Highly selective enrichment of phosphopeptides using poly(dibenzo‐18‐crown‐6) as a solid‐phase extraction material

A poly(dibenzo‐18‐crown‐6) was used as a new solid‐phase extraction material for the selective enrichment of phosphopeptides. Isolation of phosphopeptides was achieved based on specific ionic interactions between poly(dibenzo‐18‐crown‐6) and the phosphate group of phosphopeptides. Thus, a method was developed and optimized, including loading, washing and elution steps, for the selective enrichment of phosphopeptides. To assess this potential, tryptic digest of three proteins (α‐ casein, β‐casein and ovalbumin) was applied on poly(dibenzo‐18‐crown‐6). The nonspecific products were removed by centrifugation and washing. The spectrometric analysis was performed using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Highly selective enrichment of both mono‐ and multiphosphorylated peptides was achieved using poly(dibenzo‐18‐crown‐6) as solid‐phase extraction material with minimum interference from nonspecific compounds. Furthermore, evaluation of the efficiency of the poly(dibenzo‐18‐crown‐6) was performed by applying the digest of egg white. Finally, quantum mechanical calculations were performed to calculate the binding energies to predict the affinity between poly(dibenzo‐18‐crown‐6) and various ligands. The newly identified solid‐phase extraction material was found to be a highly efficient tool for phosphopeptide recovery from tryptic digest of proteins.     

Enrichment specificity of micro and nano‐sized titanium and zirconium dioxides particles in phosphopeptide mapping

Owning to their anion‐exchange properties, titanium and zirconium dioxides are widely used in phosphopeptide enrichment and purification protocols. The physical and chemical characteristics of the particles can significantly influence the loading capacity, the capture efficiency and phosphopeptide specificity and thus the outcome of the analyses. Although there are a number of protocols and commercial kits available for phosphopeptide purification, little data are found in the literature on the choice of the enrichment media. Here, we studied the influence of particle size on the affinity capture of phosphopeptides by TiO₂ and ZrO₂. Bovine milk casein derived phosphopeptides were enriched by micro and nanoparticles using a single‐tube in‐solution protocol at different peptide‐to‐beads ratio ranging from 1 : 1 to 1 : 200. Unsupervised hierarchical cluster analysis based on the whole set of Matrix Assisted Laser Desorption/Ionization time‐of‐flight mass spectra of the phosphopeptide enriched samples revealed 62 clustered peptide peaks and shows that nanoparticles have considerably higher enrichment capacity than bulk microparticles. Moreover, ZrO₂ particles have higher enrichment capacity than TiO₂. The selectivity and specificity of the enrichment was studied by monitoring the ion abundances of monophosphorylated, multiphosphorylated and non‐phosphorylated casein‐derived peptide peaks at different peptide‐to‐beads ratios. Comparison of the resulting plots enabled the determination of the optimal peptide‐to‐beads ratios for the different beads studied and showed that nano‐TiO₂ have higher selectivity for phosphopeptides than nano‐ZrO₂ particles. Copyright © 2013 John Wiley & Sons, Ltd.     

Ti (IV) attached‐phosphonic acid functionalized capillary monolith as a stationary phase for in‐syringe‐type fast and robust enrichment of phosphopeptides

In this study, poly(vinylphosphonic acid‐co‐ethylene dimethacrylate), poly(VPA‐co‐EDMA) capillary monolith was synthesized as a starting material for obtaining a stationary phase for microscale enrichment of phosphopeptides. The chelation of active phosphonate groups with Ti (IV) ions gave a macroporous monolithic column with a mean pore size of 5.4 μm. The phosphopeptides from different sources were enriched on Ti (IV)‐attached poly(VPA‐co‐EDMA) monolith using a syringe‐pump. The monolithic capillary columns exhibited highly sensitive/selective enrichment performance with phosphoprotein concentrations as low as 1.0 fmol/mL. Six different phosphopeptides were detected with high intensity by the treatment of β‐casein digest with the concentration of 1.0 fmol/mL, using Ti (IV)@poly(VPA‐co‐EDMA) monolith. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts, in a complex mixture of digested proteins (molar ratio of β‐casein to bovine serum albumin, 1:1500) and three phosphopeptides were successfully detected. Four highly intense signals of phosphopeptides in human serum were also observed with high signal‐to‐noise ratio and a clear background after enrichment with Ti (IV)@poly(VPA‐co‐EDMA) monolith. It was concluded that the capillary microextraction system enabled fast, efficient and robust enrichment of phosphopeptides from microscale complex samples. The whole enrichment process was completed within 20 min, which was shorter than in the previously reported studies.     

Matrix solid‐phase dispersion with chitosan‐zinc oxide nanoparticles combined with flotation‐assisted dispersive liquid–liquid microextraction for the determination of 13 n‐alkanes in soil samples

In this study, chitosan‐zinc oxide nanoparticles were used as a sorbent of miniaturized matrix solid‐phase dispersion combined with flotation‐assisted dispersive liquid–liquid microextraction for the simultaneous determination of 13 n‐alkanes such as C₈H₁₈ and C₂₀H₄₂ in soil samples. The solid samples were directly blended with the chitosan nanoparticles in the solid‐phase dispersion method. The eluent of solid‐phase dispersion was applied as the dispersive solvent for the following flotation‐assisted dispersive liquid–liquid microextraction for further purification and enrichment of the target compounds prior to gas chromatography with flame ionization detection. Under the optimum conditions, good linearity with correlation coefficients in the range 0.9991 < r² < 0.9995 and low detection limits between 0.08 to 2.5 ng/g were achieved. The presented procedure combined the advantages of chitosan‐zinc oxide nanoparticles, solid‐phase dispersion and flotation‐assisted dispersive liquid–liquid microextraction, and could be applied for the determination of n‐alkanes in complicated soil samples with acceptable recoveries.     

Surface-modified TiO(2) nanoparticles as affinity probes and as matrices for the rapid analysis of phosphopeptides and proteins in MALDI-TOF-MS

We utilized three different types of TiO₂ nanoparticles (NPs) namely TiO₂‐dopamine, TiO₂‐CdS and bare TiO₂ NPs as multifunctional nanoprobes for the rapid enrichment of phosphopeptides from tryptic digests of α‐ and β‐casein, milk and egg white using a simplified procedure in MALDI‐TOF‐MS. Surface‐modified TiO₂ NPs serve as effective matrices for the analysis of peptides (gramicidin D, HW6, leucine‐enkephalin and methionine‐enkephalin) and proteins (cytochrome c and myoglobin) in MALDI‐TOF‐MS. In the surface‐modified TiO₂ NPs‐based MALDI mass spectra of these analytes (phosphopetides, peptides and proteins), we found that TiO₂‐dopamine and bare TiO₂ NPs provided an efficient platform for the selective and rapid enrichment of phosphopeptides and TiO₂‐CdS NPs efficiently acted as the matrix for background‐free detection of peptides and proteins with improved resolution in MALDI‐MS. We found that the upper detectable mass range is 17 000 Da using TiO₂‐CdS NPs as the matrix. The approach is simple and straightforward for the rapid analysis of phosphopeptides, peptides and proteins by MALDI‐MS in proteome research.     

Dispersion of TiO2 on high‐surface‐area mesoporous silica: functionalization with tungstophosphoric acid and application in solvent‐free,aerobic oxidation of n‐hexadecane

Imidazole type ionic liquid, 1‐hexadecyl‐3‐methylimidazolium chloride, was used to template the synthesis of high‐surface‐area mesoporous silica under acidic conditions and crystalline titanium dioxide (TiO₂) nanoparticles of anatase phase were inserted utilizing a solvent evaporation‐induced method. The surface area of more than 700 m² g^?1 was obtained after TiO₂ impregnation. Further, the polyoxometalate, 12‐tungstophosphoric acid (PW₁₂) was dispersed on the surface of TiO₂ to form PW₁₂–TiO₂–silica hybrid catalytic materials. The catalytic activity of this hybrid material was tested for solvent‐free, aerobic oxidation of n‐hexadecane. The experimental investigation shows that PW₁₂–TiO₂ nanocrystals did not block the pore channels and gave good conversion. Copyright © 2012 John Wiley & Sons, Ltd.     

Magnetic solid‐phase extraction based on a polydopamine‐coated Fe3O4 nanoparticles absorbent for the determination of bisphenol A,tetrabromobisphenol A, 2,4,6‐tribromophenol,and (S)‐1,1’‐bi‐2‐naphthol in environmental waters by HPLC

Polydopamine‐coated Fe₃O₄ magnetic nanoparticles synthesized through a facile solvothermal reaction and the self‐polymerization of dopamine have been employed as a magnetic solid‐phase extraction sorbent to enrich four phenolic compounds, bisphenol A, tetrabromobisphenol A, (S)‐1,1′‐bi‐2‐naphthol and 2,4,6‐tribromophenol, from environmental waters followed by high‐performance liquid chromatographic detection. Various parameters of the extraction were optimized, including the pH of the sample matrix, the amount of polydopamine‐coated Fe₃O₄ sorbent, the adsorption time, the enrichment factor of analytes, the elution solvent, and the reusability of the nanoparticles sorbent. The recoveries of these phenols in spiked water samples were 62.0–112.0% with relative standard deviations of 0.8–7.7%, indicating the good reliability of the magnetic solid‐phase extraction with high‐performance liquid chromatography method. In addition, the extraction characteristics of the magnetic polydopamine‐coated Fe₃O₄ nanoparticles were elucidated comprehensively. It is found that there are hydrophobic, π–π stacking and hydrogen bonding interactions between phenols and more dispersible polydopamine‐coated Fe₃O₄ in water, among which hydrophobic interaction dominates the magnetic solid‐phase extraction performance.     

Application of zein‐modified magnetite nanoparticles in dispersive magnetic micro‐solid‐phase extraction of synthetic food dyes in foodstuffs

A simple method for the simultaneous and trace analysis of four synthetic food azo dyes including carmoisine, ponceau 4R, sunset yellow, and allura red from some foodstuff samples was developed by combining dispersive μ‐solid‐phase extraction and high‐performance liquid chromatography with diode array detection. Zein‐modified magnetic Fe₃O₄ nanoparticles were prepared and used for μ‐solid‐phase extraction of trace amounts of mentioned food dyes. The prepared modified magnetic nanoparticles were characterized by scanning electron microscopy and FTIR spectroscopy. The factors affecting the extraction of the target analytes such as pH, amount of sorbent, extraction time, type and volume of the desorption eluent, and desorption time were investigated. Under the optimized conditions, the method provided good repeatability with relative standard deviations lower than 5.8% (n = 9). Limit of detection values ranged between 0.3 and 0.9 ng/mL with relatively high enrichment factors (224–441). Comparing the obtained results indicated that Fe₃O₄ nanoparticles modified by zein biopolymer show better analytical application than bare magnetic nanoparticles. The proposed method was also applied for the determination of target synthetic food dyes in foodstuff samples such as carbonated beverage, snack, and candy samples.     

Core‐shell structured magnetic mesoporous carbon nanospheres derived from metal‐polyphenol coordination polymer‐coated Fe3O4 and its application in the enrichment of phthalates from water samples

In this work, core‐shell structured magnetic mesoporous carbon nanospheres were fabricated from the carbonization of metal‐polyphenol coordination polymer‐coated Fe₃O₄ nanoparticles. The preparation method is simple, fast, versatile, and easy to scale up. Magnetic mesoporous carbon nanospheres exhibit a high specific surface area, high superparamagnetism, and high adsorption efficiencies for phthalates. Four phthalates were extracted from aqueous solutions by using magnetic mesoporous carbon nanospheres via magnetic solid phase extraction. Subsequent analysis was performed by using high‐performance liquid chromatography with ultraviolet detection. The analytical method has good linearity in the concentration range of 1–200 ng/mL for diethyl phthalate, diisobutyl phthalate, and dicyclohexyl phthalate, and 3–200 ng/mL for dipropyl phthalate. The limits of detection were in the range of 0.10–0.62 ng/mL. Compared with previous methods, this method has a lower detection limit, wider linearity range, and faster adsorption and desorption rates. The results indicate that magnetic mesoporous carbon nanospheres are suitable for the enrichment of hydrophobic substances from aqueous solutions.     

Imprinted silica nanoparticles coated with N‐propylsilylmorpholine‐4‐carboxamide for the determination of m‐cresol in synthetic and real samples

m‐Cresol‐imprinted silica nanoparticles coated with N‐propylsilylmorpholine‐4‐carboxamide have been developed that contain specific pockets for the selective uptake of m‐cresol. Silica nanoparticles were synthesized by a sol–gel process followed by functionalization of their surface with N‐propylsilylmorpholine‐4‐carboxamide. The formation of m‐cresol‐imprinted silica nanoparticles was confirmed by UV‐Vis spectrophotometry, infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy and transmission electron microscopy. Electron microscopic studies revealed the formation of monodispersed imprinted silica nanoparticles with spherical shape and an average size of 83 nm. The developed nanoparticles were filled in a syringe and used for the extraction of m‐cresol from aqueous samples followed by quantification using high‐performance liquid chromatography with diode array detection. Various adsorption experiments showed that developed m‐cresol‐imprinted silica nanoparticles exhibited a high adsorption capacity and selectivity and offered a fast kinetics for rebinding m‐cresol. The chromatographic quantification was achieved using mobile phase consisting of acetonitrile/water (70:30 v/v) at an isocratic flow rate of 1.0 mL/min using a reversed‐phase C₁₈ column and detection at λ_max = 275 nm. The limits of detection and quantification were 1.86 and 22.32 ng/mL, respectively, for the developed method. The percent recoveries ranged from 96.66–103.33% in the spiked samples. This combination of this nanotechnique with molecular imprinting was proved as a reliable, sensitive and selective method for determining the target from synthetic and real samples.     

The use of liquid phase deposition prepared phosphonate grafted silica nanoparticle‐deposited capillaries in the enrichment of phosphopeptides

In our current work, we describe how open tubular‐immobilized metal‐ion affinity chromatography (OT‐IMAC) capillary columns connected to a solid phase microextraction (in‐tube SPME) device can be used for the enrichment of phosphopeptides. A phosphonate modified silica nanoparticle (NP)‐deposited capillary was prepared by liquid phase deposition (LPD), and used for the immobilization of Fe³⁺, Zr⁴⁺ or Ti⁴⁺. The enrichment capacities of three different OT‐IMAC capillary columns were compared by using tryptically digested α‐casein as sample. The improved extraction efficiency in our technique was demonstrated by comparing to a directly modified capillary, and a comparison of phosphopeptide extraction from simple and complex samples was tested for both modes. Our results show that the NP‐IMAC‐Zr⁴⁺ capillary column can be used to selectively isolate phosphopeptides from real samples, and can enrich for β‐casein phosphopeptides from concentrations as low as 1.7×10^?9 M.     

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.     

Graphene‐coated magnetic‐sheet solid‐phase extraction followed by high‐performance liquid chromatography with fluorescence detection for the determination of aflatoxins B1, B2, G1, and G2 in soy‐based samples

A new method named graphene‐coated magnetic‐sheet solid‐phase extraction based on a magnetic three‐dimensional graphene sorbent was developed for the extraction of aflatoxins prior to high‐performance liquid chromatography with fluorescence detection. The use of a perforated magnetic‐sheet for fixing the magnetic nanoparticles is a new feature of the method. Hence, the adsorbent particles can be separated from sample solution without using an external magnetic field. This made the procedure very simple and easy to operate so that all steps of the extraction process (sample loading, washing, and desorption) were carried out continuously using two lab‐made syringe pumps. The factors affecting the performance of extraction procedure such as the extraction solvent, adsorbent dose, sample loading flow rate, ionic strength, pH, and desorption parameters were investigated and optimized. Under the optimal conditions, the obtained enrichment factors and limits of detection were in the range of 205–236 and 0.09–0.15 μg/kg, respectively. The relative standard deviations were <3.4 and 7.5% for the intraday (n = 6) and interday (n = 4) precisions, respectively. The developed method was successfully applied to determine aflatoxins B₁, B₂, G₁, and G₂ in different soy‐based food samples.     

Extraction and preconcentration of tylosin from milk samples through functionalized TiO2 nanoparticles reinforced with a hollow fiber membrane as a novel solid/liquid‐phase microextraction technique

The aim of this study was to introduce a novel, simple, and highly sensitive preparation method for determination of tylosin in different milk samples. In the so‐called functionalized TiO₂ hollow fiber solid/liquid‐phase microextraction method, the acceptor phase is functionalized TiO₂ nanoparticles that are dispersed in the organic solvent and held in the pores and lumen of a porous polypropylene hollow fiber membrane. An effective functionalization of TiO₂ nanoparticles has been done in the presence of aqueous H₂O₂ and a mild acidic ambient under UV irradiation. This novel extraction method showed excellent extraction efficiency and a high enrichment factor (540.2) in comparison with conventional hollow fiber liquid‐phase microextraction. All the experiments were monitored at λ_max = 284 nm using a simple double beam UV‐visible spectrophotometer. A Taguchi orthogonal array experimental design with an OA₁₆ (4⁵) matrix was employed to optimize the factors affecting the efficiency of hollow fiber solid/liquid‐phase microextraction such as pH, stirring rate, salt addition, extraction time, and the volume of donor phase. This developed method was successfully applied for the separation and determination of tylosin in milk samples with a linear concentration range of 0.51–7000 μg/L (r² = 0.991) and 0.21 μg/L as the limit of detection.     

Magnetic mixed hemimicelles solid‐phase extraction coupled with high‐performance liquid chromatography for the extraction and rapid determination of six fluoroquinolones in environmental water samples

In this study, a mixed hemimicelle solid‐phase extraction method based on Fe₃O₄ nanoparticles coated with sodium dodecyl sulfate was applied for the preconcentration and fast isolation of six fluoroquinolones in environmental water samples before high‐performance liquid chromatography determination. The main factors affecting the extraction efficiency of the analytes, such as amount of surfactant, amount of Fe₃O₄ nanoparticles, extraction time, sample volume, sample pH, ionic strength, and desorption conditions, were investigated and optimized. The method has detection limits from 0.05 to 0.1 ng/mL and good linearity (r ≥ 09948) in the range 0.1–200 ng/mL depending on the fluoroquinolone. The enrichment factor is ～200. The recoveries (at spiked levels of 1, 5, and 50 ng/mL) are in the range of 79–120%.     

Phenyl‐functionalized silica‐coated magnetic nanoparticles for the extraction of chlorobenzenes,and their determination by GC‐electron capture detection

We have prepared a solid phase for the extraction of chlorobenzenes (CBs) by coating magnetic (Fe₃O₄) nanoparticles with silica via a sol‐gel process using a mixture of tetraethoxysilane and triethoxyphenylsilane. The nanoparticles were characterized by SEM, energy‐dispersive spectroscopy, and X‐ray diffractometry. The nanoparticles were used for the extraction of 1,4‐dichlorobenzene (1,4‐DCB), 1,2,3‐trichlorobenzene (1,2,3‐TCB), 1,2,4‐trichlorobenzene (1,2,4‐TCB), and 1,2,3,4‐tetrachlorobenzene (1,2,3,4‐TeCB) from water, followed by their determination by GC‐electron capture detection. Under optimal conditions, enrichment factors ranging from 220 to 360 were obtained. All determination coefficients (r²) are >0.99, and linear response is found in range 0.025–1.5 μg/L (at the lower end), and 6–120 μg/L (at the higher end). Detection limits are 6, 10, 11, and 500 ng/L for 1,2,3,4‐TeCB, 1,2,4‐TCB, 1,2,3‐TCB, and 1,4‐DCB, respectively. All RSDs are <6% (for n = 5). The method was successfully applied to the determination of CBs in environmental water samples.     

Pre‐concentration of trace amount of bisphenol A in water samples by palm leaf ash and determination with high‐performance liquid chromatography

Palm leaf ash was characterized and used as low‐cost adsorbent for solid‐phase extraction and preconcentration of bisphenol A (BPA) in real water samples. Analysis of BPA was carried out using HPLC involving Eurospher 100–5‐C₁₈ (25 cm × 4.5 mm, particle size 5 μm) column and water–acetonitrile (40:60, v/v) as mobile phase. The adsorption was achieved quantitatively at a pH of 6 with elution by 3 mL acetonitrile. The limits of detection and enrichment factor were 0.02 μg L^?1 and 333, respectively. Under optimum conditions the relative standard deviation (RSD) was 2% (n = 10). Comparison of qualification criteria of presented preconcentration procedure with other research indicated that palm leaf ash adsorbent was better than many of the adsorbents in terms of cost and reusability. Also, the limit of detection, precision and enrichment factor were comparable and even better than the previously reported methods. Finally, the efficiency of method was computed by determination of trace amounts of BPA in sea, river, mineral and tap waters with recoveries of 93.3–105.5% and RSDs of 0.61–3.12%.. Briefly, the developed solid‐phase extraction and Preparative layer chromatography (PLC) methods may be used for bisphenol A monitoring in any environmental water sample. Copyright © 2016 John Wiley & Sons, Ltd.     

TiO2 nanotube arrays prepared by anodization as an adsorbent in micro‐solid‐phase extraction to preconcentrate and determine nitrogen‐containing polycyclic aromatic hydrocarbons in water samples

In this study, a simultaneous determination method for nitrogen‐containing polycyclic aromatic hydrocarbons including 7‐methylquinoline, acridine, 5,6‐benzoquinoline, carbazole, and 9‐methylcarbazole was developed. This method is based on a micro‐solid phase extraction using TiO₂ nanotube arrays as an adsorbent in combination with HPLC. Some factors that had an effect on the enrichment were optimized, such as sample pH, surfactant concentration, ion strength, type of eluent, equilibrium time, and desorption time. Under the optimized conditions, the linear ranges and LODs were in the range of 0.01–100 and 0.0035–0.81 μg/L, respectively. The precisions of the proposed method were <9.51% (RSD, n = 6). The developed method was validated with four real samples, and the spiked recoveries were in the range of 77–109.6%. All these results demonstrated that this novel micro‐solid‐phase extraction technique was a reliable alternative to conventional preconcentration method for the extraction and analysis of such nitrogen‐containing polycyclic aromatic hydrocarbons in complex samples.     

Determination of Amantadine Residue in Honey by Solid‐phase Extraction and High‐performance Liquid Chromatography with Pre‐column Derivatization and Fluorometric Detection

Amantadine (AMA) is an anti‐viral drug used in apiculture to protect honeybee against the sacbrood virus (Morator aetatulae). This study described a reliable high‐performance liquid chromatographic (HPLC) method for analyzing AMA in honey using a solid‐phase extraction (SPE) cartridge (Plexa PCX) for purification, 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F) as a pre‐column derivatization agent, and fluorometric detection (λ_ex=470 nm, λ_em=530 nm). The chromatographic separation was performed on an XDB C18 column (150×4.6 mm i.d.) using 0.1% trifluoroacetic acid/acetonitrile (35:65,V/V) as the mobile phase at a flow rate of 1.0 mL·min⁻¹ with a run time of 20 min. Under these optimal conditions, a linear relationship was observed in the range of 0.025–1.0 µg· mL⁻¹ with a good correlation coefficient (0.998) and low limit of detection (0.0080 µg·g⁻¹), the recoveries were all above 90%, and the intra‐day and inter‐day precision (RSD) ranged from 3.4%–5.1%.     

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.