Synthesis of Fe3O4-graphene-TiO2 ternary composite networks for enhanced capture of phosphopeptides

Fe(3)O(4)-graphene-TiO(2) ternary composite networks were first synthesized, which exhibited high selectivity and capacity in the capture of phosphopeptides, due to the enhanced contact to phosphopeptides given by the graphene scaffold.     

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.     

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.     

Cyclic phosphopeptides for interference with Grb2 SH2 domain signal transduction prepared by ring-closing metathesis and phosphorylation

Cyclic phosphopeptides were prepared using ring-closing metathesis followed by phosphorylation. These cyclic phosphopeptides were designed to interact with the SH2 domain of Grb2, which is a signal transduction protein of importance as a target for antiproliferative drug development. Binding of these peptides to the Grb2 SH2 domain was evaluated by a surface plasmon resonance assay. High affinity binding to the Grb2 SH2 domain was maintained upon macrocyclization, thus indicating that this method can be used to assemble high affinity cyclic phosphopeptides that interfere with signal transduction cascades.     

Coupling of TiO(2)-mediated enrichment and on-bead guanidinoethanethiol labeling for effective phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry

Titanium dioxide (TiO2)-mediated phosphopeptide enrichment has been introduced as an effective method for extracting phosphopeptides from highly complex peptide mixtures. Chemical labeling by beta-elimination/Michael addition is also useful for increasing mass intensity in phosphopeptide analysis. Both of these methods were coupled in order to simultaneously enrich phosphopeptides and allow for detection and sequencing of the enriched peptides with high mass sensitivity. Phosphopeptides were successfully enriched on TiO2 beads without the use of any hydroxy acid additives like 2,5-dihydroxybenzoic acid. Labeling was accomplished on-bead with a guanidinoethanethiol (GET) tag containing a guanidine moiety. These GET-labeled derivatives were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). GET labeling converted phosphoserine into guanidinoethylcysteine, a structural arginine-mimic. In particular, GET-labeled lysine-terminated phosphopeptides showed dramatically increased peak intensities compared to those of the corresponding intact phosphopeptides. Additionally, the on-bead labeling minimized manipulation steps and sample loss. The coupled technique was also further validated by applying to the analysis of phosphopeptides from complex tryptic digests of phosphoprotein mixtures.     

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.     

Preparation of a TiO2 nanoparticle-deposited capillary column by liquid phase deposition and its application in phosphopeptide analysis

Analysis of phosphopeptides from complex mixtures derived from proteolytic digestion of biological samples is a challenging yet highly important task. Since phosphopeptides are usually present in small amounts, enrichment is often necessary prior to their characterization by mass spectrometry. In this study, a thin layer of titanium dioxide (TiO2) nanoparticles (NPs) was deposited onto the surface of capillary column by liquid phase deposition (LPD) technique and applied to selectively concentrate phosphopeptides from protein digest products. This is, to our knowledge, the first demonstration of using liquid phase deposition to construct in-tube solid phase microextraction devices for biological analysis. By coupling the device off-line or on-line with mass spectrometry analysis, experiments for systematic optimization of loading and washing conditions were carried out, and good trapping selectivity of TiO2 NP-deposited capillary columns towards phosphopeptides was demonstrated.     

Chemo-affinity of titania for the column-switching HPLC analysis of phosphopeptides.

A column-switching high-performance liquid chromatography (HPLC) system for the determination of phosphopeptides has been developed. The method is based on the selective adsorption of phosphopeptides on a titania (TiO2) precolumn and successive HPLC separation of the phosphopeptides on an anion-exchange column with a UV detector (215 nm). The recoveries of phosphopeptides were tested using authentic phosphopeptides [Gln-Ile-Ser(p)-Val-Arg, Ile-Ser(p)-Val-Arg and Lys-Gln-Ile-Ser(p)-Val-Arg] at an injection amount of 1 microg. The recoveries were 74.3, 79.6, and 82.6%, respectively, while the corresponding dephosphopeptides were not retained on the titania precolumn.     

Sequential Fe3O4/TiO2 enrichment for phosphopeptide analysis by liquid chromatography/tandem mass spectrometry

Protein phosphorylation regulates a wide range of cellular functions and is associated with signaling pathways in cells. Various strategies for enrichment of phosphoproteins or phosphopeptides have been developed. Here, we developed a novel sequential phosphopeptide enrichment method, using magnetic iron oxide (Fe₃O₄) and titanium dioxide (TiO₂) particles, to detect mono‐ and multi‐phosphorylated peptides. In the first step, phosphopeptides were captured on Fe₃O₄ particles. In a subsequent step, any residual phosphopeptides were captured on TiO₂ particles. The particles were eluted and rinsed to yield phosphopeptide‐enriched fractions that were combined and analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The validity of this sequential Fe₃O₄/TiO₂ enrichment strategy was demonstrated by the successful enrichment of bovine α‐casein phosphopeptides. We then applied the sequential Fe₃O₄/TiO₂ enrichment method to the analysis of phosphopeptides in L6 muscle cell lysates and successfully identified mono‐ and multi‐phosphorylated peptides. Copyright © 2010 John Wiley & Sons, Ltd.     

Facile fabrication of hydrophilic PAA-Ti/TiO2 nanocomposite for selective enrichment and detection of phosphopeptides from complex biological samples

Highly selective enrichment of trace phosphorylated proteins or peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. In this study, a novel affinity material has been synthesized to improve the enrichment specificity for phosphopeptides by using PAAS as coupling molecule. In the resulting materials, highly abundant titanium is available for selective enrichment of phosphopeptides, with plenty of carboxylate groups that can inhibit nonspecific adsorption. The enrichment results demonstrated that the hydrophilic PAA-Ti/TiO₂ composite possesses excellent selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/non-phosphopeptides (1:1000), extreme sensitivity (the detection limit was at the fmol level), and high recovery of phosphopeptides (as high as 78%). Moreover, the as-prepared nanocomposite provides effective enrichment of phosphopeptides from real samples (mouse liver), showing great potential in the detection of low-abundance phosphopeptides in biological samples.     

Improved detection of phosphopeptides by negative ion matrix-assisted laser desorption/ionization mass spectrometry using a proton sponge co-matrix

Analysis of phosphopeptides is an important task in proteomic studies. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a technique very commonly used for such a purpose. Analysis of phosphopeptides by MALDI-MS is, however, still a challenging task due to the low ionization efficiency of phosphopeptides. In this study, we reported that by using a proton sponge 1,8-bis(dimethyl-amino)naphthalene (DMAN) as a co-matrix, detection of phosphopeptides by negative ion MALDI-MS could be greatly improved. Combination of DMAN with another matrix 6-aza-2-thiothymine (ATT) and additive diammonium hydrogen citrate (DHC) allowed much lower limit of detection, significantly reduced signal suppression effects and improved position-to-position reproducibility for detection of phosphopeptides by negative ion MALDI-MS. Potential applications of the matrix system in qualification of phosphopeptides and analysis of proteolytic digests of phosphorylated proteins were also demonstrated in this study.     

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.     

Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis

Large-scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria.     

Mesoporous TiO2 aerogel for selective enrichment of phosphopeptides in rat liver mitochondria

The enrichment of low abundance phosphopeptides before MS analysis is a critical step for in-depth phosphoproteome research. In this study, mesoporous titanium dioxide (TiO₂) aerogel was prepared by precipitation and supercritical drying. The specific surface area up to 490.7 m² g⁻¹ is achieved by TiO₂ aerogel, much higher than those obtained by commercial TiO₂ nanoparticles and by the latest reported mesoporous TiO₂ spheres. Due to the large specific surface area and the mesoporous structure of the aerogel, the binding capacity for phosphopeptides is six times higher than that of conventional TiO₂ microparticles (173 vs 28 μmol g⁻¹). Because of the good compatibility of enrichment procedure with MALDI-TOF-MS and the large binding capacity of TiO₂ aerogel, a detection limit as low as 30 amol for analyzing phosphopeptides in β-casein digest was achieved. TiO₂ aerogel was further applied to enrich phosphopeptides from rat liver mitochondria, and 266 unique phosphopeptides with 340 phosphorylation sites, corresponding to 216 phosphoprotein groups, were identified by triplicate nanoRPLC-ESI-MS/MS runs, with false-positive rate less than 1% at the peptide level. These results demonstrate that TiO₂ aerogel is a kind of promising material for sample pretreatment in the large-scale phosphoproteome study.     

纳米氧化锆沉积棉花纤维的制备及其在磷酸化多肽快速富集中的应用

采用液相沉积法在棉花纤维的表面成功沉积了纳米氧化锆颗粒,并将其装填在移液枪的吸头内,通过移液枪的抽吸实现对磷酸化多肽的萃取,萃取过程只需要2 min,方法简单、快速。该材料不仅可以从β-酪蛋白酶解物这种简单的体系中萃取出9个磷酸化多肽,还可以从物质的量比为1∶100的β-酪蛋白酶解物和牛血清白蛋白（BSA）酶解物混合物这类含有大量非磷酸化多肽的复杂样品中萃取出4种磷酸化多肽,且没有非磷酸化多肽被检出,表现出较好的萃取选择性。将该材料应用于人血清和脱脂牛奶这两种复杂实际样品的酶解物中磷酸化多肽的快速富集萃取,分别检测出5种和9种磷酸化多肽,均表现出较好的选择性。     

Enrichment of phosphopeptides using bare magnetic particles

Magnetic iron(II, III) oxide (magnetite, Fe(3)O(4)) nanoparticles were used to selectively enrich phosphopeptides from tryptic digests of bovine beta-casein and from tryptic digest mixtures containing bovine beta-casein, cytochrome c, bovine serum albumin, and horse heart myoglobin. The magnetic property of the particles permits an easy and speedy enrichment process. No enrichment of phosphopeptides was observed from ferric magnetic iron(III) oxide (Fe(2)O(3)) nanoparticles. These data collectively demonstrate that the enrichment of phosphopeptides using magnetic iron(II, III) oxide nanoparticles is a practical method for the selective analysis of phosphopeptides and could be helpful in isolating and analyzing phosphorylated peptides from complex biological samples.     

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.     

Multifunctional ZrO2 nanoparticles and ZrO2-SiO2 nanorods for improved MALDI-MS analysis of cyclodextrins, peptides, and phosphoproteins

Multifunctional ZrO₂ nanoparticles (NPs) and ZrO₂-SiO₂ nanorods (NRs) have been successfully applied as the matrices for cyclodextrins and as affinity probes for enrichment of peptides (leucine-enkephalin, methionine-enkephalin and thiopeptide), phosphopeptides (from tryptic digestion products of β-casein) and phosphoproteins from complex samples (urine and milk) in atmospheric pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and MALDI time-of-flight (TOF) MS. The results show that the ZrO₂ NPs and ZrO₂-SiO₂ NRs can interact with target molecules (cyclodextrins, peptides, and proteins), and the signal intensities of the analytes were significantly improved in MALDI-MS. The maximum signal intensities of the peptides were obtained at pH 4.5 using ZrO₂ NPs and ZrO₂-SiO₂ NRs as affinity probes. The limits of detection of the peptides were found to be 75-105 fmol for atmospheric pressure MALDI-MS and those of the cyclodextrins and β-casein were found to be 7.5-20 and 115-125 fmol, respectively, for MALDI-TOF-MS. In addition, these nanomaterials can be applied as the matrices for the analysis of cyclodextrins in urine samples by MALDI-TOF-MS. ZrO₂ NPs and ZrO₂-SiO₂ NRs efficiently served as electrostatic probes for peptide mixtures and milk proteins because 2–11 times signal enhancement can be achieved compared with use of conventional organic matrices. Moreover, we have successfully demonstrated that the ZrO₂ NPs can be effectively applied for enrichment of phosphopeptides from tryptic digestion of β-casein. Comparing ZrO₂ NPs with ZrO₂-SiO₂ NRs, we found that ZrO₂ NPs exhibited better affinity towards phosphopeptides than ZrO₂-SiO₂ NRs. Furthermore, the ZrO₂ and ZrO₂-SiO₂ nanomaterials could be used to concentrate trace amounts of peptides/proteins from aqueous solutions without tedious washing procedures. This approach is a simple, straightforward, separation-and washing-free approach for MALDI-MS analysis of cyclodextrins, peptides, proteins, and tryptic digestion products of phosphoproteins.      

Two-step on-particle ionization/enrichment via a washing- and separation-free approach: multifunctional TiO2 nanoparticles as desalting, accelerating, and affinity probes for microwave-assisted tryptic digestion of phosphoproteins in ESI-MS and MALDI-MS: comparison with microscale TiO2

We introduce a simplified sample preparation method using bare TiO₂ nanoparticles (NPs) to serve as multifunctional nanoprobes (desalting, accelerating, and affinity probes) for effective enrichment of phosphopeptides from microwave-assisted tryptic digestion of phosphoproteins (α-casein, β-casein and milk) in Electrospray Ionization Mass Spectrometry (ESI-MS) and Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). The results demonstrate that TiO₂ NPs can effectively enrich and accelerate the digestion reactions of phosphoproteins in aqueous solutions and also from complex real samples. After the microwave experiments, we directly injected the resulting solutions into the ESI-MS and MALDI-MS systems for analysis, and excellent sensitivity was achieved without the need for any washing procedure or separation process. The reasons are attributed to the high binding affinity and selectivity of TiO₂ NPs toward phosphopeptides. Thus, phosphopeptides can be adsorbed onto the TiO₂ NP surface. The digested or partially digested phosphoproteins can be concentrated onto the TiO₂ NP surface. This results in the effective or complete digestion of phosphoproteins in a short period of time (45 s). In addition, high sensitivity and sequence coverage of phosphopeptide can be obtained using TiO₂ NPs as microwave absorbers and affinity probes in MALDI-MS and ESI-MS. This is due to the photocatalytic nature of the TiO₂ NPs because the absorption of microwave radiation that can accelerate the activation of trypsin for efficient digestion of phosphoproteins and enhances the ionization of phosphopeptides. The lowest concentrations detected for ESI-MS and MALDI-MS were 0.1 μM and 10 fmol, respectively, for α-casein. Comparing the two-step approach of TiO₂ NPs with microscale TiO₂ particles, the microscale TiO₂ particles shows no effect on the microwave-assisted tryptic digestion of phosphoproteins. The current approach offers multiple advantages, such as great simplicity, high sensitivity and selectivity, straightforward and separation/washing-free technique for phosphorpeptide enrichment analysis.     

Development of core–shell structure Fe3O4@Ta2O5 microspheres for selective enrichment of phosphopeptides for mass spectrometry analysis

Protein phosphorylation is one of the most important post-translational modifications. Due to the dynamic nature and low stoichiometry of the protein phosphorylation, enrichment of phosphopeptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Many metal oxides such as titanium dioxide and zirconium dioxide have been successfully applied to isolation and enrichment of phosphopeptides. Recently, niobium pentoxide was proved to have the ability for selective enrichment of phosphopeptides. Considering the proximity of tantalum to niobium, we supposed that Ta₂O₅ can be used as affinity probes for phosphopeptide enrichment. In the work, we synthesized Fe₃O₄@Ta₂O₅ magnetic microspheres with core–shell structure for selective enrichment of phosphopeptides. To demonstrate its ability for selective enrichment of phosphopeptides, we applied Fe₃O₄@Ta₂O₅ magnetic microspheres to isolation and enrichment of the phosphopeptides from tryptic digestion of standard proteins and real samples, and then the enriched peptides were analyzed by matrix-assisted laser desorption mass spectrometry analysis (MALDI-MS) or liquid chromatography coupled to electrospray ionization mass spectrometry (LC–ESI-MS). Experiment results demonstrate that Ta₂O₅ coated-magnetic microspheres show the excellent potential for selective enrichment of phosphopeptides.