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Titania coated magnetic mesoporous hollow silica microspheres: fabrication and application to selective enrichment of phosphopeptidesw Jian-Hong Wu,a Xiao-Shui Li,a Yong Zhao,b Qiang Gao,ac Lin Guoab and Yu-Qi Feng*a Received 23rd July 2010, Accepted 27th September 2010 DOI: 10.1039/c0cc02763d Titania coated magnetic hollow mesoporous silica spheres with high surface area were created, which can be used in efficient and rapid capture of phosphopeptides from peptide mixtures. Mesoporous materials are traditionally used in heterogeneous catalysis, adsorption, host–guest chemistry and drug delivery because of their remarkable features,1,2 but recently, they are also used in proteomics studies, especially in phosphoproteomics studies.3,4 Meanwhile, magnetic materials have long been adopted in biological experiments, such as cell labeling, enzyme immobilization and drug delivery.5,6 By combining mesoporous texture and magnetical separation properties, magnetic mesoporous spheres represent a new class of separation materials with great application potentials.7–9 Reversible protein phosphorylation regulates many cellular processes such as proliferation, differentiation and apoptosis.10 Many proteins with regulatory function are not abundantly expressed and the stoichiometry of their phosphorylation can be quite low. Thus, phosphorylated protein or peptide enrichment is normally required for analysis of protein phosphorylation.11 Based on Lewis acid–base interaction, several metal oxides (TiO2, ZrO2 and Fe2O3) have been widely used for phosphopeptide enrichment.12–14 Very recently, mesoporous TiO2 has been exploited as the adsorbent of phosphopeptides and showed high binding capacity due to its surface area.15 In this work, we immobilized TiO2 on magnetic hollow mesoporous silica spheres to prepare magnetically separable adsorbent for capturing phosphopeptides. The fabrication strategy and working principle of this composite material are illustrated in Scheme 1. The hollow mesoporous silica spheres (HMSS) were prepared according to a previously reported method.16 Then, the magnetic particles were introduced into the hollow core of HMSS through a vacuum impregnation of Fe(NO3)3.9 The Fe(NO3)3-loaded powders were impregnated in ethylene glycol (EG) up to incipient wetness. The impregnated sample was then subjected to heat treatment under nitrogen. It was believed that the EG played an important role in the conversion of iron salt into magnetic
iron oxides (see ESIw) Finally, titania were loaded to the asprepared magnetic hollow mesoporous silica spheres (MHMSS) by either the sol–gel method17 (TiO2-1/MHMSS-2) and or by liquid-phase deposition (LPD) method18 (TiO2-2/MHMSS-2) (see ESIw for details). The morphology and structure of the as-synthesized spheres were investigated by scanning electron microscopy (SEM). The HMSS exhibit monodisperse, uniform and spherical morphologies with an average diameter of 2–4 mm (Fig. 1a). The hollow structure of the HMSS has been confirmed by broken spheres crushed by high pressure (Fig. 1b). Loading of Fe and Ti has no effect on the morphology of HMSS (Fig. 1c–e). The magnetic separable property of TiO2-2/ MHMSS-2 was tested in water by placing or removing using an external magnet. As shown in Fig. 1f, in the absence of an external magnetic field, these magnetic microspheres dispersed well in the aqueous solution. When the external magnetic field was applied, all magnetic microspheres went straight towards the magnet and adhered tightly to the side wall of the bottle tightly, and the liquid phase became clear and transparent immediately. The N2 adsorption–desorption isotherms and pore size distributions of HMSS, MHMSS and TiO2/MHMSS were measured (Fig. S1, ESIw). All the samples yielded typical IV adsorption isotherms and H1 hysteresis loops, which are characteristic of a mesoporous material. As listed in Table 1, the surface area decreased gradually in the order of HMSS, MHMSS, TiO2/MHMSS, and the pore volume also decreased in the same order. Compared with HMSS, the pore volume of MHMSS-1 decreased sharply, but the pore size distribution showed little change. This indicated that most of magnetic particles has been introduced into the cavities of HMSS, consistent with a previous observation.9 Three MHMSS samples, MHMSS-1, MHMSS-2 and MHMSS-3, were
a
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China. E-mail:
[email protected]; Fax: +86-27-68755595; Tel: +86-27-68755595 b College of Life Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072, P. R. China c Faculty of Material Science & Chemistry Engineering, China University of Geosciences, Wuhan 430074, P. R. China w Electronic supplementary information (ESI) available: Experimental details and further characterization data. See DOI: 10.1039/ c0cc02763d
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Scheme 1 Schematic illustration of the synthesis strategy of TiO2/ MHMSS and the working principle for phosphopeptide adsorption.
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Fig. 1 SEM images of HMSS (a, b), MHMSS-2 (c), TiO2-1/MHMSS-2 (d) and TiO2-2/MHMSS-2 (e). Table 1 Surface area (As) and pore volume (Vp) of the materials Sample
As/m2 g
HMSS MHMSS-1 MHMSS-2 MHMSS-3 TiO2-1/MHMSS-2 TiO2-2/MHMSS-2
608 505 453 387 438 369
1
Vp/cm3 g
1
2.05 1.56 1.47 1.27 1.17 0.98
prepared with different amounts of Fe(NO3)3 and their saturation magnetization values were found to be 1.7, 7.9 and 8.8 emu g 1, respectively (Fig. S2, ESIw). Taking both the magnetic property and specific surface area into consideration, MHMSS-2 was used in subsequent experiments. The X-ray diffraction (XRD) patterns of as-prepared HMSS and MHMSS were investigated (Fig. S3, ESIw). A broad band centered at 2y = 221 for both materials were observed, which may be assigned to the characteristic reflection from amorphous silica (JCPDS 29-0085). The diffraction peaks at 33.3, 35.8, 54.1, 57.4 and 62.71 in patterns of MHMSS indicated the existence of crystalline ion oxide. Because of the similar spinel structure of Fe3O4 and g-Fe2O3, it was hard to distinguish these two iron oxides. The magnetic property of MHMSS should be derived from one of, or a combination of, Fe3O4 and g-Fe2O3.19 In fact, the iron oxide loaded samples showed a red–brown color (Fig. 1f), which is characteristic for g-Fe2O3.19 Additionally, in the XPS spectra of MHMSS (Fig. S4, ESIw) a binding energy at about 711 eV was found and could be assigned to 2p3/2 of Fe3+ ions while the characteristic peak (about 709 eV) of 2p3/2 of Fe2+ was absent, suggesting that the Fe3O4 phase was at low concentration or was absent.19 On the basis of XRD and XPS analysis, it could be inferred that the magnetic particles of MHMSS were mainly composed of g-Fe2O3. To exactly represent the composition of MHMSS, it was advisable to denote the iron oxide of MHMSS as FeIII2FeII1 xO4 x.20 X-Ray photoelectron scanning microscopy (XPS) was carried out to confirm the existence of titania in the as-prepared TiO2-1/ MHMSS-2 and TiO2-2/MHMSS-2 (Fig. S5, ESIw). As a control sample, MHMSS had no peak in the Ti binding energy region. In contrast, both TiO2-1/MHMSS-2 and TiO2-2/MHMSS-2 had peaks in the Ti 2p and Ti 3p binding energy regions, indicating the presence of titania. 9032
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The capacity of TiO2 or TiO2-based materials to capture phosphopeptides can be attributed to the Lewis acid-basic interaction between phosphate groups and Ti ions. As enrichment efficiency of these materials is affected by the crystallinity,15 the crystallinity of the spheres were analyzed by XRD and Raman spectra (Fig. S3 and S6, ESIw). The X-ray diffraction pattern of TiO2-1/MHMSS-2 is similar to that of MHMSS, indicating that the titania in TiO2-1/MHMSS-2 was amorphous and did not crystallize, or that the particles were too small to show Bragg diffraction.21 However, several new diffraction peaks were found in the X-ray diffraction pattern of TiO2-2/MHMSS-2, indicating the existence of crystalline titania. The Raman spectra of TiO2-2/MHMSS-2 was typical of the anatase TiO2 phase, but with the Raman peaks slightly broader and shifted comparing to those of single crystals. The results showed that the TiO2 nanoparticles loaded on TiO2-2/ MHMSS-2 by LPD method were well crystallized in the anatase structure. The efficiency of TiO2-1/MHMSS-2 and TiO2-2/MHMSS-2 in phosphopeptide enrichment was studied by using the trypsin digests of a-casein (3 pmol) (Fig. S7, ESIw) followed by matrixassisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Because Fe also shows binding ability to phosphopeptides, MHMSS-2 spheres were also studied for comparison. Compared with MHMSS-2 (Fig. 2a), more phosphopeptides were captured by TiO2-1/ MHMSS-2 (Fig. 2b). Non-phosphopeptides were detected as a minor component after enrichment in MHMSS-2 and TiO2-1/ MHMSS-2. Their intensity decreased obviously in TiO2-2/ MHMSS-2 (Fig. 2c)), indicating superior selectivity for TiO2-2/ MHMSS-2. Besides the obvious advantage in separation procedure, our magnetic mesoporous spheres provided
Fig. 2 Mass spectra obtained using MHMSS-2 (a), TiO2-1/MHMSS2 (b), TiO2-2/MHMSS-2 (c) and commercial TiO2 (d) to selectively enrich phosphorylated peptides from a tryptic digest of a-casein at concentration of 1.0 10 7 M (3 pmol). The phosphopeptides are indicated by ‘‘an’’. The internal standard is marked with ‘‘#’’.
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successfully synthesized and can be used in the highly efficient and rapid enrichment of phosphopeptides. The large surface area of TiO2-2/MHMSS-2 provided the foundation for its impressive phosphopeptide enrichment capacity. Compared to nanoparticle-based TiO2, TiO2-2/MHMSS-2 does not need centrifugation during the enrichment procedure, reducing potential loss of the material-phosphopeptides conjugate during sample preparation. This work was supported by grants from the National Science Fund for Distinguished Young Scholars (No.20625516), the Science Fund for Creative Research Groups (No. 20921062) and the National 973 project of China (2007CB914200). Fig. 3 Mass spectra obtained using TiO2-2/MHMSS-2 to selectively enrich phosphorylated peptides from a tryptic digest of b-casein and BSA with ratio of 1 : 1 (a), 1 : 10 (b) and 1 : 100 (c). b-casein was at concentration of 1.0 10 7 M (3 pmol). The phosphopeptides are indicated by ‘‘bn’’. The internal standard is marked with ‘‘#’’.
comparable selectivity towards phosphopeptides compared to widely used commercial TiO2 nanoparticles (Fig. 2d). Due to its nano-size restraint, the separation procedure for TiO2 nanoparticles is usually difficult. Because centrifugation (15 000 g) was used for the separation of TiO2 nanoparticles from supernatant in our experiment, it is likely some phosphopeptides were lost during the enrichment procedure. This likely explained why the relative ratio of an added internal standard peptide at m/z 1210 to other casein phosphopeptides was lower in TiO2-2/MHMSS-2 compared to that observed in the commercial TiO2. Based on the above investigation, TiO2-2/MHMSS-2, the best performer in our study, was subjected to further testing with a more complex peptide mixtures consisting of BSA and b-casein (Fig. S8 showed the obtained mass spectra of tryptic digest of the mixture of b-casein and non-phosphoprotein BSA with a molar ratio of 1 : 1, 1 : 10 and 1 : 100, ESIw). Identification of phosphopeptides from b-casein was impossible before enrichment because of the interference of the abundant non-phosphorylated peptides. After enrichment by TiO2-2/MHMSS-2, three b-casein phosphopeptides can be clearly detected (Fig. 3). For clarity, details of the observed phosphopeptides are listed in Table S1 (ESIw). These results indicated the reliable and rapid performance of the TiO2-2/ MHMSS-2 in the selective isolation of phosphorylated peptides from a complex peptide mixture. In summary, two magnetic mesoporous materials, TiO2-1/ MHMSS-2 and TiO2-2/MHMSS-2, with large surface area, high pore volume and large pore diameter, have been
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