Preparation of Bi0.15Fe0.15TiO2 Nanocomposites for the Highly

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Preparation of Bi0.15Fe0.15TiO2 Nanocomposites for the Highly Selective Enrichment of Phosphopeptides Deshuai Zhen, Chan Gao, Baode Zhu, Qian Zhou, Chenyi Li, Ping Chen, and Qingyun Cai Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00606 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 11, 2018

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Analytical Chemistry

Preparation of Bi0.15Fe0.15TiO2 Nanocomposites for the Highly Selective Enrichment of Phosphopeptides Deshuai Zhen,†,‡,∥ Chan Gao,†,∥ Baode Zhu,§ Qian Zhou,§ Chenyi Li,† Ping Chen,§,* and Qingyun Cai†,∗ †

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of

Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China ‡

College of Chemistry and Chemical Engineering, Qiannan Normal University for

Nationalities, Duyun, 558000, P. R. China §

State Key Laboratory of Developmental Biology of Freshwater Fish, The National &

Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of Life Sciences, Hunan Normal University, Changsha, 410081, P. R. China

∗

Corresponding author. Fax.: +86-0731-88821848 E-mail address: [email protected]; [email protected] 1

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ABSTRACT: A novel Bi0.15Fe0.15TiO2 nanocomposites (B0.15F0.15TNs) were synthesized for the first time by a modified sol-gel technology, and successfully applied to selective extraction and enrichment of phosphopeptides from digested protein mixture solutions and real samples (tissue protein extract from human liver). The co-doping of Bi and Fe into TiO2 results in a significant enhancement in both the enrichment efficiency and selectivity. Compared with the commercial available TiO2 extractant, the proposed B0.15F0.15TNs possess a lower detection limit (2 × 10-9 M) and higher selectivity at a low weight ratio of phosphopeptides/nonphosphopeptides (1:1200). Additionally, a total of 223 phosphorylation sites were identified from the human liver lysate after enrichment by the B0.15F0.15TNs. Besides, the synthesis of B0.15F0.15TNs is quite easy, of high yield and inexpensive. KEYWORDS: TiO2, enrichment, phosphopeptides, Mass Spectrometry, Nanomaterials

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Protein phosphorylation is a common post-translational modifications (PTMs) in the proteome and plays an important role in adjusting and controlling the vitality and function of protein. It regulates almost all the life activities, including cell signal transduction, differentiation, growth, apoptosis and so on. However，the dysfunction of protein phosphorylation will induce many serious diseases, such as diabetes and cancer.1-4 Herein, studies of protein phosphorylation might contribute to better understand the cell life activities and pathogenesis of some disease. Given the significance of protein phosphorylation, various mass spectrometry (MS)-based methods have been used to analyze protein phosphorylation, for example, LC/MS/MS, MALDI/MS/MS and LC-FAIMS-MS/MS.5-9 Although the high power of MS in the identification of the expression proteins, analysis of phosphopeptides with MS still faces big challenges including the low stoichiometry, poor ionization efficiency and the suppression effect by nonphosphopeptides. Thus, the selective enrichment of phosphopeptides is particularly important because phosphopeptides account for only a small part of the digested portions and their identification is easily covered by the nonphosphopeptides. To achieve this goal, various approaches have been developed for the extraction and enrichment of phosphopeptides, including chemical-modification strategies, strong cation exchange chromatography, immunoprecipitation, immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC). Among these techniques, IMAC and MOAC are the most convenient and common tools in phosphoproteomics research.10-13 A wide variety of metal oxides (MOs) have been proposed as affinity probes (APs), such as TiO2,14-18 ZrO2,19-22 SnO2,23-25 Al2O3,26-27 Nb2O5,28 Fe2O329 etc. These materials demonstrated to be effective in the enrichment of phosphopetides because of the bidentate interactions between the MOs 3



and phosphate groups, and have higher selectivity for phosphopetides because of the reduced nonspecific binding as compared with IMAC.30-32 As for MOAC, the most popular MOs are TiO2 and ZrO2, which can be used in analytical column-, miniaturized column-, or batch-format.33,34 However, no approaches can enrich all the phosphopeptides because of the low adsorption efficiency and significant losses of phosphorylated proteins during the washing steps.35,36 To overcome this limitation, combined strategies have been developed to enrich phosphopetides. SiO2/TiO2,37 ZnO/TiO2 nanorod arrays,38 Graphene/TiO2,39 Fe3O4/TiO2,40 TiO2-ZrO2/SiO2,41 Fe3O4/ZrO2,42 SiO2/La2O3,43 and Fe3O4/TiO2-ZrO244 have been successfully applied to the enrichment of phosphopeptides. Despite these successful cases, it is still appearing to design and prepare functional anchors for phosphopetides with high specificity and efficiency. In this work, a new material B0.15F0.15TNs was prepared by co-doping Bi and Fe into TiO2 for the efficient enrichment of the low abundance phosphopeptides. The enrichment specificity toward phosphopeptides was assessed by the enrichment of phosphopeptide from complex protein mixtures and tissue protein extract from a human liver.

EXPERIMENTAL SECTION Reagents and Materials. Bovine β-casein, bovine serum albumin, 2, 5dihydroxybenzoic acid (DHB), tetrabutyl titanate (TBT, C16H36O4Ti), bismuth nitrate hydrate (Bi(NO3)3·5H2O), ferric nitrate nonahydrate (Fe(NO3)3·9H2O), acetic acid (HOAc, CH3COOH), ethanol (ETOH), dithiothreitol (DTT), iodoacetamide (IAA), ammonium hydroxide (28% NH3 in water), acetonitrile (ACN) and trifluoroacetic acid (TFA) of HPLC-MS grade, Urea and Tris(hydroxymethyl)-aminomethane (Tris) were purchased from Sigma-Aldrich Co. RC-DCTM kit was from Bio-Rad 4


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Laboratories (Hercules, CA, USA). Complete protease inhibitors cocktail was from Roche (Mannheim, Germany). Proteomics sequencing grade trypsin, and sucrose were obtained from Promega (Madison, WI, USA). All reagents and solvents of analytical grade were used without further purification. Commercially available TiO2 column (P25) purchased from GL Sciences B.V, Japan was selected as the reference for the comparative studies. Ultrapure water (Millipore, 18.2MOre-sistivity) was used throughout the experiments.

Scheme 1. Schematic illustration of the enrichment of phosphopeptides.

Synthesis of BFTNs. The B0.15F0.15TNs with the molar ratio of Bi/Fe/Ti =0.15:0.15:1.0 were prepared based on the previous reported work with slight modifications.45 Briefly, solution A, a transparent yellow sol, was obtained by dissolving 20 ml TBT in 30 ml ETOH. Solution B was obtained by dissolving a 1.78 g Fe(NO3)3·9H2O and 2.14 g Bi(NO3)3·5H2O in 20 ml ETOH at pH 2-3 adjusted by addition of HOAc. 2.2 ml ultrapure water was added into the solution B which was then mixed with solutions A and kept continuously stirring at 30 °C overnight. After 5



aging for one day, a gel was formed. The gel was dried in a vacuum oven at 80 °C overnight, and calcined at 700 °C for 2 h to obtain the final nanocrystals B0.15F0.15TNs. Bare TiO2 and the BFTNs with the different molar contents of Bi and Fe were synthesized via a similar procedure. Characterization of BFTNs. The Brunauer-Emmett-Teller (BET) approach using N2 adsorption-desorption apparatus (Tristar 3000, Micromeritics instrument Co.) was utilized to measure the specific surface areas and pore volume of the nanocomposite over the relative pressure ranging from 0.05 to 0.35. The sample was degassed at 90 °C for 3 h before measured. Scanning electron microscopy (SEM) images and Transmission electron microscopy (TEM) images were recorded on a SU8010 microscope (Japan) and a JEM-2100F microscope (Japan), respectively. The crystal composition and structure of the BFTNs were analyzed through X-ray diffraction (D8 Advance, Brucker, German) with Cu K radiation (λ= 0.15418 nm) under the operating conditions of 40 kV and 80 mA and in the range from 10° to 80° with a scan speed of 1.00° min-1. Protein Digestion. The human liver was obtained from the Third Xiangya Hospital of Central South University with the protocol (PN: 010008) approved by the Ethics Committee of the University of Hunan Normal University. And the samples (100 mg) were washed, minced and suspended in precooled lysis buffer (8 M urea, 75 mM NaCl, 1 mM PMSF, 50 mM NaF, 10 mM Na2P2O4, 50 mM Tris, pH 8.2) in ice. 30 min later, the samples and supernatant were centrifuged at 800 × g for 10 min then 14, 000 × g for 15 min at 4 °C. The resulting supernatant contained the total liver proteins. The protein concentration of the supernatant was measured using a RC-DCTM kit. Bovine β-casein (mw: 25382) was dissolved in 50 mM NH4HCO3 solution and reduced with 10 mM dithiothreitol at 60 °C and alkylated with 55 mM iodoacetamide 6


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at room temperature in the dark. The denatured proteins were digested with trypsin (1:50 protease/protein, w/w) overnight at 37 °C. The final concentration of β-casein digestion was 1 µg/µL or 4×10-5 M calculated based on the molecular weight. Bovine serum albumin (BSA, 1 µg/µL, MW: 66430) and proteins of human liver (0.2 µg/µL) were treated the same way. The work solution was prepared by diluting the digestion with loading buffer to get the desired concentration. Enrichment of phosphopeptides. Scheme 1 illustrates the enrichment process. The BFTNs (10 mg) were firstly activated by washing buffer (30% ACN, 0.1% TFA) and loading buffer (80% ACN, 5% TFA, 1 M Glycolic acid), then 50 µg BFTNs were added into a tryptic peptide mixture which was dissolved in 2-fold volume of loading buffer. After sonicating and standing for 10 min at room temperature for phosphopeptide enrichment. The BFTNs were collected by centrifugation and washed one time with loading buffer and three times with washing buffer. Finally, the peptides captured by the BFTNs were eluted with 5% ammonium aqueous solution and analyzed by MALDI-TOF MS. The eluate from human liver sample was analyzed by LC-ESI MS. Mass Spectrometry. 0.5 µL sample and 0.5 µL DHB aqueous solution (20 mg/mL in 50% ACN aqueous solution containing 1% H3PO4) were mixed, deposited on a plate, and dried naturally. MALDI-TOF MS analysis were performed in positive ion mode within the mass range of 1000–3500 Da on a AB SCIEX 5800 MALDI TOF/TOF™ system (AB SCIEX, Foster City, USA) with a N2 laser at 337 nm, a pulse width of 20 ms, a vacuum degree of 4×10-7 Torr and an acceleration voltage of 20 kV. The Nano-LC-ESI-MS/MS analysis of human liver sample was performed on a LTQ-Orbitrap Velos MS/MS (ThermoFisher Scientific Inc.) equipped with an ESI nanospray source. The enriched peptides by the BFTNs were desalted, lyophilized, 7



and re-suspended in 5% ACN aqueous solution containing 0.1% formic acid. The resuspended peptides were fractionated on a Nano LC column (50 µm ×15 cm, 100 Å pore size, 2 µm particle size, Pepmap C18, ThermoFisher Scientific Inc.) with an HPLC system (Shimadzu) at a flow rate of 400 nL/min. The HPLC was performed using the buffer A (0.1% formic acid) and buffer B (99.9% ACN, 0.1% formic acid) as eluents. The gradient was set as follows: from 0 to 5% buffer B in 10 min, 5% to 30% buffer B in 30 min, 30% to 60% buffer B in 5 min, and 60% to 80% buffer B in 3 min. Holding on 80% buffer B for 7 min, then decrease to 5% buffer B in 3 min and hold for 7 min. The MS was operated in positive ion mode and the data-dependent MS/MS mode for survey scans (m/z 300 to 1800) with a mass resolution of 100,000, choosing up to seven most intense precursor ions MS/MS for analysis. Database Searching. The peak list generation and peptide identification were performed using Mascot 2.3.01 program. The main parameters were set as follows: (1) precursor mass tolerance, ±15 ppm; product ion mass tolerance 0.5 Da; (2) trypsin digestion, up to one missed cleavages; (3) fixed modification, carbamidomethyl (C); (4) variable modification, Ser, Thr and Tyr. False discovery rates (FDR) of the identified peptides and proteins were estimated by searching against the database with the reversed amino acid sequence. Only peptides with at least six amino acids in length and an FDR of 1% were considered to be identified successfully.

RESULTS AND DISCUSSION Characterization of the B0.15F0.15TNs material. The SEM images of Figures 1A and B show the size distribution of the TiO2 and B0.15F0.15TNs calcined at 700 °C for 2 h, respectively. The as-prepared B0.15F0.15TNs are basically spherical particles at an average particle size of ~40 nm in diameter, while the TiO2 nanocrystal has an irregular shape at an average particle size of ~60 8


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nm. The B0.15F0.15TNs are relatively more homogenous with smaller particle size than TiO2, properly due to the Fe and Bi’s restraining the growth of particles, which induces a large surface area accounting for more binding sites.46,47 The mean sizes (D) of the B0.15F0.15TNs nanocrystal were estimated to be 34 nm based on the Scherrer equation (1).

D=

Kλ β cosθ

…………………………………………………………………… (1)

Where, K (k=0.89): Scherrer constant, λ: the wavelength of X rays, β: the half width of the diffraction peak, θ: the diffraction Angle. The element mapping analysis of the B0.15F0.15TNs indicates that the doped Bi and Fe are uniformly distributed in the TiO2 nanocrystal (Figure 1C). The EDX spectrum (Figure 1E) indicates the existing of Ti, O, Fe and Bi, without contamination elements. The Cu peaks originate from the Cu TEM grid. The XRD patterns of Figures 1D and S1 show that the B0.15F0.15TNs nanocrystal calcined at 700 °C is a mixed crystal types of anatase and rutile TiO2 phases with presence of Bi2O3 phase (JCPDS NO. 010731374) and rhombohedral α-Fe2O3 phase (JCPDS NO.000011053). The obvious diffractional peaks indicative of Bi2O3 phase suggests that Bi presents as a separate phase on the surface of TiO2. In the contrast, the diffractional peaks indicative of α-Fe2O3 phase are rarely observed, implying that Fe mainly exists inside the TiO2 particles due to the smaller radius of Fe than Ti that will be discussed immediatelly. The morphology of B0.15F0.15TNs was further investigated with the high-angle annular dark-field scanning TEM (HAADF-STEM) (Figure 1C(a)) and TEM (inset in Figure 1E), it clearly shows that B0.15F0.15TNs is in particles at a size of ~ 30 nm. The 9



acquired selected area diffraction (SAD) pattern shown in Figure 1C (b) displays a typical lattice spacing of 0.1069 nm and 0.1947 nm, ascribed to the energetically stable crystal facets of rutile TiO2 (222) and (120), respectively. The lattice spacing is less than 0.1094 nm and 0.2045 nm, the lattice spacing of pure rutile TiO2 (JCPDS NO. 969007433), probably due to the doping of Fe in TiO2 lattice. The ionic radius of Ti4+ (68 pm) is smaller than that of Bi3+ (110 pm) and larger than that of Fe3+ (63 pm). As a result, Fe3+ can incorporate into TiO2 lattice while Bi3+ can not. The replacement of Ti4+ with smaller Fe3+ causes the contract of TiO2 lattice (decrease in lattice spacing), and meanwhile the shift of diffractional peaks to higher angle, according to Bragg’s law.48-51 The 101 and 110 peaks of TiO2 shift from 25.28o (anatase) to 25.51o and 27.43o (rutile) to 27.54o in B0.15F0.15TNs, respectively (Figure 1D).

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Figure 1. The SEM images of prepared TiO2 (A) and B0.15F0.15TNs (B), the inset shows the corresponding size distribution. C, the high-angle annular dark-field scanning TEM image (a), Selected Area Diffraction pattern (b), and EDS element mapping of Ti, O, Bi and Fe. D, the XRD patterns, and E, the EDX spectrum (inset, TEM image) of B0.15F0.15TNs. F, Raman spectrum of the B0.15F0.15TNs (a) and TiO2 (b).

Raman spectroscopic analysis (Figure 1F) was used to further investigate the crystalline structure characters of the B0.15F0.15TNs. The major characteristic peaks at around 140, 197, 394, 447, 513, 612 and 635 cm-1 corresponding to the Eg, Eg, B1g, Eg, 11



A1g+B1g, A1g and Eg vibrational modes of anatase or rutile TiO2, as indicated in Fig. 1F, respectively.52,53 The doping of Bi and Fe results in a decrease in the intensity and broadening of the characteristic peaks, as well as the shift of the Eg mode (140 cm-1) to high frequency (149 cm-1). These phenomena can be assigned to the formation of Ti-O-Fe.54

Figure 2. Nitrogen adsorption−desorption isotherms of (A) prepared TiO2 and (B) B0.15F0.15TNs (Insets, the corresponding pore size distribution).

Figure 2 shows the N2 adsorption–desorption isotherm and the corresponding pore size distribution for the prepared TiO2 and B0.15F0.15TNs nanocrystal. The specific surface area (10.8 m2/g) and pore volume (0.57 cm3/g) of B0.15F0.15TNs are much higher than those of TiO2 (4.7 m2/g and 0.019 cm3/g), respectively, indicating that the doping of Bi and Fe increases the surface area and pore volume. The higher specific surface area of B0.15F0.15TNs means more contacting area for phosphate groups benefiting a high enrichment capacity toward phosphopeptides. Selective enrichment and detection of phosphopeptides from biological samples. The selective enrichment ability of the BFTNs toward phosphorylated peptides was firstly investigated with the tryptic peptide mixture of 2 × 10-8 M β-casein (this

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standard

phosphorylated

protein

containing

trace

amount

of

α-casein).

Phosphorylated peptides identified in β-casein and α-casein are listed in Table 1.46,47 Table S1 reveals the enrichment abilities of the BFTNs with different molar ratio of Bi/Fe/Ti. The enrichment ability depends on the Bi and Fe content. The highest abundance at m/z 2061.7 and 3122.6 were observed on the B0.15F0.15TNs (700 °C) adsorbent which was used in following researches, Figure 3F(b). Figure 3F(a) exhibits the enrichment abilities of the B0.15F0.15TNs calcined at different temperatures. The highest abundance at m/z 2061.7 and 3122.6 are achieved on the B0.15F0.15TNs calcined at 700 °C, at which mixed crystal types of anatase and rutile TiO2 phases are formed, indicating that the mixed anatase-rutile TiO2 phases benefit the adsorption of phosphopeptides. The enrichment ability of the B0.15F0.15TNs was then compared with the prepared TiO2 and commercial P25 column, Figure 3. Only low intensity peaks of phosphorylated peptides are detected by MS without enrichment (Figure 3A). With enrichment by B0.15F0.15TNs (Figure 3B), P25 column (Figure 3C) and prepared TiO2 (Figure 3D), multiple phosphorylated peptides are detected. Four phosphopeptides from β-casein and even three α-casein phosphopeptides are detected with B0.15F0.15TNs (Figure 3B). Nevertheless, two phosphopeptides from β-casein are detected with TiO2 (Figure 3D), and three phosphopeptides are identified even with the expensive P25 column (Figure 3C). Moreover, the relative abundance at m/z 2061.7 and 3122.6 observed on the Fe2O3 (Figure 3F(b)) are significantly lower than those on the B0.15F0.15TNs, indicating the higher selectivity of B0.15F0.15TNs. The high enrichment ability of B0.15F0.15TNs toward phosphopeptides is due to the high specific surface area resulted from the Fe doping as well as the high affinity of Bi3+ toward phosphate group.29,31,42,53 The solubility product (Ks) of BiPO4 and FePO4 13



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are 1.3 × 10-23 and 9.91 × 10-16, respectively,56,57 indicating the strong affinity of Bi3+ to phosphate group.58,59 Meanwhile, the doped Fe inhibits the growth of TiO2 nanocrystal inducing a high specific surface area benefiting the high adsorption capacity, and the doped Bi has a strong reversible bridging bidentate interaction with phosphate group favoring the selectively binding of PO43- from phosphopeptides. To further explore the detection limit of B0.15F0.15TNs toward phosphopeptides, the digested β-casein solutions was diluted gradually then enriched by B0.15F0.15TNs and analyzed by MS. There is still a phosphopeptide peak detectable as far as the concentration of β-casein digests is decreased to 2 × 10-9 M (Figure 3E).

Table 1. Detailed Information on the Observed Phosphopeptides from Tryptic Digestion of α-Casein S1 , α-Casein S2 and β-Casein a Peak number

Theoretical m/z

Observed m/z

aa

Peptide sequence

1

1466.6

1466.4

α-S2/153−164

TVDMSTEVFTK

2

1660.8

1660.8

α-S1/106−119

VPQLEIVPNSAEER

3

1952.0

1952.9

α-S1/104−119

YKVPQLEIVPNSAEER

4

2061.7

2061.8

β/33–48

FQSEEQQQTEDELQDK

5

2432.6

2432.0

β/30-48

IEKFQSEEQQQTEDELQDK

6

2556.1

2554.1

β/33–52

FQSEEQQQTEDELQDKIHPF

7

3122.6

3122.2

β/1–25

RELEELNVPGEIVESLSSSEESIT R

a

The phosphorylation sites are underlined.

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Figure 3. MALDI-TOF mass spectrum of peptides derived from 2 × 10-8 M β-casein without (A) and with enrichment by B0.15F0.15TNs (B), P25 column (C), and prepared TiO2 (D), as well as derived from 2 × 10-9 M peptides enriched by B0.15F0.15TNs (E). (F) compared the peak intensity at m/z 2061.7 and 3122.6 derived from 2×10-8 M βcasein enriched by B0.15F0.15TNs prepared at different temperatures (a), and the B0.15F0.15TNs, TiO2, P25 column, and Fe2O3 (b). # represent the metastable losses of phosphoric acid. Selective Enrichment of Phosphopeptide from Semi-complex Sample. The enrichment specificity of the B0.15F0.15TNs toward phosphopeptides was further evaluated with semi-complex sample, the mixture of β-casein digestion and bovine serum albumin (BSA) in weight ratios of 1:1, 1:10, 1:100, 1:500, 1:1000 and 1:1200, at the final concentration of β-casein digestion of 2 × 10-7 M, 2 × 10-8 M, 2 × 10-9 M, 4 × 10-10 M, 2 × 10-10 M and 1.7 × 10-10 M, respectively. As shown in Figure 4 A, no phosphopeptides are detected without enrichment due to the existence of 10 times 15



non-phosphopeptides. After enrichment by the B0.15F0.15TNs, seven phosphopeptides are detected at the ratio of 1:1 (Figure 4B). The ion signals of phosphopeptides are stronger than those nonphosphopeptides at the ratio ranges from 1:1 to 1:100 (Figure 4 B-D). There are five phosphopeptides peaks being detected at the ratio of 1:100 (Figure 4D). Nevertheless, only one peak of phosphopeptides is detected while it is enriched by the prepared TiO2 at the ratio of 1:100 (Figure 4E). There are still two phosphopeptide peaks being detected when the β-casein concentration is diluted to 1:500 (Figure 4F). With further diluting the β-casein concentration to 1:1000, there is still a strong characteristic phosphopeptide peak (Figure 4G), and phosphopeptide can be detectable at the ratio of 1:1200 (Figure 4H), indicating a high specificity of B0.15F0.15TNs in mimic complex biological samples.

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Figure 4. MALDI-TOF mass spectrum of the mixture of β-casein digestion and BSA at weight ratio (β-casein to BSA) of (A) 1:10 without enrichment, (B) 1:1, (C) 1:10, (D) 1:100, (F) 1:500, (G) 1:1000 and (H) 1:1200 with enrichment by B0.15F0.15TNs. (E) Enriched by prepared TiO2 at ratio (β-casein to BSA) of 1:100. # represent the metastable losses of phosphoric acid.

Selective Enrichment of Phosphopeptide from Complex Biological Sample Finally, a complex and real biological sample (human liver cancer tissue) was used to evaluate the enrichment ability of the B0.15F0.15TNs. As shown in Figure 5A, the B0.15F0.15TNs give a higher specificity with 75% phosphopetides in enrichment 17



compared to prepared TiO2 with 51% phosphopetides. In total, 38 singlephosphopetides and 78 multi-phosphopeptides were identified from the human liver lysate after enrichment by B0.15F0.15TNs (Table S2). Among them, 223 phosphorylation sites were included, with 171 (76.7%) on serines, 37 (16.6%) on threonines, and 15 (6.7%) on tyrosines. The identification also shows that the B0.15F0.15TNs is useful in profiling disease-related signaling pathways. However, only approximately 104 phosphorylation sites from human liver lysate were identified by using prepared TiO2. As shown in Figure 5B, the percentage of multiphosphopeptides enriched by B0.15F0.15TNs is almost two times that by TiO2, with 69% to 36%. The B0.15F0.15TNs is more efficient for enriching multi-phosphopeptides than the pure TiO2, which are attributed to the high specific surface area resulted from the Fe3+ doping and the strong affinity of Bi3+ to phosphate group. The selective and effective enrichment of phosphopeptides for phosphoprotome analysis of human liver with B0.15F0.15TNs can proposed an approach to understand the possible changes of phosphoproteins in signaling pathways.

Figure 5. The percentages of phosphopeptides and non-phosphopeptides (A), and percentages of single-phosphopetides and multi-phosphopeptides (B) enriched by B0.15F0.15TNs and TiO2 samples.

CONCLUSIONS 18


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Novel B0.15F0.15TNs nanoparticles were synthesized for the first time for the highly efficient enrichment of phosphopeptides. The B0.15F0.15TNs show much higher efficiency and specificity in the enrichment of phosphopeptides from simple to complex phosphopeptide-containing samples as compared to the unmodified TiO2 nanoparticles and even the commercial P25 TiO2 column. The B0.15F0.15TNs possess a low detection limit (2 × 10-9 M) and high selectivity at a low weight ratio of phosphopeptides/nonphosphopeptides (1:1200). The high enrichment efficiency and specificity are ascribed to the synergistic effect of doped Bi and Fe, which not only inhibit the growth of TiO2 nanocrystals inducing a high specific surface area, but also have strong affinity and coordination ability toward phosphate groups of peptides.

ASSOCIATED CONTENT Supporting Information. XRD patterns of B0.15T0.15Ns and TiO2 calcined at 700 °C; MALDI-TOF mass spectrum of peptides derived from β-casein (2×10-8 M) enriched by B0.15F0.15TNs prepared at different temperatures; The intensity of phosphopeptides from Tryptic Digestion at m/z=2061.7 and 3122.6 enriched by the BFTNs with different molar ratio; Detail information of the phosphopeptides enriched from tryptic digests of human liver lysis by using preparation of B0.15F0.15TNs. This is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *Qingyun Cai, Ph.D. College of Chemistry & Chemical Engineering, Hunan University, Changsha 410082, P.R. China, E-mail: [email protected] 19



*Ping Chen, Ph.D. College of Life Sciences, Hunan Normal University, Changsha 410081, P.R. China, E-mail: [email protected] Author Contributions ∥ D.Z.

and C.G. contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by a grant from National Natural Science Foundation of China (21874038, 21235002, 31670838, and 31370817). REFERENCES (1) Gruber, W.; Scheidt, T.; Aberger, F.; Huber, C.G. Cell Communication and Signaling. 2017, 15, 12. (2) Schunter, A.J.; Yue, X.; Hummon, A.B. Anal. Bioanal. Chem. 2017, 409, 17491763. (3) Thingholm, T.E.; Jensen, O.N.; Larsen, M.R. Proteomics. 2009, 9, 1451-1468. (4) Yi, T.; Zhai, B.; Yu, Y.; Kiyotsugu, Y.; Raschle, T.; Etzkorn, M.; Seo, H.; Nagiec, M.; Luna, R.E.; Reinherz, E.L.; Blenis, J.; Gygi, S.; Wangner, G. Proc Natl Acad Sci U S A. 2014, 111, 2182-2190. (5) Chen, B.; Hwang, L.; Ochowicz, W.; Lin, Z.; Guardado-Alvarez T.M.; Cai, W.; Xiu, L.; Dani, K.; Colah, C.; Jin, S.; Ge, Y. Chem Sci. 2017, 8, 4306−4311.

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Preparation of Bi0.15Fe0.15TiO2 Nanocomposites for the Highly

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