Fe3O4@Al2O3 magnetic core-shell microspheres for rapid and highly specific capture of phosphopeptides with mass spectrometry analysis

Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres with phosphopeptides has been developed. With a well-defined core-shell structure, the Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres not only have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy isolation by positioning an external magnetic field. The prepared Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres have been successfully applied to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and ovalbumin. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:50 as well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres over Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC (immobilized metal affinity chromatography) resin, and TiO(2) beads. Finally, the Al(2)O(3) coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.     

Preparation of monodisperse immobilized Ti affinity chromatography microspheres for specific enrichment of phosphopeptides

This study presented an approach to prepare monodisperse immobilized Ti⁴⁺ affinity chromatography (Ti⁴⁺-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8 μm were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13 μm were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti⁴⁺ ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti⁴⁺-IMAC microspheres to phosphopeptides was demonstrated by selective enrichment of phosphopeptides from mixture of tryptic digests of α-casein and bovine serum albumin (BSA) at molar ratio of 1 to 500 by MALDI-TOF MS analysis. The sensitivity of detection for phosphopeptides determined by MALDI-TOF MS was as low as 5 fmol for standard tryptic digest of β-casein. The Ti⁴⁺-IMAC microspheres were compared with commercial Fe³⁺-IMAC adsorbent and homemade Zr⁴⁺-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe³⁺-IMAC, Zr⁴⁺-IMAC and Ti⁴⁺-IMAC methods were 26, 114, 127 and 181, 11, 11 respectively for the same tryptic digest samples. The results indicated that the Ti⁴⁺-IMAC had the best performance for enrichment of phosphopeptides.     

Mesoporous Fe2O3 microspheres: rapid and effective enrichment of phosphopeptides for MALDI-TOF MS analysis

Mesoporous Fe(2)O(3) microspheres have been successfully synthesized by the polymerization (urea and formaldehyde)-induced ferric hydroxide colloid aggregation. The urea-formaldehyde resin was removed by calcination in air. The obtained mesoporous Fe(2)O(3) materials have spherical morphology with uniform particle size of approximately 3.0 microm and porous surface with large inter-particle pores of approximately 48.0 nm. The surface area is as large as approximately 33.3 m(2)/g and the pore volume is 0.31 cm(3)/g. The mesoporous Fe(2)O(3) microspheres were used for the enrichment of phosphopeptides for the first time, in which high sensitivity, selectivity and capacity of specifically enriched phosphopeptides were achieved under a mild condition in a relative short time. After enriched from tryptic digest products of beta-casein by the novel mesoporous Fe(2)O(3) microspheres, phosphopeptides can be selectively detected with high intensity in MALDI-TOF mass spectrometry. Elimination of "shadow effect" was observed by using mesoporous Fe(2)O(3) microspheres, and the detectable limitation is 5x10(-10) M. This material is also effective for enrichment of phosphopeptides from the complex tryptic digests of commercial phosphoprotein casein, with much more phosphorylated sites (26 in 27 of total) and higher signal/noise ratio in the MALDI-TOF mass spectrometry, compared to commercial Fe(2)O(3) nanoparticles. It shows a great potential application in the field of rapid and effective isolation of phosphopeptides.     

Ti-SBA-15介孔材料用于磷酸化肽的高效富集

结合基质辅助激光解吸飞行时间质谱(MALDI-TOF MS)检测技术,考察了Ti-SBA-15介孔材料对β-酪蛋白酶解产物中磷酸化肽的选择性富集性能。实验结果显示,含Ti和Si物质的量比为0.08的Ti-SBA-15介孔材料可选择性地对β-酪蛋白酶解产物中的磷酸化肽进行选择性富集;对于β-酪蛋白和牛血清白蛋白物质的量比为1:100的蛋白质酶解混合液,Ti-SBA-15仍能实现对其磷酸化肽的有效富集。上述结果表明,作为一种多孔、高比表面积的磷酸化多肽的选择性吸附材料,Ti-SBA-15有望在磷酸化蛋白质组的分析中得到广泛的应用。     

Synthesis of novel Fe3O4@SiO2@CeO2 microspheres with mesoporous shell for phosphopeptide capturing and labeling

Fe(3)O(4)@SiO(2)@CeO(2) microspheres with magnetic core and mesoporous shell were synthesized, and the multifunctional materials were utilized to capture phosphopeptides and catalyze the dephosphorylation simultaneously, thereby labeling the phosphopeptides for rapid identification.     

Highly efficient enrichment of phosphopeptides by magnetic nanoparticles coated with zirconium phosphonate for phosphoproteome analysis

The location of phosphorylation plays a vital role for the elucidation of biological processes. The challenge of low stoichiometry of phosphoproteins and signal suppression of phosphopeptides by nonphosphopeptides in mass spectrometry (MS) analysis makes the selective enrichment of phosphopeptides prior to MS analysis necessary. Besides the immobilized metal affinity chromatography (IMAC) method, some affinity methods based on nanoparticles displayed a higher enrichment efficiency for phosphopeptides such as Fe(3)O(4)/TiO2 and Fe(3)O(4)/ZrO(2) nanoparticles. To further improve the selectivity and compatibility of the affinity methods, a novel strategy based on magnetic nanoparticles coated with zirconium phosphonate for the enrichment of phosphopeptides has been developed in this study. Under optimized experimental conditions, 1 x 10(-9) M phosphopeptides in 50 microL tryptic digest of beta-casein could be enriched and identified successfully. Reliable results were also obtained for 1 x 10(-8) M phosphopeptides in 50 microL tryptic digest of beta-casein in the presence of nonphosphopeptides from a tryptic digest of bovine serum albumin (BSA) over 20 times in concentration. The performance of nanoparticles for use in a real sample was further demonstrated by employing the strong cation-exchange chromatography (SCX) fraction of a tryptic digest of a protein extract from Chang liver cells as a model sample. Experimental results show that the nanoparticles can be easily and effectively used for enrichment of phosphopeptides in low concentration. Most importantly, our approach is more compatible with commonly used SCX strategies than Fe(3+)-IMAC. The proposed method thus has great potential for future studies of large-scale phosphoproteomes.     

磁性Fe_3O_4微粒表面有机改性

在分散聚合法制备复合磁性微球过程中 ,采用硅烷偶联剂 KH- 570对磁性 Fe_3O_4微粒进行表面改性 .红外光谱 (FTIR)、光电子能谱 (XPS)分析结果表明 ,偶联剂与磁性微粒表面以化学键形式结合 .改性后 ,Fe_3O_4微粒与单体及其聚合物之间具有良好的亲和性 ,采用改性后的磁性微粒可以显著改善磁性微球的性能指标 .     

磁性Fe3O4微粒表面有机改性

在分散聚合法制备复合磁性微球过程中,采用硅烷偶联剂KH 570对磁性Fe3O4微粒进行表面改性.红外光谱(FTIR)、光电子能谱(XPS)分析结果表明,偶联剂与磁性微粒表面以化学键形式结合.改性后,Fe3O4微粒与单体及其聚合物之间具有良好的亲和性,采用改性后的磁性微粒可以显著改善磁性微球的性能指标.     

Magnetic microspheres modified with Ti(IV) and Nb(V) for enrichment of phosphopeptides

Magnetic microspheres (Fe₃O₄) were coated with polydopamine (PDA) and loaded with the metal ions Ti(IV) and Nb(V) to give a material of type Fe₃O₄@PDA-Ti/Nb. It is shown to be useful for affinity chromatography and for enrichment of phosphopeptides from both standard protein solutions and real samples. For comparison, such microspheres loaded with single metal ions only (Fe₃O₄@PDA-Ti and Fe₃O₄@PDA-Nb) and their physical mixtures were also investigated under identical conditions. The binary metal ion-loaded magnetic microspheres display better enrichment efficiency than the single metal ion-loaded microspheres and their physical mixture. Both multiphosphopeptides and monophosphopeptides can be extracted. The Fe₃O₄@PDA-Ti/Nb microspheres exhibit ultra-high sensitivity (the lowest detection amount being 2 fmol) and selectivity at a low mass ratio such as in case of β-casein/BSA (1:1000).

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Graphical abstract Magnetic microspheres (Fe₃O₄) were coated with polydopamine (PDA) and loaded with the metal ions Ti(IV) and Nb(V) to give a material of type Fe₃O₄@PDA-Ti/Nb. Results showed its great potential as an affinity probe in phosphoproteome research due to rapid magnetic separation of phosphopeptides, ultrahigh sensitivity and selectivity, and remarkable reusability.

     

Zirconium arsenate-modified magnetic nanoparticles: preparation, characterization and application to the enrichment of phosphopeptides

Phosphorylation, one of the most important post-translational modifications of protein, plays a crucial role in a large number of biological processes. Large-scale identification of protein phosphorylation by mass spectrometry is still a challenging task because of the low abundance of phosphopeptides and sub-stoichiometry of phosphorylation. In this work, a novel strategy based on the specific affinity of zirconium arsenate to the phosphate group has been developed for the effective enrichment of phosphopeptides. Zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe(3)O(4)@SiO(2)) were prepared by covalent immobilization of zirconium arsenate on Fe(3)O(4)@SiO(2) magnetic nanoparticles under mild conditions, and characterized by transmission electron microscope (TEM), Fourier transform infrared (FT-IR) spectroscopy, energy dispersive X-ray spectroscopy (EDX) and vibrating sample magnetometer (VSM). The prepared ZrAs-Fe(3)O(4)@SiO(2) was applied for the selective enrichment of phosphopeptides from the digestion mixture of phosphoproteins and bovine serum albumin (BSA). Our results demonstrated that the ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles possess higher selectivity for phosphopeptides and better capture capability towards multiply-phosphorylated peptides than commercial zirconium dioxide (ZrO(2)), which has been widely employed for the enrichment of phosphopeptides. In addition, endogenous phosphopeptides from human serum can be effectively captured by ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles. It is the first report, to the best of our knowledge, in which the zirconium arsenate-modified magnetic nanoparticles were successfully applied to the enrichment of phosphopeptides, which offers the potential application of this new material in phosphoproteomics study.     

Novel approach for the synthesis of Fe3O4@TiO2 core-shell microspheres and their application to the highly specific capture of phosphopeptides for MALDI-TOF MS analysis

A novel approach is proposed to synthesize Fe(3)O(4)@TiO(2) microspheres with a well-defined core-shell structure, and the synthesized Fe(3)O(4)@TiO(2) core-shell microspheres were successfully applied for the simple and fast enrichment of phosphopeptides via direct MALDI-TOF mass spectrometry analysis.     

Highly selective and sensitive enrichment of phosphopeptides via NiO nanoparticles using a microwave-assisted centrifugation on-particle ionization/enrichment approach in MALDI-MS

The strategy to concentrate phosphopeptides has become a critical issue for mapping protein phosphorylation sites, which are well known as posttranslational modifications in proteomics. In this study, we propose a simple and highly sensitive method for phosphopeptide enrichment on NiO nanoparticles (NPs) from a trypsin predigested phosphoprotein complex solution in a microwave oven. Furthermore, this technique was combined with centrifugation on-particle ionization/enrichment of phosphopeptides and phosphopeptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Weak magnetism of these NPs and a positive surface charge effect at low pH accomplished rapid and selective phosphopeptide enrichment within 30s. Trypsin-digested products of phosphoproteins such as α-casein and β-casein, human blood serum, nonfat milk, and egg white were also investigated to explore their phosphopeptide enrichment from complex samples by this approach. The results demonstrate that NiO NPs exhibit good affinity to trace the phosphopeptides even in the presence of 30 times higher molar concentration of complex solution of non-phosphopeptide proteolytic predigested bovine serum albumin. The detection limits of NiO NPs for α-casein and β-casein were 2.0?×?10(-9) M, with good signal-to-noise ratio in the mass spectrum. NiO NPs were found to be effective and selective for enrichment of singly and multiply phosphorylated peptides at a trace level in complex samples in a microwave oven. The cost of preparing NiO NPs is low, the NiO NPs are thermally stable, and therefore, they hold great promise for use in phosphopeptide enrichment.     

Tuning of Ti-doped mesoporous silica for highly efficient enrichment of phosphopeptides in human placenta mitochondria

Extraction of phosphopeptides from rather complex biological samples has been a tough issue for deep and comprehensive investigation into phosphoproteomes. In this paper, we present a series of Ti-doped mesoporous silica (Ti-MPS) materials with tunable composition and controllable morphology for highly efficient enrichment of phosphopeptides. By altering the molar ratio of silicon to titanium (Si/Ti) in the precursor, the external morphology, Ti content, internal long-rang order, and surface area of Ti-MPS were all modulated accordingly with certain regularity. Tryptic digests of standard phosphoprotein α- and β-casein were employed to assess the phosphopeptide enrichment capability of Ti-MPS series. At the Si/Ti molar ratio of 8:1, the optimum enrichment performance with admirable sensitivity and capacity was achieved. The detection limit for β-casein could reach 10 fmol, and 15 phosphopeptides from the digest of α-casein were resolved in the spectrum after enrichment, both superior to the behavior of commercial TiO₂ materials. More significantly, for the digest of human placenta mitochondria, 396 phosphopeptides and 298 phosphoproteins were definitely detected and identified after enrichment with optimized Ti-MPS material, demonstrating its remarkable applicability for untouched phosphoproteomes. In addition, this research also opened up a universal pathway to construct a composition-tunable functional material in pursuit of the maximum performance in applications.

A capillary column packed with a zirconium(IV)-based organic framework for enrichment of endogenous phosphopeptides

A zirconium(IV)-based metal organic framework (Zr-MOF) was deposited on polydopamine-coated silica microspheres to form microspheres of type SiO₂@PDA@Zr-MOF. These were packed into capillary columns for enrichment of phosphopeptides. The column was off-line coupled to both matrix-assisted laser desorption/ionization time of flight mass spectrometry and LC-ESI-MS/MS. The method has a detection limit as low as 4 fmol of β-casein digest and a selectivity as high as 1:1000 (molar ratio of β-casein and BSA digest). It was applied to the analysis of human saliva. In total, 240 endogenous phosphopeptides were identified in only 25 μL human saliva.

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Graphical abstract A zirconium-based metal organic framework (Zr-MOF) was modified outside of polydopamine-coated silica microspheres to form microspheres named SiO₂@PDA@Zr-MOF. Then they were packed in capillary columns for selective enrichment of phosphopeptides via interaction between Zr-O clusters and phosphate groups. The pre-concentration resulted in a better detection of phosphopeptides by mass spectrometry. Tris: Tris(hydroxymethyl)aminomethane; DMF: Dimethyl Formamide; Zr-MOF: Zirconium(IV)-organic framework; MOAC: Metal oxide affinity chromatography.

     

Isolation of phosphopeptides using zirconium-chlorophosphonazo chelate-modified silica nanoparticles

Due to the low abundance of phosphoproteins and substoichiometry of phosphorylation, the elucidation of protein phosphorylation requires highly specific materials for isolation of phosphopeptides from biological samples prior to mass spectrometric analysis. In this study, chlorophosphonazo type derivatives of chromotropic acid including p-hydroxychlorophosphonazo (HCPA) and chlorophosphonazo I (CPA I), traditionally used in the photometric determination of transition metal ions, have been employed as chelating ligands in the preparation of novel affinity materials for phosphopeptide enrichment. The chromogenic reagents of HCPA and CPA I were chemically modified on the surface of silica nanoparticles, and the functionalized materials were charged with zirconium ions through the strong complexation between chelating ligands and Zr(4+). The obtained zirconium-chlorophosphonazo chelate-modified silica nanoparticles (Zr-HCPA-SNPs and Zr-CPA I-SNPs) were applied to the selective enrichment of phosphopeptides, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The purification procedures were optimized using α-casein digest at first, and then the performance of these two affinity materials for efficient and specific enrichment of phosphopeptides was evaluated with the tryptic digests of standard proteins (α-casein, β-casein, ovalbumin and bovine serum albumin). It is found that Zr-HCPA-SNPs are superior to Zr-CPA I-SNPs in phosphopeptide enrichment. Using Zr-HCPA-SNPs to trap phosphopeptides in α-casein digest, the detection limit was close to 50fmol based on MALDI-TOF MS analysis. Finally, Zr-HCPA-SNPs were used to directly isolate phosphopeptides from diluted human serum of healthy, diabetes and hypertension persons, respectively. Our results show that the constitution and level of phosphopeptides are remarkably different among the three groups, which indicate the powerful potentials of Zr-HCPA-SNPs in disease diagnosis and biomarker screening.     

Use of polyethylenimine-modified magnetic nanoparticles for highly specific enrichment of phosphopeptides for mass spectrometric analysis

Phosphopeptides have been isolated and concentrated by use of polyethyleneimine (PEI)-modified magnetic nanoparticles as an extremely specific affinity probe. The particles specifically captured phosphopeptides from a tryptic digest of a protein mixture that contained 0.07% (mole/mole) phosphoproteins, which is the highest specificity obtained to date. The time required for enrichment of the phosphopeptides was 1 min only. PEI-modified magnetic nanoparticles carry positive charges over a wide range of pH—between 3 and 11. This feature means the particles are effectively dispersed in solution during phosphopeptide capture. Mass spectrometric analysis revealed the very high efficiency of enrichment of phosphopeptides that contain both single and multiply-phosphorylated sites. The detection limit in the analysis of phosphopeptides obtained from both bovine α-casein and β-casein by matrix-assisted laser desorption/ionization mass spectrometry was 5 fmol. This approach was also used to enrich the phosphopeptides in a protein digest obtained from non-fat milk.     

Iminodiacetic acid-modified magnetic poly(2-hydroxyethyl methacrylate)-based microspheres for phosphopeptide enrichment

Magnetic non-porous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres prepared by the dispersion polymerization and modified with iminodiacetic acid (IDA) were employed for the IMAC separation of phosphopeptides. Fe³⁺ and Ga³⁺ ions immobilized on IDA-modified magnetic microspheres were used for the enrichment of phosphopeptides from the proteolytic digests of two model proteins differing in their physico-chemical properties and phosphate group content: porcine pepsin A and bovine α-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The phosphopeptides separated from the proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ability of the prepared Fe³⁺- and Ga³⁺-IDA-modified magnetic microspheres to capture phosphopeptides from complex mixtures was shown on an example of bovine milk proteolytic digest.     

Hydrophilic Nb-immobilized magnetic core–shell microsphere – A novel immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides

Rapid and selective enrichment of phosphopeptides from complex biological samples is essential and challenging in phosphorylated proteomics. In this work, for the first time, niobium ions were directly immobilized on the surface of polydopamine-coated magnetic microspheres through a facile and effective synthetic route. The Fe₃O₄@polydopamine-Nb⁵⁺ (denoted as Fe₃O₄@PD-Nb⁵⁺) microspheres possess merits of high hydrophilicity and good biological compatibility, and demonstrated low limit of detection (2 fmol). The selectivity was also basically satisfactory (β-casein:BSA = 1:500) to capture phosphopeptides. They were also successfully applied for enrichment of phosphopeptides from real biological samples such as human serum and nonfat milk. Compared with Fe₃O₄@PD-Ti⁴⁺ microspheres, the Fe₃O₄@PD-Nb⁵⁺ microspheres exhibit superior selectivity to multi-phosphorylated peptides, and thus may be complementary to the conventional IMAC materials.     

Nanodiamond-based two-step sampling of multiply and singly phosphorylated peptides for MALDI-TOF mass spectrometry analysis

Simultaneous detection of multiply and singly phosphorylated peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging because of suppression effects during ionization. In oder to overcome this problem, this study presents a new approach to improve the detection of phosphopeptides by stepwise enrichment using polyarginine-coated (PA-coated) and titanium dioxide-coated (TiO(2)-coated) nanodiamonds for fractionation of multiply and singly phosphorylated peptides prior to on-probe MALDI MS analysis. The feasibility of this approach was demonstrated using synthetic peptides containing different numbers of phosphate groups, tryptic digests of α-casein, β-casein, and complex protein mixtures. The high specificity of the approach is shown in its effective enrichment and fractionation of phosphopeptides from the digest of β-casein and bovine serum albumin at a molar ratio as low as 1 : 1000, which out-performs the commercial Fe(3+)-IMAC and TiO(2) isolation kits. It offers a simple and effective alternative for the fractionation and identification of multiply and singly phosphorylated peptides by MALDI MS and allows for deduction of more information from limited starting materials.     

γ-Fe(2)O(3) and Fe(3)O(4) magnetic hierarchically nanostructured hollow microspheres: Preparation, formation mechanism, magnetic property, and application in water treatment

In this paper, we report the preparation of γ-Fe(2)O(3) and Fe(3)O(4) magnetic hierarchically nanostructured hollow microspheres by a solvothermal combined with precursor thermal conversion method. These γ-Fe(2)O(3) and Fe(3)O(4) magnetic hierarchically nanostructured hollow microspheres were constructed by three-dimensional self-assembly of nanosheets, forming porous nanostructures. The effects of experimental parameters including molar ratio of reactants and reaction temperature on the precursors were studied. The time-dependent experiments indicated that the Ostwald ripening was responsible for the formation of the hierarchically nanostructured hollow microspheres of the precursors. γ-Fe(2)O(3) and Fe(3)O(4) magnetic hierarchically nanostructured hollow microspheres were obtained by the thermal transformation of the precursor hollow microspheres. Both γ-Fe(2)O(3) and Fe(3)O(4) hierarchically nanostructured hollow microspheres exhibited a superparamagnetic property at room temperature and had the saturation magnetization of 44.2 and 55.4emu/g, respectively, in the applied magnetic field of 20 KOe. Several kinds of organic pollutants including salicylic acid (SA), methylene blue (MB), and basic fuchsin (BF) were chosen as the model water pollutants to evaluate the removal abilities of γ-Fe(2)O(3) and Fe(3)O(4) magnetic hierarchically nanostructured hollow microspheres. It was found that γ-Fe(2)O(3) hierarchically nanostructured hollow microspheres showed a better adsorption ability over SA than MB and BF. However, Fe(3)O(4) hierarchically nanostructured hollow microspheres had the best performance for adsorbing MB.