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.     

In‐tube solid‐phase microextraction capillary column packed with mesoporous TiO2 nanoparticles for phosphopeptide analysis

In this study, an in‐tube solid‐phase microextraction column packed with mesoporous TiO₂ nanoparticles, coupled with MALDI–TOF–MS, was applied to the selective enrichment and detection of phosphopeptides in complex biological samples. The mesoporous TiO₂ nanoparticles with high specific surface areas, prepared by a sol–gel and solvothermal method, were injected into the capillary using a slurry packing method with in situ polymerized monolithic segments as frits. Compared with the traditional solid‐phase extraction method, the TiO₂‐packed column with an effective length of 1 cm exhibited excellent selectivity (α‐casein/β‐casein/BSA molar ratio of 1:1:100) and sensitivity (10 fmol of a β‐casein enzymatic hydrolysis sample) for the enrichment of phosphopeptides. These performance characteristics make this system suitable for the detection of phosphorylated peptides in practical biosamples, such as nonfat milk.     

Identification of phosphorylation sites of equine β‐casein isoforms

Equine β‐casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P–7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion‐exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro‐column. This method turned out to be an efficient tool to separate the CPPs Arg¹–Lys³⁴ and Glu⁴–Lys³⁴ from non‐phosphorylated peptides. Purification was achieved by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and each CPP was hydrolyzed by endoproteinase Glu‐C. Finally, the digests were analyzed by RP‐HPLC/electrospray ionization mass spectrometry (RP‐HPLC/ESI‐MS) and identified by nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) to locate the phosphorylated sites of the β‐casein isoforms 4P–7P with accuracy. Thus, the isoform 4P was found to be phosphorylated on residues Ser⁹, Ser²³, Ser²⁴, and Ser²⁵. Addition of phosphate groups on Ser¹⁸, Thr¹², and Ser¹⁰ led to the formation of the isoforms 5P–7P, respectively. The results indicated that the in vivo phosphorylation of the equine β‐casein follows a sequential way and is not randomly performed. Copyright © 2010 John Wiley & Sons, Ltd.     

Highly Water‐Dispersible Surface‐Modified Gd2O3 Nanoparticles for Potential Dual‐Modal Bioimaging

Water‐dispersible and luminescent gadolinium oxide (GO) nanoparticles (NPs) were designed and synthesized for potential dual‐modal biological imaging. They were obtained by capping gadolinium oxide nanoparticles with a fluorescent glycol‐based conjugated carboxylate (H L ). The obtained nanoparticles (GO‐ L ) show long‐term colloidal stability and intense blue fluorescence. In addition, L can sensitize the luminescence of europium(III) through the so‐called antenna effect. Thus, to extend the spectral ranges of emission, europium was introduced into L‐ modified gadolinium oxide nanoparticles. The obtained Eu^III‐doped particles (Eu:GO‐ L ) can provide visible red emission, which is more intensive than that without L capping. The average diameter of the monodisperse modified oxide cores is about 4 nm. The average hydrodynamic diameter of the L ‐modified nanoparticles was estimated to be about 13 nm. The nanoparticles show effective longitudinal water proton relaxivity. The relaxivity values obtained for GO‐ L and Eu:GO‐ L were r₁=6.4 and 6.3 s^?1 mM ^?1 with r₂/r₁ ratios close to unity at 1.4 T. Longitudinal proton relaxivities of these nanoparticles are higher than those of positive contrast agents based on gadolinium complexes such as Gd‐DOTA, which are commonly used for clinical magnetic resonance imaging. Moreover, these particles are suitable for cellular imaging and show good biocompatibility.     

Site‐specific separation and detection of phosphopeptide isomers with pH‐mediated stacking capillary electrophoresis‐electrospray ionization‐tandem mass spectrometry

This study reported a pH‐mediated stacking CE coupled with ESI MS/MS method to determine the phosphorylation sites of three synthetic phosphopeptides containing structural isomers. These phosphopeptides mimic the phosphopeptides (amino acid residues 12–25) derived from the trypsin‐digested products of human lamin A/C protein. The LODs were determined to be 118, 132 and 1240 fmol for SGAQASS¹⁹TpPL²²SPTR, SGAQASS¹⁹TPL²²SpPTR, and SGAQASS¹⁹TpPL²²SpPTR, respectively. The established method was employed to analyze the phosphorylation sites of the trypsin‐digested products of glutathione S‐transferase‐lamin A/C (1–57) fusion protein that had been phosphorylated in vitro by cyclin‐dependent kinase 1. The results indicated that this method is feasible to specifically determine the phosphorylation site from phosphopeptide isomers in the trypsin‐digested products of a kinase‐catalyzed phosphoprotein, which should benefit the investigation of protein kinase‐mediated cellular signal transduction.     

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.     

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.     

Titanium dioxide nanoparticle coating of polymethacrylate-based chromatographic monoliths for phosphopetides enrichment

Metal oxide affinity chromatography has been one of the approaches for specific enrichment of phosphopeptides from complex samples, based on specific phosphopeptide adsorption forming bidentate chelates between phosphate anions and the surface of a metal oxide, such as TiO₂, ZrO₂, Fe₂O₃, and Al₂O₃. Due to convective mass transfer, flow-independent resolution and high dynamic binding capacity, monolith chromatographic supports have become important in studies where high resolution and selectivity are required. Here, we report the first synthesis and characterization of immobilisation of rutile TiO₂ nanoparticles onto organic monolithic chromatographic support (CIM-OH-TiO₂). We demonstrate the specificity of CIM-OH-TiO₂ column for enrichment of phosphopeptides by studying chromatographic separation of model phosphorylated and nonphosphorylated peptides as well as proving the phosphopeptide enrichment of digested bovine α-casein. The work described here opens the possibility for a faster, more selective enrichment of phosphopeptides from biological samples that will enable future advances in studying protein phosphorylation.     

The highly selective capture of phosphopeptides by zirconium phosphonate-modified magnetic nanoparticles for phosphoproteome analysis

The highly selective capture of phosphopeptides from proteolytic digests is a great challenge for the identification of phosphoproteins by mass spectrometry. In this work, the zirconium phosphonate-modified magnetic Fe₃O₄/SiO₂ core/shell nanoparticles have been synthesized and successfully applied for the selective capture of phosphopeptides from complex tryptic digests of proteins before the analysis of MALDI-TOF mass spectrometry with the desired convenience of sample handling. The ratio of magnetic nanoparticle to protein and the incubation time for capturing phosphopeptides from complex proteolytic digests were investigated, and the optimized nanoparticle-to-protein ratio and incubation time were between 15:1 to 30:1 and 30 min, respectively. The excellent detection limit of 0.5 fmol β-casein has been achieved by MALDI-TOF mass spectrometry with the specific capture of zirconium phosphonate-modified magnetic Fe₃O₄ nanoparticles. The great specificity of zirconium phosphonate-modified magnetic Fe₃O₄ nanoparticles to phosphopeptides was demonstrated by the selective capture of phosphopeptides from a complex tryptic digest of the mixture of α-casein and bovine serum albumin at molar ratio of 1 to 100 in MALDI-TOF-MS analysis. An application of the magnetic nanoparticles to selective capture phosphopeptides from a tryptic digest of mouse liver lysate was further carried out by combining with nano-LC-MS/MS and MS/MS/MS analyses, and a total of 194 unique phosphopeptides were successfully identified.     

Recovery and separation of erythromycin from industrial wastewater by imprinted magnetic nanoparticles that exploit β‐cyclodextrin as the functional monomer

A type of surface imprinting over magnetic Fe₃O₄ nanoparticles utilizing erythromycin‐A as a template for use in the separation and recovery of erythromycin was developed and investigated. As the intermolecular forces play a key role in the performance of imprinted materials, differential scanning calorimetry, and ¹H NMR spectroscopy was employed to evaluate the interactions between erythromycin and the functional monomer β‐cyclodextrin. To synthesize the surface imprinted polymers, magnetic Fe₃O₄ nanoparticles, the core materials, were modified with a free radical initiator to initialize polymerization in a “grafting from” manner. Then using acryloyl‐modified β‐cyclodextrin as the functional monomer and ethyleneglycol dimethacrylate as the cross‐linker, thin erythromycin‐imprinted films were fabricated by the radical‐induced graft copolymerization of monomers on the surface of the Fe₃O₄ nanoparticles. Selectivity experiments showed that the erythromycin‐A‐imprinted materials had recognition ability toward erythromycin derivatives. Finally, these magnetic molecularly imprinted particles were successfully used for the separation and enrichment of erythromycin from the mother liquor. The recovery, detected by high‐performance liquid chromatography and differential pulse voltammetry, approached 97%. The combination of the specific selectivity of the imprinted material and the magnetic separation provided a powerful tool that is simple, flexible, and selective for the separation and recovery of erythromycin.     

磷酸铵盐作为基质添加物促进磷酸化肽在MALDI-MS分析中的离子化效率

本文建立了以磷酸铵盐为添加剂的基质新系统,增强了磷酸化肽在MALDI正离子模式下的离子化。系统地考察了不同的磷酸盐以及不同的盐浓度对磷酸化肽离子化效率的影响。考察了两种适合于磷酸化肽离子化的基质类型2,5-二羟基苯甲酸和2,4,6-三羟基苯乙酮。用2,5-二羟基苯甲酸作为基质时,当加入10 mM 磷酸氢二铵时,磷酸化蛋白质β-casein的磷酸肽 48FQ[pS]EEQQQTEDELQDK63的离子化效率可以增强5-8倍,当加入10 mM磷酸二氢胺时,磷酸肽的离子化效率可以增强3-4倍。用2,4,6-三羟基苯乙酮作为基质时,当加入5mM磷酸氢二铵时,磷酸化肽的离子化效率比文献报道的最有利于磷酸化肽离子化的基质体系增强了2倍。并探讨了铵根离子和磷酸根离子促进磷酸化肽在MALDI的正离子模式下离子化效率的机理。     

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.     

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.     

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.     

Conductive carbon tape as a sample platform for microwave-based MALDI MS detection of proteins and phosphoproteins

In this study, we developed a novel microwave-assisted protein preparation and digestion method for matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry analysis and identification of proteins that involves using conductive carbon tape as a sample platform for sample preparation (reduction and alkylation) and digestion under microwave heating and as a plate for MALDI analysis. This method allows for the enzymatic digestion products of proteins to be directly analyzed by MALDI mass spectrometry and results in a marked reduction in sample loss. Our protocol requires only a small volume (1 μL) of reaction solvent, which increases the frequency of enzyme-to-protein contact, thereby resulting in more efficient digestion of sample than conventional in-solution digestion methods. To test this protocol, we used magnetic iron (II, III) oxide nanoparticles as concentrating probes to enrich phosphopeptides from a mixture of peptides in enzymatically digested protein samples. We found that the one-pot on-tape-based protein preparation and digestion under microwave heating combined with the on-tape-based enrichment method not only dramatically reduced the time required for phosphopeptides analysis but also allowed for the simultaneous identification of phosphoproteins. The advantages of our protocol include ease of use, high digestion efficiency, high specificity, and rapid (15 min) identification of proteins and enrichment of phosphopeptides in a mixture of enzymatically digested protein samples.     

Methyl orange degradation enhanced by hydrogen spillover onto platinum nanocatalyst surface

Following a thermal reduction method, platinum nanoparticles were synthesized and stabilized by polyvinylpyrrolidone. The colloidal platinum nanoparticles were stable for more than 3 months. The micrograph analysis unveiled that the colloidal platinum nanoparticles were well dispersed with an average size of 2.53 nm. The sol–gel‐based inverse micelle strategy was applied to synthesize mesoporous iron oxide material. The colloidal platinum nanoparticles were deposited on mesoporous iron oxide through the capillary inclusion method. The small‐angle X‐ray scattering analysis indicated that the dimension of platinum nanoparticles deposited on mesoporous iron oxide (Pt‐Fe₂O₃) was 2.64 nm. X‐ray photoelectron spectroscopy (XPS) data showed that the binding energy on Pt‐Fe₂O₃ surface decreased owing to mesoporous support–nanoparticle interaction. Both colloidal and deposited platinum nanocatalysts improved the degradation of methyl orange under reduction conditions. The activation energy on the deposited platinum nanocatalyst interface (2.66 kJ mol^?1) was significantly lowered compared with the one on the colloidal platinum nanocatalyst interface (40.63 ± 0.53 kJ mol^?1).     

Iron oxide/tantalum oxide core–shell magnetic nanoparticle-based microwave-assisted extraction for phosphopeptide enrichment from complex samples for MALDI MS analysis

A new type of metal-oxide-coated magnetic nanoparticles (NPs)—tantalum-oxide-coated magnetic iron oxide (Fe₃O₄@Ta₂O₅) NPs—which are used as affinity probes for selectively trapping phosphopeptides from complex samples, is demonstrated in this study. In this approach, phosphopeptide enrichment was achieved by incubating the NPs with sample solutions under microwave heating within 1 min. The NP–target species conjugates were readily isolated from samples by magnetic separation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis. When using human serum as the sample, phosphorylated fibrinopeptide-A-derived ions are the only ions observed in the MALDI mass spectra after enrichment by the Fe₃O₄@Ta₂O₅ NPs. Furthermore, only phosphopeptides appear in the MALDI mass spectra after using the affinity probes to selectively trap target species from the tryptic digest of a cell lysate and milk sample. The results demonstrated that the Fe₃O₄@Ta₂O₅ NPs have the capability of selectively trapping phosphorylated peptides from complex samples. The detection limit of this approach for a phosphopeptide (FQpSEEQQQTEDELQDK) was ~10 fmol. Figure For the first time, tantalum oxide-coated magnetic iron oxide (Fe₃O₄@Ta₂O₅) NPs were demonstrated as suitable affinity-probes for selectively trapping phosphopeptides from complex samples. To shorten the analysis time, phosphopeptide enrichment was achieved by incubating the NPs with sample solutions under microwave-heating within 1 min. MALDI MS was employed for characterization of the species trapped by the NPs.     

Facile preparation of titanium phosphate‐modified chitosan for selective capture of phosphopeptides

A facile two‐step method for preparing chitosan‐based immobilized metal ion affinity chromatography was developed. First, chitosan was phosphorylated by esterification with phosphoric acid, and then titanium was chelated onto the phosphorylated chitosan. The obtained chitosan‐based titanium immobilized metal ion affinity chromatography was ultrafine microparticles and had good dispersibility in acidic buffer. The selectivity and sensitivity were evaluated by phosphopeptide enrichment of mixtures of α‐casein and bovine serum albumin. The enriched peptides were analyzed by mass spectrum. Enrichment protocols were optimized and the optimum‐loading buffer was 80% acetonitrile with 1% trifluoroacetic acid. With α‐casein concentration as low as 2 pmol, 12 phosphopeptides were detected with considerably high intensity from the digest mixtures of α‐casein and bovine serum albumin with molar ratio of 1:200. The microparticles was also applied in real biological samples, 29 phosphoproteins containing 40 phosphorylated sites were identified from salt‐stressed Arabidopsis thaliana leaves.     

High‐performance liquid chromatography separation of unsaturated organic compounds by a monolithic silica column embedded with silver nanoparticles

The optimization of a porous structure to ensure good separation performances is always a significant issue in high‐performance liquid chromatography column design. Recently we reported the homogeneous embedment of Ag nanoparticles in periodic mesoporous silica monolith and the application of such Ag nanoparticles embedded silica monolith for the high‐performance liquid chromatography separation of polyaromatic hydrocarbons. However, the separation performance remains to be improved and the retention mechanism as compared with the Ag ion high‐performance liquid chromatography technique still needs to be clarified. In this research, Ag nanoparticles were introduced into a macro/mesoporous silica monolith with optimized pore parameters for high‐performance liquid chromatography separations. Baseline separation of benzene, naphthalene, anthracene, and pyrene was achieved with the theoretical plate number for analyte naphthalene as 36 000 m^?1. Its separation function was further extended to cis/trans isomers of aromatic compounds where cis/trans stilbenes were chosen as a benchmark. Good separation of cis/trans‐stilbene with separation factor as 7 and theoretical plate number as 76 000 m^?1 for cis‐stilbene was obtained. The trans isomer, however, is retained more strongly, which contradicts the long‐ established retention rule of Ag ion chromatography. Such behavior of Ag nanoparticles embedded in a silica column can be attributed to the differences in the molecular geometric configuration of cis/trans stilbenes.     

Two‐step Immobilized Metal Affinity Chromatography (IMAC) for Phosphoproteomics Using Mass Spectrometry

In this study, a new strategy named two‐step IMAC is demonstrated as a novel prelude to MS analysis of phosphoproteome by increasing the enrichment factor of phosphoproteins/phosphopeptides from a protein mixture. In this method, the first IMAC was performed at the protein level to extract the minute amount of phosphoproteins present in the sample. During this step, nonphosphoproteins and other undesired chemicals or inhibitors were excluded. After tryptic digestion, the second IMAC was performed at the peptide level to enrich phosphopeptides present in the tryptic digest, and the eluent from the second IMAC was analyzed by MALDI‐MS. It is particularly noticeable that the eluent from the first IMAC can be directly digested by trypsin without buffer exchange. Our results revealed that β‐casein that was spiked in a protein mixture can be successfully extracted by the first IMAC at a concentration of less than 1–3%, and the two phosphopeptides of β‐casein with single and four phosphorylation sites, respectively, can be captured by the second IMAC. It was found that the two‐step IMAC method could significantly reduce non‐specific bindings from unwanted proteins and greatly enhance the MALDI‐MS signal of phosphopeptide ions compared to the typical one‐step IMAC, by which only IMAC at the peptide level was performed. Two‐step IMAC was also found to tolerate a greater amount and a greater concentration range of proteins than one‐step IMAC, which is especially important when analyzing complicated unknown samples. Furthermore, the MS signal of phosphopeptide ions did not appear to be degraded by the presence of biological matrixes, such as the cell lysate in which the β‐casein was spiked in.