/N ratio (Figure8b) after enrichment by DMSNs@PDA-Ti4+ (peptide

sequences are listed in Table S1), whereas the number ofphosphopeptides enriched by the other two materials wassignificantly reduced (Figure 4a). The number of thephosphopeptides identified by DMSNs@PDA-Ti4+ was alsohigher than those in many previously reported IMACnanomaterials.53−55 For healthy human serum enrichment(Figure 8d and Figure S7c, d), since human serum containedonly four endogenous phosphopeptides, the phosphopeptidesidentified by the three materials had no significant difference in

Figure 6. Comparison of the number and signal intensity of identifiedphosphopeptides enriched by DMSNs@PDA-Ti4+, mSiO2@PDA-Ti4+,and commercial TiO2 from the mixture of α-casein and BSA digests ata molar ratio of 1:5000 (loading buffer: 50% ACN, 6% TFA).

Figure 7. MALDI-TOF mass spectra of phosphopeptides enrichedfrom α-casein tryptic digest with different concentrations usingDMSNs@PDA-Ti4+.

Figure 8. MALDI-TOF mass spectra of the tryptic digest of nonfatmilk (a) before and (b) after enrichment by DMSNs@PDA-Ti4+.MALDI-TOF mass spectra of human serum (c) before and (d) afterenrichment by DMSNs@PDA-Ti4+. # indicates dephosphorylatedpeptides.

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number but a slight difference in abundance. The detailedinformation on the four endogenous phosphopeptides fromhuman serum is given in Table S2.Furthermore, HeLa cell extracts, which contain a number of

abundant phosphopeptides, were used to demonstrate theexcellent enrichment performance of DMSNs@PDA-Ti4+. Theenrichment experiments of DMSNs@PDA-Ti4+ and commer-cial TiO2 were performed under their respective optimalenrichment conditions (see details in the ExperimentalSection). Figure 9 gives an overview of the enrichmentperformance of DMSNs@PDA-Ti4+ and commercial TiO2(the detailed enrichment results of three runs are listed inTable S3). After database searching, a total of 1707phosphopeptides (860 monophosphopeptides and 847 multi-phosphopeptides with the detailed information are given inTable S4) were identified after enrichment with commercialTiO2. The enrichment specificity (the ratio of the number ofdetected phosphopeptides to that of all identified peptides) wascalculated to be 88.3%. However, for DMSNs@PDA-Ti4+, 2422phosphopeptides were detected (1429 monophosphopeptidesand 993 multiphosphopeptides with the detailed informationare given in Table S5) with an ultrahigh specificity (96.3%),indicating that the DMSNs@PDA-Ti4+ could reduce thenonspecific adsorption to a large extent. This specificity is thehighest among reported materials used for phosphopeptideenrichment from the lysate of real biological samples (see TableS6). Among the identified phosphopeptides, 1153 phospho-peptides were found in both materials (Figure 9c). In addition,the peak area values were also used to quantify the amount ofidentified phosphopeptides. As shown in Figure 9e, the amountof phosphopeptides captured by DMSNs@PDA-Ti4+ was 1.6times that by commercial TiO2, indicating that the DMSNs@PDA-Ti4+ have a larger phosphopeptide loading capacity.Therefore, DMSNs@PDA-Ti4+ can be used as a powerful

enrichment approach for phosphopeptide enrichment in cancercells, which is useful in elucidating the regulatory mechanismsof disease-related signaling pathways.In summary, DMSNs@PDA-Ti4+, a novel porous IMAC

nanomaterial with high density of immobilized Ti4+ andexcellent hydrophilicity, have been successfully synthesized.The large amount of immobilized Ti4+ could provide moreaffinity sites for phosphopeptide enrichment, and the excellenthydrophilicity could synergistically improve the selectivity ofphosphopeptides. Besides, the large pore sizes could effectivelyprevent the “shadow effect”, thereby facilitating the diffusionand release of phosphopeptides. Therefore, the DMSNs@PDA-Ti4+ performed better than mSiO2@PDA-Ti4+, commercialTiO2, and other reported materials in phosphopeptideenrichment. Furthermore, a total of 2422 phosphopeptideswere identified from HeLa cell extracts with an ultrahighspecificity (96.3%). It is anticipated that the DMSNs@PDA-Ti4+ can be used as a promising IMAC material for highselective enrichment of phosphopeptides in phosphoproteomeanalysis.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.anal-chem.8b01369.

Optimization of PDA modification content; TEM imagesand N2 adsorption−desorption isotherms of mSiO2@PDA-Ti4+; MALDI-TOF mass spectra of the enrichmentresults of mSiO2@PDA-Ti4+ and commercial TiO2;detailed information on phosphopeptides obtainedfrom α-casein digest, nonfat milk digests, human

Figure 9. Comparison of commercial TiO2 and DMSNs@PDA-Ti4+ for peptides enrichment from HeLa cell extracts. Overlap of phosphopeptidescaptured by (a) commercial TiO2 and (b) DMSNs@PDA-Ti4+ in three runs. (c) Overlap of total phosphopeptides identified by the two materials.The number (d) and peak area values (e) of identified peptides. Mono-P, Multi-P, and Non-P represent singly, multiply, and nonphosphorylatedpeptides, respectively.

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serum, and HeLa cell extracts; comparison of DMSNs@PDA-Ti4+ with the previous materials (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: minbolan@ecust.edu.cn (M.L).*E-mail: c.yu@uq.edu.au (C.Y).*E-mail: honglizhao@ecust.edu.cn (H.Z).

ORCIDMingliang Ye: 0000-0002-5872-9326Chengzhong Yu: 0000-0003-3707-0785Minbo Lan: 0000-0002-4247-0006NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research was financially supported by the Science andTechnology Commission of Shanghai Municipality (STCSM,No. 16520710800), the Fundamental Research Funds for theCentral Universities (No. 222201817022), the Natural ScienceFoundation of Shanghai (No. 16ZR1407600), and theShanghai Sailing Program (No. 16YF1402400). C.Y. acknowl-edges the support from Australian Research Council, AustralianNational Fabrication Facility at The University of Queensland.

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