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.     

Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry

Titanium dioxide metal oxide affinity chromatography (TiO₂‐MOAC) is widely regarded as being more selective than immobilized metal‐ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO₂‐MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO₂‐MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non‐phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5‐dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO₂‐MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO₂‐MOAC showed higher specificity than immobilized gallium (Ga³⁺), immobilized iron (Fe³⁺), or zirconium dioxide (ZrO₂) affinity chromatography for phosphopeptide enrichment. Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS), which was more efficient for smaller, singly phosphorylated peptides. Copyright © 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.     

Identification of uranyl binding proteins from human kidney-2 cell extracts by immobilized uranyl affinity chromatography and mass spectrometry

To improve our knowledge on protein targets of uranyl ion (UO₂²⁺), we set up a proteomic strategy based on immobilized metal-affinity chromatography (IMAC). The successful enrichment of UO₂²⁺-interacting proteins from human kidney-2 (HK-2) soluble cell extracts was obtained using an ion-exchange chromatography followed by a dedicated IMAC process previously described and designed for the uranyl ion. By mass spectrometry analysis we identified 64 proteins displaying varied functions. The use of a computational screening algorithm along with the particular ligand-based properties of the UO₂²⁺ ion allowed the analysis and categorization of the protein collection. This profitable approach demonstrated that most of these proteins fulfill criteria which could rationalize their binding to the UO₂²⁺-loaded phase. The obtained results enable us to focus on some targets for more in-depth studies and open new insights on its toxicity mechanisms at molecular level.     

Purification of a Lectin from M. rubra Leaves Using Immobilized Metal Ion Affinity Chromatography and Its Characterization

Lectins represent a heterogeneous group of proteins/glycoproteins with unique carbohydrate specificity, with wide range of biomedical applications. The multi-step purification protocols generally used for purification of lectin result in a significant reduction in the final yield and activity. In the present study, Morus rubra lectin (MRL) was purified to homogeneity from the leaves using a single-step immobilized metal ion affinity chromatography (IMAC) procedure. The approximate molecular weight of purified MRL resolved as a single band on SDS-PAGE was 52?kDa. Final percentage yield of purified lectin by IMAC was calculated as 74.7?%. Purified MRL was specific to three sugars, galactose, d-galactosamine and N-acetyl-d-galactosamine, and rendered haemagglutination (HA) activity towards different human blood group RBCs. MRL showed stability over a wide range of temperature (up to 80?°C) and pH (4?C11). Chelation of the lectin with EDTA did not alter HA which indicates that metal ion is not required for activity. In the presence of Fe²⁺, Ca²⁺, Zn²⁺, Ni²⁺, Mn²⁺, Na⁺ and K⁺, HA activity was reduced to 50?%, whereas the presence of trivalent metal ions (Fe3⁺ and Al³⁺) and Cu²⁺ did not affect the activity. In the presence of Mg²⁺ and Hg²⁺, only 25?% of HA activity remained.     

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

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

Immobilized metal ion affinity chromatography of proteins on IDA-Fe3+

Several proteins, selected for their varied isoelectric points within pH range ca. 4 to ca. ll, bind to immobilized (chelated to iminodiacetate) ferric ion, IDA-Fe³⁺, when applied in a 50 mM buffer, pH 6.0. These proteins can be displaced from IDA-Fe³⁺ columns by an increase of pH from 6 to 8, an increase of sodium chloride from 0 to 1 Malar and, in particular, by both. Apotransferrins, in contrast to other proteins, are able to scavenge Fe³⁺ ion from IDA-Fe³⁺. Interestingly, two proteins, both quite acidic, behave quite differently on IDA-Fe³⁺: α₁-acid glycoprotein (sialic acid) does not bind, whereas phosvitin (phosphate 1) binds avidly. IDA-Fe³⁺ sorbent, due to its unusual sorptive properties, represents a new addition of particular significance to the family of chromatographic sorbents available for protein purification.     

Synthesis,Characterization and Applications of Immobilized Iminodiacetic Acid-Modified Silica

Porous immobilized iminodiacetic acid modified silica of the general formula S—(CH₂)₃—N(CH₂COOH)₂, (where S represents [Si—O] _n siloxane network) has been prepared by replacement of the iodide in 3-iodopropyl modified silica with diethyliminodiacetate. The immobilized-diethyliminodiacetate ligand system (S-DIDA) was then hydrolyzed by hydrochloric acid to produce the immobilized iminodiacetic acid ligand system (S-IDA). The iodo functionalized modified silica (S-I) was prepared by polycondensation of Si(OEt)₄ and (MeO)₃Si(CH₂)₃I. The XPS and CP/MAS ¹³C NMR spectra showed that not all iodine atoms are replaced and that the hydrolysis of ethyl acetate groups are incomplete upon treatment with HCl. The immobilized iminodiacetic acid ligand system exhibits high potential for the uptake of various di- and trivalent metal ions such as (Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺). Complexation of the iminodiacetate ligand system for the metal ions at the optimum conditions was found in the order: Cu²⁺ > Fe³⁺ > Ni²⁺ > Co²⁺ > Mn²⁺ > Zn²⁺. Stability studies of the iminodiacetate ligand system showed that a degradation of the siloxane network and leaching of some species occurred upon treatment with strong acid and base aqueous solutions.     

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.     

Analysis of Nonionic Surfactants by High Performance Liquid Chromatography

Abstract

This review discusses the principles of immobilized metal ion affinity chromatography (IMAC) and its applications to protein separations. IMAC functions by binding the accessible electron-donating pendant groups of a protein - such as histidine, cysteine, and tryptophan - to a metal ion which is held by a chelating group covalently attached on a stationary support. A common chelating group is iminodiacetate. The ions commonly used are of borderline or soft metals, such as Cu²⁺, Ni²⁺, Co²⁺, and Zn²⁺. Protein retention in IMAC depends on the number and type of pendant groups which can interact with the metal. The interaction is affected by a variety of independent variables such as pH, temperature, solvent type, salt type, salt concentration, nature of immobilized metal and chelate, ligand density, and protein size. Proteins are usually eluted by a decreasing pH gradient or by an increasing gradient of a competitive agent, such as imidazole, in a buffer. There are still several unresolved issues in IMAC. The exact structures of protein-immobilized metal complexes need to be known so that retention behavior of proteins can be fully understood and sorbent structures can be optimized. Engineering parameters, such as adsorption/desorption rate constants, sorbent capacities, and intraparticle diffusivities, need to be developed for most protein systems. Engineering analysis and quantitative understanding are also needed so that IMAC can be used efficiently for large scale protein separations.     

Facile synthesis of gallium ions immobilized and adenosine functionalized magnetic nanoparticles with high selectivity for multi-phosphopeptides

Despite recent advances in phosphoproteome research, detection and characterization of multi-phosphopeptides have remained a challenge. Here we present a novel IMAC strategy for effective extracting multi-phosphopeptides from complex samples, through Ga³⁺ chelation to the adenosine tri-phosphate (ATP)-functionalized magnetic nanoparticles (Ga³⁺-ATP-MNPs). The high specificity of Ga³⁺-ATP-MNPs was demonstrated by efficient enriching multi-phosphopeptides from the digest mixture of β-casein and BSA with molar ratio as low as 1:5000. Ga³⁺-ATP-MNPs were also successfully applied for the phosphoproteome analysis of rat liver mitochondria, resulting in the identification of 193 phosphopeptides with 331 phosphorylation sites from 158 phosphoproteins. In other words, 54.4% of the phosphopeptides trapped by Ga³⁺-ATP-MNPs were observed with more than one phosphorylated sites, resulting in significant improvement on the identification of peptides with multi-phosphorylated sites. The high specificity of Ga³⁺-ATP-MNPs towards multi-phosphopeptides may be due to the synergistic effect of the strong hydrophilic surface functionalized by ATP and the proper chelating strength provided by Ga³⁺. Moreover, the unique magnetic core of Ga³⁺-ATP-MNPs also facilitates the isolation process and on-plate enrichment for direct MALDI MS analysis with limit of detection as low as 30 amol. This new affinity-based protocol is expected to provide a powerful approach for characterizing multiple phosphorylation sites on proteins in complex and dilute analytes, which may be explored as complementary technique for improving the coverage of phosphoproteome.     

Synthesis,characterization and applications of polysiloxane networks with immobilized pyrogallol ligands

A porous, solid insoluble polysiloxane‐immobilized ligand system bearing pyrogallol active sites of the general formula P? (CH₂)₃? NH(CH₂)₃OC₆H₃(OH)₂ (where P represents [Si? O]_n siloxane network) has been prepared by the reaction of 3‐aminopropylpolysiloxane with 1,3‐dibromopropane followed by the reaction with pyrogallol. ¹³C CP‐MAS NMR and X‐ray photoelectron spectroscopy confirmed that the pyrogallol is chemically bonded to the siloxane backbone. Thermal analysis showed that the ligand system is stable under nitrogen at relatively high temperature. The polysiloxane–pyrogallol ligand system exhibits high potential for the uptake of the metal ions (Fe³⁺, Co²⁺, Ni²⁺ and Cu²⁺). Complexation of the pyrogallol ligand system for the metal ions at the optimum conditions was found to be in the order Fe³⁺ > Cu²⁺ > Ni²⁺ > Co²⁺. Copyright © 2005 John Wiley & Sons, Ltd.     

Study of the Photodegradation Kinetics and Pathways of Hexaflumuron in Liquid Media

Hexaflumuron, one of the benzoylphenylurea insect growth regulators, can be leached into surface water and thus having a potential impact on aquatic organisms. In this study, the photodegradation processes of hexaflumuron under high‐pressure mercury lamp irradiation were assessed. The photodegradation kinetics were studied, as were the effects of pH, different light sources, organic solvents and environmental substances, including nitrate ions (NO₃^?), nitrite ions (NO₂^?), ferrous ions (Fe²⁺), ferric ions (Fe³⁺), humic acid, sodium dodecyl sulfate (SDS) and hydrogen peroxide (H₂O₂). Three photodegradation products in methanol were identified by gas chromatography‐mass spectrometry (GC‐MS). In general, the degradation of hexaflumuron followed first‐order kinetics. In the four media studied, the photodegradation rate order was n‐hexane > methanol > ultrapure water > acetone. Faster degradation was observed under high‐pressure mercury lamp irradiation than under xenon lamp irradiation. The pH had a considerable effect, with the most rapid degradation occurring at pH 5.0. The photodegradation rate of hexaflumuron was promoted in the presence of NO₃^?, NO₂^?, Fe²⁺, humic acid, SDS and H₂O₂, but inhibited by Fe³⁺. Moreover, the presumed photodegradation pathway was proposed to be the cleavage of the urea linkage.     

Selective ion-exchangers containing phosphorus in their functional groups

A series of selective ion-exchangers was synthetized, containing phosphinic or phosphonic acid functional groups. The selective sorption of Sc³⁺, Fe³⁺, Ga³⁺, In³⁺, Al³⁺, La³⁺, Pb²⁺, Co²⁺ and Ca²⁺ ions in 0.1–4.0M HNO₃ medium was investigated using a batch experiment technique. The selectivity of these exchangers generally decreased in the order: Sc>Fe>In>Ga>Al>La>Pb>Cu>Co>Ca. The observed large differences in selectivity resulted in effective separation procedures of scandium from lanthanides, and gallium (or indium) from bivalent elements. The selective separation of these ions from their different mixtures is also possible.     

Macrocyclic Polysiloxane Immobilized Ligand System and Its Structural Characterization

A new porous solid macrocyclic 1,4,7,10,14,17,20‐heptaazadocosane‐3,21‐dione polysiloxane ligand system of the general formula P‐(CH₂)₃‐C₁₅H₃₂O₂N₅, (where P represents [Si‐O]n siloxane network) has been prepared by the reaction of immobilized iminobis(N‐diethylenediamineacetamide)polysiloxane with 1,3 dibromopropane. The new macrocyclic polysiloxane ligand system exhibits high potential for the uptake of metal ions (Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺). Complexation with copper ions exhibits a high selectivity in which two copper ions were involved per one macrocyclic ligand group.     

Extraction of uranium by amine, amide and benzamide grafted covalently on silica gel

Organic complexing moieties such as amine, 2°-amide and benzamide functional groups were covalently linked to silica gel by performing organomodification on commercially available silica gel. The extraction of uranium as a function of pH, time, concentration of uranium and the effect of ions such as Fe²⁺, Co²⁺, Ni²⁺ and CO₃ ^2- on the distribution coefficient of uranium by sorbent was studied. The data was fitted into Langmuir adsorption isotherm. The selectivity of the sorbents towards uranium were found to decrease in the order gel-amine >> gel-benzamide ≅ gel-2°-amide at all pHs. The presence of metal ions such as Fe²⁺, Co²⁺, Ni²⁺ and CO₃ ^2- were found to decrease the distribution coefficients (K _d, ml/g) of uranium and the decrease was very high when Fe²⁺ was present. The performance of the sorbent, gel-amine, under a given column condition was tested by following the breakthrough curve of uranium up to C/C ₀ = 0.025. The sorbent was found to be good for the quantitative pre-concentration of uranium from a large amount of sodium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhancement of Mercury Sorbent Using Metal Aluminate Carbonates with Chloride under Hydrothermal Conditions

Mercury sorbents M–Al–CO₃ (M=Mg²⁺, Mn²⁺, Fe²⁺, Cu²⁺, or Zn²⁺) were prepared by the coprecipitation of M^{2 +} and Al³⁺ in an alkaline NaOH/Na₂CO₃ solution. The formation of a layered double hydroxide structure significantly enhanced Hg removal, uniquely with a suitable binder as a support. Both Hg(NO₃ )₂ and HgCl₂ were used to perform the capture test. The calcined sorbent exhibited superior efficiency, particularly for materials having bivalent states of mixed oxide (MO), such as calcined Mn and Fe. Chloride ions effectively raised the mercury scrubber efficiency, regardless of the type of sorbent. The results show that synthetic sorbents can provide 96% removal of Hg at 200°C. In addition, a suitable binder such as TiO₂ can be used as a support for dispersing metal oxides, which significantly decreases the MO content while maintaining a high equivalent capture capacity.     

高效液相色谱法同时测定水体中的环丙沙星和氟甲喹

为了满足实验室测试和野外测试需要,也为其他学者研究不同抗生素之间的相互作用提供相关的资料,本研究选用环丙沙星(CIP)和氟甲喹(FLU)作为代表,建立了一种简单、稳定、易普及的高效液相色谱法用于同时快速测定水体中两种氟喹诺酮类抗生素的含量,并讨论了不同流动相及其比例和水样中几种常见阴、阳离子(Ca²⁺、Mg²⁺、Fe³⁺、Al³⁺、SO₄^2-和HCO₃^-)对抗生素测定的影响。结果表明:三乙胺对改善柱效有明显效果;低浓度离子对测试影响不大,但Fe³⁺和Al³⁺可能与固定相的表面羟基或测试组分发生配合作用,造成基线不稳。实验结果对其他研究者对于流动相的选择与优化具有借鉴意义。     

Properties of surface acid sites of ZrO2 and SO4ZrO2-based systems studied by diffuse-reflectance Fourier-transform IR spectroscopy: adsorption of acetonitrile-d3

The properties of acid sites of ZrO₂ and SO₄/ZrO₂-based systems modified by metal ions were studied by DRIFT spectroscopy using acetonitrile-d₃ as a probe molecule. In the case of ZrO₂. CD₃CN interacts with the Lewis acid sites (LAS) with moderate strength. Adsorption on the Brönsted acid sites (BAS) is very weak, which indicates the absence of strong BAS on the surface of ZrO₂. Modification of the surface by SO₄ groups results in the appearance of a new type of BAS that are capable of adsorbing CD₃CN in the polycoordinated form,i.e., stronger complexes with the adsorbate. Addition of metal ions (Fe, Ga, Zn, or Co) leads to the formation of a new type of LAS connected with Fe³⁺, Ga³⁺, Zn²⁺, and Co²⁺ promoter ions.     

Selective removal of heavy metal ions using sol-gel immobilized and SPE-coated thiacrown ethers

Sorbent materials based on three thiacrown ethers, 1,4,7,10-tetrathiacyclododecane (12S4), 1,4,7,10,13-pentathiacyclopentadecane (15S5) and 1,4,7,10.13,16-hexathiacyclooctadecane (18S6) were prepared either by immobilizing the ligands into sol-gel (SG) matrix or coating on commercial solid phase extraction (SPE) column. SG sorbents were characterized by FT-IR, energy dispersive X-ray microanalysis (EDX) and thermogravimetric analysis/derivative thermogravimetric analysis (TGA/DTG). A marked thermal stability of the ligands when immobilized in sol-gel matrix was noted. The competitive sorption characteristics of a mixture of eleven metal ions (Mg²⁺, Zn²⁺, Cd²⁺, Co²⁺, Mn²⁺, Ca²⁺, Cu²⁺, Ni²⁺, Ag⁺, V⁴⁺, Hg²⁺) using: (i) batch method with ligands trapped in SG matrices, and (ii) off-line SPE column containing coated ligands were studied using ICP-MS. The extraction of metals were optimized for key parameters such as pH, contact time/flow rate, particle size (for SG sorbents) and ligand concentration. Under the optimized conditions, all the immobilized thiacrown ethers exhibited highest selectivity toward Ag⁺, with lesser responses to Hg²⁺ while the extraction of other metal ions were negligible. Among the SG sorbents, 18S6-SG offer the highest capacity and the best selectivity over Hg²⁺. However, for practical applications such as for selective isolation and preconcentration of Ag⁺, the SPE type especially based on 18S6 is preferred as analysis time and recoveries are favorable. The sorbents can be repeatedly used three times as there was no significant deterioration in the metal uptake (%E > 90%) or interference from other metal ions. The optimized procedures were successfully applied for the separation and preconcentration of traces Ag⁺ in different water samples.     

Study of the ropinirole hydrochloride interactions with human holo-transferrin in the presence of common metal ions

The binding of ropinirole hydrochloride (REQUIP) to human holo-transferrin (hTf) in the absence and presence of common ions has been investigated by fluorescence spectroscopy combined with fluorescence anisotropy, time-resolved fluorescence and circular dichroism (CD) under simulative physiological conditions. The quenching of the fluorescence intensity and the red shift in the maximum wavelength revealed an increased polarity of the microenvironment of the tryptophan and tyrosine residues. The number of binding sites and the apparent binding constants of REQUIP with hTf in the presence of Co²⁺, Fe³⁺, Cr³⁺, Al³⁺, Ca²⁺, Pb²⁺, Co ₃ ^2? , Cu²⁺, Mg²⁺ and K⁺ ions were determined. Time-resolved fluorescence decay profile gives the lifetime components reduce from 4.20 to 3.57?ns. Steady-state and time-resolved fluorescence data illustrated that the fluorescence quenching of complexes are static mechanism. Gradual addition of hTf led to marked increase in fluorescence anisotropy. From the value of anisotropy, it is argued that the REQUIP is located in a restricted environment of hTf. The quantitative analysis of CD spectra indicated that, in the presence of Co²⁺ and Fe³⁺ ions, the ??-helical structure content of hTf increased and for the other common ions, the ??-helical content decreased. In addition, thermodynamic analysis demonstrated that the van der Waals forces and hydrogen bond interactions stabilized the hTf?CREQUIP complex. These experimental results revealed that REQUIP could bind to hTf and those common ions, therefore could be a useful guideline for further therapeutic projects.