Facile synthesis of Ti4+‐immobilized affinity silica nanoparticles for the highly selective enrichment of intact phosphoproteins

Currently, great challenges to top‐down phosphoproteomics lie in the selective enrichment of intact phosphoproteins from complex biological samples. Herein, we developed a facile approach for synthesis of Ti⁴⁺‐immobilized affinity silica nanoparticles and applied them to the selective separation and enrichment of intact phosphoproteins based upon the principle of metal(IV) phosphate/phosphonate chemistry. The as‐prepared affinity materials exhibited high selectivity and adsorption capacities for model phosphoproteins (328.9 mg/g for β‐casein, 280.5 mg/g for ovalbumin, and 225.8 mg/g for α‐casein), compared with nonphosphoproteins (79.28 mg/g for horseradish peroxidase, 72.70 mg/g for BSA, and 27.28 mg/g for lysozyme). In addition, the resuability of the affinity silica nanoparticles was evaluated, and the results demonstrated a less than 10% loss of adsorption capacity after six adsorption–regeneration cycles. The practicability of the affinity materials was demonstrated by separating phosphoproteins from protein mixtures and drinking milk samples, and the satisfactory results indicated its potential in phosphoproteomics analysis.     

Evaluation and optimization of the metal‐binding properties of a complex ligand for immobilized metal affinity chromatography

The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu²⁺ to l ‐glutamic acid were measured, the metal ion‐binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal‐binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum‐binding conditions of Cu²⁺ on the l ‐glutamic acid‐silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal‐binding behaviors.     

基于天冬氨酸的固定化金属离子亲和色谱材料用于磷酸化肽选择性富集

采用点击化学的方法将自然界中的天冬氨酸(aspartic acid)键合到硅球上(命名为Click Asp),并将Fe3+配位到Click Asp上,合成固定金属离子亲和色谱(IMAC)材料(Fe3+-Click Asp);采用红外光谱、X射线电子能谱和扫描电镜等表征证明Fe3+-Click Asp成功合成。将此IMAC材料用于蛋白质酶解液和牛奶中的磷酸化肽的富集,实现了磷酸化肽的高选择性富集。本研究为磷酸化蛋白质组学提供了新材料和新方法。     

Optimizing the performance of tin dioxide microspheres for phosphopeptide enrichment

Phosphopeptide enrichment based on metal oxide affinity chromatography is one of the most powerful tools for studying protein phosphorylation on a large scale. To complement existing metal oxide sorbents, we have recently introduced tin dioxide as a promising alternative. The preparation of SnO₂ microspheres by the nanocasting technique, using silica of different morphology as a template, offers a strategy to prepare materials that vary in their particle size and their porosity. Here, we demonstrate how such stannia materials can be successfully generated and their properties fine-tuned in order to obtain an optimized phosphopeptide enrichment material. We combined data from liquid chromatography-mass spectrometry experiments and physicochemical characterization, including nitrogen physisorption and energy-dispersive X-ray spectroscopy (EDX), to explain the influence of the various experimental parameters.     

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.     

Functionalized diamond nanopowder for phosphopeptides enrichment from complex biological fluids

Diamond is known for its high affinity and biocompatibility towards biomolecules and is used exclusively in separation sciences and life science research. In present study, diamond nanopowder is derivatized as Immobilized Metal Ion Affinity Chromatographic (IMAC) material for the phosphopeptides enrichment and as Reversed Phase (C-18) media for the desalting of complex mixtures and human serum profiling through MALDI-TOF-MS. Functionalized diamond nanopowder is characterized by Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectroscopy. Diamond-IMAC is applied to the standard protein (β-casein), spiked human serum, egg yolk and non-fat milk for the phosphopeptides enrichment. Results show the selectivity of synthesized IMAC-diamond immobilized with Fe³⁺ and La³⁺ ions. To comprehend the elaborated use, diamond-IMAC is also applied to the serum samples from gall bladder carcinoma for the potential biomarkers. Database search is carried out by the Mascot program (www.matrixscience.com) for the assignment of phosphorylation sites. Diamond nanopowder is thus a separation media with multifunctional use and can be applied to cancer protein profiling for the diagnosis and biomarker identification.     

Efficient fabrication of high‐capacity immobilized metal ion affinity chromatographic media: The role of the dextran‐grafting process and its manipulation

Novel high‐capacity Ni²⁺ immobilized metal ion affinity chromatographic media were prepared through the dextran‐grafting process. Dextran was grafted to an allyl‐activated agarose‐based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran‐grafted agarose microspheres and partly dextran‐grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran‐grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran‐grafted microspheres, while just distributed around the outside of the partly dextran‐grafted ones. Flow hydrodynamic properties were improved greatly after the dextran‐grafting process, and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran‐grafting process, and partly dextran‐grafted Ni²⁺ chelating medium had a maximum binding capacity for His‐tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high‐efficiency and large‐scale protein purification.     

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.     

Study of pepsin phosphorylation using immobilized metal affinity chromatography

The interactions of pepsin with immobilized trivalent metal ions and the participation of the enzyme phosphate group in this process were investigated using high performance immobilized metal affinity chromatography. Two different sorbents were used: the newly prepared one, consisting of Ga(3+ )chelate of (6-amino-1-hydroxyhexane-1,1-diyl) bis(phosphonic acid) covalently bound to a methacrylate support (BP-Ga(3+)), and the commercial one, containing immobilized Fe(3+ )ions (POROS MC20-Fe(3+)). The comparison of the behavior of porcine pepsin A and its partially dephosphorylated form on both sorbents showed that both forms of pepsin were adsorbed under the same conditions. To eliminate the participation of free carboxyl groups in pepsin adsorption, both enzyme forms were modified by amidation or esterification. Native enzyme and its partially dephosphorylated form both with modified carboxyl groups differed in their interaction with immobilized Ga(3+ )and Fe(3+). Phosphorylated pepsin molecules with esterified carboxyl groups were adsorbed on both sorbents while nonphosphorylated ones with esterified carboxyl groups were not adsorbed.     

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification

Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl(2)-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification.     

Application of liquid phase deposited titania nanoparticles on silica spheres to phosphopeptide enrichment and high performance liquid chromatography packings

A novel core-shell composite (SiO(2)-nLPD), consisting of micrometer-sized silica spheres as a core and nanometer titania particles as a surface coating, was prepared by liquid phase deposition (LPD). Here, we show the resulting core-shell composite to have better efficient and selective enrichment for mono- and multi-phosphopeptides than commercially available TiO(2) spheres without any enhancer. The material exhibited favorable characteristics for HPLC, which include narrow pore size distribution, high surface area and pore volume. We also show that the core-shell composite can efficiently separate adenosine phosphate compounds due to the Lewis acid-base interaction between titania and phosphate group when used as HPLC packings. After coating the silica sphere with titania by LPD, the silanol of silica spheres will be shielded and that the stationary phase, C(18) bonded SiO(2)-3LPD, could be used under extreme pH condition.     

聚多巴胺修饰棉花固定金属离子用于磷酸化多肽的富集

本文通过多巴胺自聚合在天然的棉花纤维表面,构建了仿生聚多巴胺(PDA)膜层,然后利用儿茶酚羟基固定Ti~(4+),设计并合成了一种固定金属亲合色谱(Immobilized Metal Ion Affinity Chromatography,IMAC)材料Cotton@PDA-Ti~(4+),并将其用于磷酸化多肽的富集。该材料机械性能好,化学性能稳定和生物相容性好,且制备过程简单,通过简易的In-pipet-tip固相萃取(SPE)装置使整个富集操作过程简便快速。实验结果表明,Cotton@PDA-Ti~(4+)不仅可以从简单的蛋白酶解物(β-casein)中富集磷酸化多肽,并且在含有大量非磷酸化多肽的复杂体系样品中对磷酸化多肽也表现出良好的选择性。另外,利用Cotton@PDA-Ti~(4+)对磷酸化多肽进行富集也有较高的效率。我们将该材料应用于实际样品,如人体血清以及脱脂牛奶酶解物中磷酸化多肽的富集,均表现出了较好的选择性。说明该方法有可能用于磷酸化蛋白质组的全分析。     

Use of phage display methods to identify heptapeptide sequences for use as affinity purification 'tags' with novel chelating ligands in immobilized metal ion affinity chromatography

This study describes the screening of a peptide phage display library for amino acid sequences that bind with different affinities to a novel class of chelating ligands complexed with Ni2+ ions. These chelating ligands are based on the 1,4,7-triazacyclononane (TACN) structure and have been chosen to allow enhanced efficiency in protein capture and decreased propensity for metal ion leakage in the immobilized metal ion affinity chromatographic (IMAC) purification of recombinant proteins. Utilising high stringency screening conditions, various peptide sequences containing multiple histidine, tryptophan, and/or tyrosine residues were identified amongst the different phage peptide sequences isolated. The structures, and particularly the conserved locations of these key amino acid residues within the selected heptapeptides, form a basis to design specific peptide tags for use with these novel TACN ligands as a new mode of IMAC purification of recombinant proteins.     

Preparation and application of polyvinyl alcohol‐decorated cell membrane chromatography for screening anti‐osteoporosis components from Liuwei Dihuang decoction‐containing serum

Cell membrane chromatography is a powerful tool for screening active components from complex matrices. New cell membrane carriers need to be developed to increase the coverage of cell membranes on the surface of stationary phases, thereby improving cell membrane chromatographic retention. In this work, we prepared polyvinyl alcohol‐poly dimethyl diallyl ammonium chloride modified silica gel as a cell membrane carrier. Osteoblast cell was used as cell membrane source, which was widely used to evaluate the osteogenic activity of drugs. The new cell membrane chromatographic stationary phase was used to screen anti‐osteoporosis components in Liuwei Dihuang decoction‐containing serum. The chemical structures of the new modified materials were characterized by scanning electronic microscopy, Fourier transform infrared spectroscopy, and elemental analysis characterization. Compared with the common cell membrane column, the cell membrane coverage of this modified material was increased by 30%, and thus provided a stronger retention effect in positive drugs. Nineteen metabolites in rat serum samples were retained on the cell membrane chromatography and identified by ultra‐high‐performance liquid chromatography/time‐of‐flight mass spectrometry. Among those, four components (morroniside, catalpol, loganin, and acteoside) were selected for in vitro pharmacodynamics validation. They significantly increased the osteoblast proliferation. The new modified material was successfully applied to screen anti‐osteoporosis components from Liuwei Dihuang Decoction‐containing serum.     

Design of a ruthenium(III) immobilized affinity material based on a β‐cyclodextrin‐functionalized poly(glycidyl methacrylate‐ethylene dimethacrylate) monolith for the enrichment of hippuric acid

Immobilized metal affinity chromatography has drawn great attention as a widespread separation and purification approach. In this work, ruthenium was firstly introduced into the preparation of immobilized metal affinity chromatography considering its affinity to N,O‐donor ligands. A β‐cyclodextrin‐functionalized poly(glycidyl methacrylate‐ethylene dimethacrylate) monolith was designed and employed as the supporting material in immobilized metal affinity chromatography. Thiosemicarbazide was introduced into the synthesis process, which not only acted as a bridge between β‐cyclodextrin and glycidyl methacrylate, but also chelated with ruthenium because of its mixed hard‐soft donor characteristics. The developed monolithic ruthenium(III)‐immobilized metal affinity chromatography column was utilized for the adsorption and separation of hippuric acid, a biological indicator of toluene exposure. To achieve high extraction capacity, the parameters affecting the extraction efficiency were investigated with an orthogonal experiment design, L9 (3⁴). Under the optimized conditions, the enrichment factor of hippuric acid was 16.7‐fold. The method reproducibility was investigated in terms of intra‐ and interday precisions with relative standard deviations lower than 8.7 and 9.5%, respectively. In addition, ruthenium(III)‐immobilized metal affinity chromatography material could be used for up to 80 extractions without an apparent change in extraction recovery.     

Preparation of an iminodiacetic acid-modified capillary and its performance in capillary liquid chromatography and immobilized metal chelate affinity capillary electrophoresis

We prepared iminodiacetic acid (IDA)-modified and Cu(II)-IDA-modified capillaries through polymerization of N-(vinylbenzylimino) diacetic acid. The fundamental performance of these capillaries was examined in capillary liquid chromatography (LC) and immobilized metal chelate affinity capillary electrophoresis (IMACE). Copper(II), cobalt(II), and hematin were detected at different retention times by means of capillary LC with a chemiluminescence detector, during which the IDA-modified capillary was used. The difference in the retention times was attributed to the difference in the interaction between metal ions or complex and IDA moieties on the inner wall of the capillary. In addition, human serum albumin (HSA) and human serum gamma-globulin (HgammaG) were separated and detected using IMACE with an absorption detector, during which the Cu(II)-IDA-modified capillary was used. The separation of HSA and HgammaG was achieved through the interaction between proteins and Cu(II) chelate moieties on the inner wall of this capillary.     

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.     

Preparation and characterization of polyvinyl alcohol‐grafted Fe3O4 magnetic nanoparticles through glutaraldehyde

Magnetic nanoparticles (MNPs) are of great interest owing to their numerous existing and potential biomedical applications. To further explore the potential of MNPs in biomedical and other fields, we have designed and synthesized polyvinyl alcohol (PVA) polymer grafted Fe₃O₄ MNPs through glutaraldehyde (GLA) link. The success of this process has been ascertained using Fourier transform infrared (FT‐IR) analysis, thermogravimetric analysis (TGA), X‐ray diffraction (XRD) analysis and scanning electron microscopy (SEM) analysis. The FT‐IR analysis of resultant MNPs show infrared peak characteristics of PVA. TGA analysis clearly shows two major stages of thermal degradation, one corresponding to organic phase of PVA and GLA and another corresponding to Fe₃O₄ nanoparticles. XRD results and SEM images further support the FT‐IR and TGA results and confirm the presence of PVA layer surrounding Fe₃O₄ MNP surface. Under SEM examination, the magnetic cores exhibit somewhat irregular shapes varying from spherical, to oval to cubic. Copyright © 2012 John Wiley & Sons, Ltd.     

Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography

This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein–CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing β-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6–0.8 mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency.