TiO2 Composite Monolithic Capillary

Article pubs.acs.org/ac

Facile Preparation of SiO2/TiO2 Composite Monolithic Capillary Column and Its Application in Enrichment of Phosphopeptides Shao-Ting Wang, Meng-Ya Wang, Xin Su, Bi-Feng Yuan, and Yu-Qi Feng* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China S Supporting Information *

ABSTRACT: A novel SiO2/TiO2 composite monolithic capillary column was prepared by sol−gel technology and successfully applied to enrich phosphopeptides as a metal oxide affinity chromatography (MOAC) material. For the monolith preparation, tetramethoxysilane (TMOS) and tetrabutoxytitanium (TBOT) were used as silica and titania source, respectively, and glycerol was introduced to attenuate the activity of titanium precursor, which provided a mild synthetic condition. The prepared monolith was characterized by energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The results revealed an approximate 1/2 molar ratio of titanium to silica as well as an atom-scale homogeneity in the framework. The scanning electron microscopy (SEM) results demonstrated an excellent anchorage between the column and the inner capillary wall, and nitrogen adsorption−desorption experiments showed a bimodal porosity with a narrow mesopore distribution around 3.6 nm. The prepared monolith was then applied for selective enrichment of phosphopeptides from the digestion mixture of phosphoproteins and bovine serum albumin (BSA) as well as human blood serum, nonfat milk, and egg white using an in-tube solid phase microextraction (SPME) system. Our results showed that SiO2/TiO2 composite monolithic capillary column could efficiently enrich the phosphopeptides from complex matrixes. To the best of our knowledge, this is the first attempt for preparing the silica−metal composite monolithic capillary column, which offers the promising application of the monolith on phosphoproteomics study.

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control and may lead to serious shrinkage of the framework. Another challenge was the weak anchorage of the titania structure to the capillary wall. In the latest study, a relatively sophisticated premodification of capillary wall was used to form adequate Ti−O−Si bonds between the column and the naked capillary wall during sol−gel process.10 For the fact that glycerol could be utilized as the stabilizer to create a mild condition and silica components could provide easy anchorage toward naked capillary wall, the glycerol-involving preparation of SiO2/TiO2 composite monolith may be a promising alternative for preparing TiO2-based in-capillary monolithic materials. Moreover, the related reports showed that, due to the interfacial electronic and structural interactions, the SiO2/TiO2 composite materials have better performance on enrichment of phosphorylated targets than pure TiO2 materials.16−18 Protein phosphorylation, one of the most significant posttranslational modifications (PTMs), is involved in a variety of biological processes.19 Generally, mass spectrometry (MS), including electrospray ionization-mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), is widely applied in such field to

ince the concept of monolithic column was introduced, this kind of bimodal porous material has been widely used as both extraction and separation media in analytical chemistry.1−3 The mesopores on the skeleton give the monolith relatively high specific surface area, and the continuous macropores in the framework provide rapid mass transportation. Generally, this kind of material could be divided into silica-based, organicbased, and silica−organic hybrid monoliths. Recently, increasing efforts have been devoted to introduce metal oxides into the monolithic structure due to their amphoteric property and unique chemical affinity. To this end, many pure metal oxide4−11 and composite12−15 monolithic materials have been fabricated. However, unlike the traditional ones, these metal oxide-based monoliths are fairly difficult to prepare within confined geometries such as capillary columns, which is quite unfavorable for their development in minimized systems. Titania is the most comprehensively studied and widely used metal oxide; however, only a few studies achieved the preparation of pure TiO2 monolithic capillary columns,7,10 and the TiO2-based composite monolith have even not been successfully synthesized in capillary so far. The challenge for the preparation of TiO2-based monolith could be largely ascribed to the ultramoisture sensitivity of the titanium precursors (generally, titanium isopropoxide and tetrabutoxytitanium). A strong acidic condition was always utilized to slow down the reaction rate during the preparation, which was difficult to © 2012 American Chemical Society

Received: May 9, 2012 Accepted: August 17, 2012 Published: August 17, 2012 7763

dx.doi.org/10.1021/ac301258q | Anal. Chem. 2012, 84, 7763−7770

Analytical Chemistry

Article

purchased from Promega (Madison, WI, USA). Purified water was obtained with a Milli-Q apparatus (Millipore, Bedford, MA, USA). Preparation of SiO2/TiO2 Monolithic Capillary Column. The fused silica capillaries were washed with 1 mol/L NaOH (2 h), water (30 min), 1 mol/L HCl (1 h), and methanol (10 min) successively to activate the silanol groups on the wall. Then, the capillaries were allowed to dry under nitrogen flow at 150 °C for 5 h. The monolithic SiO2/TiO2 gels were prepared from a solution containing TBOT, glycerol, EtOH, TMOS, 0.01 mol/L acetic acid, and PEG. The optimal preparation conditions were as follows. 100 mg of TBOT and 500 mg of glycerol were mixed in a 1.5 mL Eppendorf vial obtaining a white milk-like solution.6 Subsequently, 100 mg of TMOS and 150 mg of EtOH were added into the above mixture followed by vortexing for several minutes and ultrasonication for 30 min. After ice-cooling for 20 min, 70 mg of 0.1 mol/L acetic acid with 20 mg of PEG was introduced to the mixture. The resulting sol was then filled into the pretreated capillary with a certain length by a syringe. After being sealed at both ends with silicone rubber, the capillary was allowed to further react at 35 °C for 12 h and 120 °C for 3 h. In the end, the capillary was rinsed with EtOH and water to remove unreacted compounds and soluble hydrolysis products followed by drying under room temperature before use. Sample Preparation. Bovine α-casein and β-casein were made up into stock solutions of 1 mg/mL using milli-Q purified water. Proteins were digested in trypsin (enzyme to substrate ratio of 1:50 (w/w) in 100 mM Tris−HCl, pH 8.5) and incubated overnight at 37 °C. BSA (1 mg) was dissolved in 100 μL of denaturing buffer solution (8 M urea in 100 mM Tris− HCl, pH 8.5). The obtained protein solution was mixed with 5 μL of 100 mM tris(2-chloroethyl)phosphate (TCEP) and incubated for 20 min at room temperature to reduce protein disulfide bonding. Iodoacetamide (3 μL of 500 mM stock) was added, and the solution was incubated for an additional 30 min at room temperature in the dark. The reduced and alkylated protein mixture was diluted to 4-fold with 100 mM Tris−HCl, pH 8.5. Nine microliters of 100 mM CaCl2 was added to produce a total volume of 50 μL, and the mixture was digested by incubating overnight at 37 °C with trypsin at an enzyme to substrate ratio of 1:50 (w/w). All the tryptic digestions were lyophilized to dryness and stored at −20 °C for future use. Human serum samples were collected from healthy persons and obtained from The Hospital of Wuhan University according to the standard clinical procedures. The utilization of human serum was complied with guidelines of Ethics Committee of the Institute, and all participants gave their informed consent. The sample was stored at −80 °C until use. For in-solution digestion, the nonfat milk or egg white (both 50 μL) was first denatured by the ammonium bicarbonate solution (50 mM, 250 μL) containing urea (8 M) and incubated at 37 °C for 30 min. Then, DTT solution (200 mM, 25 μL) was introduced, and the temperature was maintained at 55 °C for 1 h. After cooling to room temperature, the IAA solution (200 mM, 50 μL) was added and the mixture was kept in the dark for 3 h. Finally, the resulting sample was incubated with trypsin (2 mg/mL, 5 μL) at 37 °C for 24 h and diluted 20 times by sampling solution before use. Extraction of Phosphopeptides. The procedure for phophopeptides enrichment was similar with the typical intube solid phase microextraction (SPME) experiments reported previously.44 As shown in Figure 1 and Table S1, Supporting

characterize the proteolytic digests of phosphoproteins. However, the direct MS analysis of phosphorylated peptides from digestion products is seriously obstructed by the substoichiometric nature of phosphorylation as well as the ion suppression by the large excess of nonphosphopeptides. Therefore, purification and enrichment of the phosphorylated targets from the complex samples are highly required before MS analysis.20 For this purpose, a number of pretreatment methods have been developed, including immunoprecipitaion,21 covalent chemical modification,20 ion exchange chromatography,22−25 immobilized metal affinity chromatography (IMAC),26−29 and metal oxide affinity chromatography (MOAC).16−18,30−35 Taking advantages of the selectivity, recovery, and relatively high salt tolerance,35,36 TiO2-based MOAC materials16,18,31,34,35 have been regarded as the most powerful and promising materials for phosphopeptide sample preparation.37 Recently, more and more studies have been focused on applying the monolithic capillary column in the enrichment of phosphopeptides.38 Most of the existed works utilized the columns as strong anion-exchangers25 or IMAC materials.39−42 Up to date, the monolithic capillary column of MOAC mode has not been reported. In the current study, we developed a facile sol−gel method for preparing a novel SiO2/TiO2 incapillary composite monolith under mild condition and investigated the performance of such monolith on the enrichment of phosphopeptides. During the preparation, we used glycerol instead of strong acids to attenuate the reactivity of titanium precursor (tetrabutoxytitanium (TBOT)) and mediate the difference of condensation reaction kinetics between TBOT and tetramethoxysilane (TMOS).6,43 The existed silica in the system could provide additional anchorage (Si−O−Si bonds) of the monolith to the capillary so that there is no need to modify the capillary wall with titania sources. The successful incorporation of titanium in silica framework was confirmed by energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The morphology and porosity of the monolith was examined by scanning electron microscopy (SEM) and nitrogen adsorption−desorption experiments, respectively. In addition, the SiO2/TiO2 in-capillary composite monolith was successfully applied to enrich the phosphopeptides from tryptic digests of α-casein and β-casein and the mixture of α-casein, β-casein, and bovine serum albumin (BSA) as well as human serum samples, nonfat milk, and egg white.

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EXPERIMENTAL SECTION Materials. Fused-silica capillaries with 100 μm I.D. × 365 μm O.D. were purchased from Yongnian Fiber Plant (Hebei, China). Tetramethoxysilane (98% TMOS) was obtained from the Chemical Factory of Wuhan University (Wuhan, China). Tetrabutoxytitanium (TBOT), glycerol, ethanol (EtOH), acetic acid, poly (ethylene glycol) (PEG, Mw = 10 000), sodium hydroxide (NaOH), hydrochloric acid (HCl, 37 wt %), and disodium hydrogenorthophosphate were all of analytical grade and purchased from Shanghai General Chemical Reagent Factory (Shanghai, China). HPLC grade acetonitrile (ACN) was purchased from Fisher Scientific (Pittsburgh, PA, USA). Phosphoric acid (H3PO4), trifluoroacetic acid (TFA), ammonia hydrate (NH3·H2O, 25%), 2,5-dihydroxybenzoic acid (2,5DHB), bovine α-casein, bovine β-casein, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nonfat milk and chicken eggs were purchased from a local supermarket in Wuhan. Sequencing grade trypsin was 7764



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Figure 1. The schematic of the devices for enrichment experiments.

Figure 2. SEM images of the in-capillary SiO2/TiO2 monolith (A), (B), and (C) and the pore diameter distribution of the material (D).

Information, the extraction process was briefly described as follows. The flow rates of pump A and pump B were 5 μL/min. The solution A and solution B were 1% TFA/ACN 50/50 (v/ v) and 0.1% NH3·H2O, respectively. All the samples were dissolved by solution A to the given concentration (10-fold dilution was used for human serum samples). The length of the monolithic capillary column was 15 cm. At the beginning, both valves A and B were set at LOAD position. Before the extraction, the solution A flew through the monolith for conditioning. At the same time, the sample loop (20 μL) was filled with sample solution by a syringe. Valve A was switched

to INJECT position for a given time interval in the extraction step (4 min in this case) and then returned to the LOAD position. The solution A then kept flowing through the monolith for another 8 min to eliminate the residual sample solution and remove unretained matrix. The solution B then desorbed the extracted phosphopeptides from the monolith for 4 min by switching valve B to INJECT position. After that, the eluted solution was lyophilized to dryness. Finally, 2 μL of matrix solution (mixture of 20 mg/mL 2,5-DHB in 50% (v/v) ACN, 1% (v/v) phosphoric acid) was introduced into the 7765



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eluate, and 1 μL of the mixture was used for MALDI-MS analysis. Apparatus. The silicon and titanium species were determined by Shimadzu EDX-720 energy-dispersive X-ray analysis (EDX, Kyoto, Japan) using Mg Kα radiation as the excitation source. The X-ray diffraction experiments were taken with a Bruker SMART APEX II X-ray diffractometer (Billerica, Germany) using Cu Kα radiation and rotating anode operated at 40 kV and 30 mA. The microscopic morphology of the monolith was examined by a Quanta 200 scanning electron microscopy (SEM) (FEI, Holand). Nitrogen sorption experiments were carried out at 77 K using JW-BK surface area and pore size analyzer (JWGB Sci. & Tech., Beijing, China). The specific surface area values were calculated according to the BET (Brunauer−Emmett−Teller) equation at P/Po between 0.05 and 0.2.45 All MALDI-TOF-MS spectra were recorded with Axima TOF2 mass spectrometry (Shimadzu, Kyoto, Japan). The instrument was equipped with a 337 nm nitrogen laser with a 3 ns pulse width. The detection was performed in positive ion reflector mode with an accelerating voltage of 20 kV. Typically, 200 laser shots were averaged to generate each spectrum.

of PEG was added into the mixture. At the same time, some dilute acetic acid (0.01 mol/L) was also utilized because it could promote the condensation of silanols between the capillary wall and the monolithic surface, which provided better anchorage. The in situ 120 °C treatment was carried out to strengthen the monolithic framework and eliminate the shrinkage during the drying process.48 This synthesis strategy was facile, and the whole procedure could be finished within one day. The composition of the material was examined by EDX experiments. As shown in Figure S1, Supporting Information, the monolith was clearly constituted by oxygen, silicon, and titanium. The atom percentage was 69.2% for oxygen, 21.1% for silicon, and 9.7% for titanium. This data indicated the molar ratio of Ti/Si was approximately 1/2, which fitted well with the original feeding amounts of titanium and silicon. To investigate the homogeneity and phase distribution, we performed XRD experiments for the monoliths prepared outside of the capillary. The patterns showed that both as synthesized and thermo treated (the heating program was 5 °C per minute to 500 °C and held for 3 h) SiO2/TiO2 monolithic materials were entirely amorphous (Figure S2, Supporting Information), which indicated that no crystallization of TiO2 could be generated even after relatively high temperature treatments. This result suggested that the titanium was incorporated and isolated in the silica frameworks so that the crystallization was considerably prevented in this case. In other words, the titanium and silicon were distributed homogeneously on the atom scale in the final composites.49 Figure 2A−C displays the SEM images of the monolithic capillary column. It can be seen that the monolith possessed a continuous skeleton with interconnecting macropores, and the framework attached tightly to the inner-wall of the capillary indicating a good anchorage. The surface area and mesopore distribution of the prepared monolith were examined by a nitrogen adsorption−desorption experiment. Our results showed that the specific surface area of the proposed material was 115 m2/g and the monolith exhibited a narrow mesopore distribution with the BJH most frequent pore diameter of 3.6 nm (Figure 2D). Taken together, this SiO2/TiO2 composite monolith owned complete column structure, favorable surface area, and typical bimodal porosity, which made it a suitable choice as extraction medium. Enrichment of Phosphopeptides by SiO2/TiO2 Monolithic Capillary Column. The enrichment performance of SiO2/TiO2 monolith for phosphopeptides was first investigated by the tryptic digests of α-casein and β-casein (3 pmol each). The direct analysis of the digested proteins (Figure 3A) showed that only very limited phosphopeptides could be detected, while the nonphosphorylated peptides dominated the spectrum. However, after the enrichment with the SiO2/TiO2 monolithic capillary column, all of the phosphopeptides (the sequences are listed in Table S2, Supporting Information) could be observed in the spectrum (Figure 3B), and the spectrum of the sampling eluate after enrichment by the column was also analyzed to check the capture capability (Figure 3C). The result showed that no phosphopeptides but only nonphosphopeptides could be detected in this case, which indicated high affinity of the proposed monolith toward phosphopeptides. For comparison, the pure TiO2 monolithic material synthesized outside the capillary was also used to enrich phosphopeptides (Supporting Information). As shown in Figure S3, Supporting Information, only nine phosphopep-

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RESULTS AND DISCUSSION Preparation and Characterization of the SiO2/TiO2 Monolithic Column. To develop a controllable and mild

Figure 3. MALDI mass spectra of tryptic digests of α-casein and βcasein with the concentration of 1.5 × 10−7 M (3 pmol for each) obtained by direct analysis (A), after enrichment with the SiO2/TiO2 monolith (B), or with the sampling solution after enrichment (C). Phosphopeptides are marked with “αn” or “βn”, and the internal standard is marked with “#”.

system, we introduced glycerol to slow down the reaction rate of TBOT toward water instead of strong acids, because glycerol can form a more stable secondary precursor by transesterifying onto the primary titanium precursor. This mechanism has been comprehensively discussed in the previous studies.6,46,47 To induce phase separation and adjust porosity, a certain amount 7766



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Figure 4. MALDI mass spectra of the tryptic digest mixtures of α-casein, β-casein, and BSA without (A, C, E) or with (B, D, F) SiO2/TiO2 monolithic capillary column enrichment. Molar ratios of α-casein and β-casein to BSA are 1:1:1 (A, B), 1:1:10 (C, D), and 1:1:100 (E, F). The concentrations of both α-casein and β-casein were 1.5 × 10−7 M (3 pmol for each). Phosphopeptides are marked with “αn” or “βn”, and the internal standard is marked with “#”.

Figure 5. MALDI mass spectra of tryptic digest of β-casein with 50 fmol (A) and 10 fmol (B) obtained after enrichment by SiO2/TiO2 monolithic capillary column.

tides (α4, α7, α8, α9, α12, α13, β1, β3, β4) could be identified in this situation. This result suggested the introduction of silica in the structure of the monolith could improve its performance on the selective enrichment of phosphopeptides, which was consistent with the previous reports.16−18 To evaluate the performance of this SiO2/TiO2 monolithic capillary column in the complex systems, the monolithic capillary column was used to extract phosphopeptides from the mixture of the tryptic digests of α-casein, β-casein, and BSA with molar ratios of 1:1:1, 1:1:10, or 1:1:100. The MALDI-TOF mass spectra of direct analysis of the samples before enrichment were shown in Figure 4A,C,E. Along with the increase of BSA (especially up to 10-fold and more), the signals of nonphosphopeptides dramatically

Figure 6. Mass spectra of human serum phosphopeptides obtained by direct analysis (A) or after enrichment with pure TiO2 material (B) or SiO2/TiO2 monolithic capillary column (C).

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(Figure 6B). However, there were still plenty of signals of nonphosphopeptides. For the SiO2/TiO2 composite monolith group, four phosphopeptides derived from fibrinopeptide A could be distinctly observed after enrichment with only very few interferences as shown in Figure 6C. The sequence information of the four phosphopeptides was listed in Table 1. This result, which was consistent with the latest related studies,50−56 demonstrated the good potential application of such novel monolithic SiO2/TiO2 material in enrichment of endogenous phosphopeptides from complex biological samples. To further study the performance of the SiO2 /TiO 2 composite monolithic material toward other real biosample matrixes, the in-solution digestions of nonfat milk and egg white were introduced as typical examples. For the milk sample, only three phosphopeptides could be identified through direct analysis (Figure S4A, Supporting Information) and five after extraction with pure TiO2 monolithic material (Figure S4B, Supporting Information). While after enrichment with the SiO2/TiO2 composite monolith, eleven phosphopeptides could be observed (eight from α-casein and three from β-casein) with good resolution.54−56 As for egg white, direct analysis (Figure 7A) led to nearly no distinct peaks because of the severe suppression of ionization caused by the high concentration of salts in the digest. After enrichment with pure TiO2 monolithic material, plenty of peaks came out in the spectrum. However, most of them were nonphosphopeptides (Figure 7B), indicating the limited specificity of pure TiO2 material. For the SiO2/TiO2 composite monolith group, three noticeable peaks at m/z 2090.71, 2902.63, and 3844.81 showed up (Figure 7C) with very few interferences. These signals belonged to phosphoprotein ovalbumin in the egg white,56 and the corresponding sequences were listed in Table 2. It was clear that the specificity of the SiO2/TiO2 composite monolith was much higher than the pure TiO2, which could be attributed to the lower Lewis acidity of titanium in the silica framework of the SiO2/TiO2 composite monolith compared with the pure TiO2.16,17 These results indicated that the proposed SiO2/TiO2 composite monolith had excellent selectivity toward phosphopeptides in the real in-solution digestion samples.

Table 1. Detailed Information of the Observed Endogenous Phosphopeptides from Human Serum No.

[M + H]+

phosphorylation site

amino acid sequence

F1 F2 F3 F4

1389.31 1460.39 1545.50 1616.57

1 1 1 1

DSGEGDFLAEGGGV ADSGEGDFLAEGGGV DSGEGDFLAEGGGVR ADSGEGDFLAEGGGVR

Figure 7. Mass spectra of egg white phosphopeptides obtained by direct analysis (A) or after enrichment with pure TiO2 material (B) or SiO2/TiO2 monolithic capillary column (C).

enhanced and the identification of phosphopeptides became impossible. However, as shown in Figure 4B,D,F, all the phosphopeptides could be easily observed after enrichment by the SiO2/TiO2 monolithic capillary column, even with 100-fold of BSA. These results indicated the excellent selectivity of the SiO2/TiO2 composite material toward phosphopeptides under interference of abundant nonphosphopeptides. In addition, βcasein digest with different amounts was used (50 fmol and 10 fmol) to evaluate the detection limit of the enrichment strategy. As shown in Figure 5, three phosphopeptides (β1, β3, β4) could be clearly identified with 50 fmol of sample, and the welldetected signal of β1 could still be obtained even at the level of 10 fmol, which demonstrated the high detection sensitivity of the strategy. The human serum sample was also used to investigate the enrichment capability of the SiO2/TiO2 composite monolith. As shown in Figure 6A, only one phosphopeptide could be identified with poor resolution by direct analysis. The situation slightly improved after enrichment with the pure TiO2 material

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CONCLUSIONS In the current study, we presented a facile preparation method to fabricate a novel SiO2/TiO2 composite monolithic capillary column and utilized it as a MOAC material for highly specific capture of phosphopeptides. This is the very first attempt for preparing silica−metal in-capillary composite monolithic columns. The synthetic condition was mild, and the cycle was less than 24 h. Silicon and titanium in the prepared SiO2/ TiO2 composite monolithic capillary column distributed homogeneously in the framework, and the molar ratio of Ti and Si was approximate 1/2. A bimodal porosity was obtained in the monolith with the most frequent mesopore diameter around 3.6 nm. This monolith was then successfully applied in an in-tube SPME system to selectively extract phosphopeptides from complex samples, like human blood serum, nonfat milk,

Table 2. Detailed Information of the Observed Phosphopeptides from Tryptic Digested Egg White [M + H]+

phosphorylation site

amino acid sequence

2090.72 2902.63 3844.81

1 1 1

EVVGSAEAGVDAASVSEEFR FDKLPGFGDSIEAQCGTSVNVHSSLR ISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFR 7768



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and egg white. The whole extraction procedure can be finished in 16 min. Taken together, we developed a facile preparation method to generate a novel SiO2/TiO2 composite monolithic capillary column which demonstrated excellent performance on efficient enrichment of phosphopeptides.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86 27 68755595. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for financial support from the Major State Basic Research Development Program of China (973 Program) (2012CB720600, 2012CB720601), the National Natural Science Foundation of China (91017013, 31070327), the Natural Science Fund for Creative Research Groups (No. 20921062), NSFC, and the Fundamental Research Funds for the Central Universities.

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Article

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