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.     

Phosphate-functionalized magnetic microspheres for immobilization of Zr ions for selective enrichment of the phosphopeptides

In this work, we developed phosphate functionalized magnetic Fe₃O₄@C microspheres to immobilize Zr⁴⁺ ions for selective extraction and concentration of phosphopeptides for mass spectrometry analysis. Firstly, we synthesized Fe₃O₄@C magnetic microspheres as our previous work reported. Then, the microspheres were functionalized with phosphate groups through a simple hydrolysis reaction using 3-(trihydroxysilyl)propyl methylphosphate. And the Zr⁴⁺ ions were immobilized on phosphate-functionalized magnetic microspheres by using phosphate chelator. Finally, we successfully employed Zr⁴⁺-phosphate functionalized magnetic microspheres to selectively isolate the phosphopeptides from tryptic digests of standard protein and real samples including rat brain. All the experimental results demonstrate the enrichment efficiency and selectivity of the method we reported here.     

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.     

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.     

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

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

Magnetic Silica‐Coated Sub‐Microspheres with Immobilized Metal Ions for the Selective Removal of Bovine Hemoglobin from Bovine Blood

Magnetic silica‐coated magnetite (Fe₃O₄) sub‐microspheres with immobilized metal‐affinity ligands are prepared for protein adsorption. First, magnetite sub‐microspheres were synthesized by a hydrothermal method. Then silica was coated on the surface of Fe₃O₄ particles using a sol–gel method to obtain magnetic silica sub‐microspheres with core‐shell morphology. Next, the trichloro(4‐chloromethylphenyl) silane was immobilized on them, reacted with iminodiacetic acid (IDA), and charged with Cu²⁺. The obtained magnetic silica sub‐microspheres with immobilized Cu²⁺ were applied for the absorption of bovine hemoglobin (BHb) and the removal of BHb from bovine blood. The size, morphology, and magnetic properties of the resulting magnetic micro(nano) spheres were investigated by using scanning microscopy (SEM), transmission electron microscopy (TEM), X‐ray diffraction (XRD), and a vibrating sample magnetometer (VSM). The measurements showed that the magnetic sub‐microspheres are spherical in shape, very uniform in size with a core‐shell, and are almost superparamagnetic. The saturation magnetization of silica‐coated magnetite (Fe₃O₄) sub‐microspheres reached about 33 emu g^?1. Protein adsorption results showed that the sub‐microspheres had a high adsorption capacity for BHb (418.6 mg g^?1), low nonspecific adsorption, and good removal of BHb from bovine blood. This opens a novel route for future applications in removing abundant proteins in proteomic analysis.     

Preparation of magnetic microspheres with thiol-containing polymer brushes and immobilization of gold nanoparticles in the brush layer

Narrow-disperse magnetic microspheres were prepared by alkaline coprecipitation of Fe²⁺ and Fe³⁺ ions within poly(acrylic acid–divinylbenzene) microspheres that were prepared by distillation–precipitation copolymerization. Magnetic microspheres with polymer brushes that contain epoxy groups were prepared by graft copolymerization of glycidyl methacrylate and glycerol monomethacrylate via atom transfer radical polymerization (ATRP) from the magnetic microsphere surfaces. Subsequently, magnetic microspheres with thiol-containing polymer brushes were prepared by treating the epoxy group-containing magnetic microspheres with sodium hydrosulfide. Gold nanoparticles were immobilized in the brush layer of the thiol-containing magnetic microspheres through Au–S coordination. The catalytic activity of the gold nanoparticle-immobilized magnetic microspheres was investigated using the reduction of 4-nitrophenol to 4-aminophenol with sodium borohydride as a model reaction. The catalyst could be reused for over 10 cycles without noticeable loss of catalytic activity.     

Octyl-functionalized hybrid magnetic mesoporous microspheres for enrichment of low-concentration peptides prior to direct analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Octyl‐functionalized hybrid magnetic mesoporous (Fe₃O₄·nSiO₂·meso‐hybrid‐C8) microspheres were synthesized and applied in the isolation and pre‐concentration of low‐concentration peptides prior to direct analysis by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Such microspheres possess high surface area (324 m²/g), hydrophobic group (C8), relatively large pore volume (0.304 cm³/g), uniform pore diameter (~3.7 nm), and magnetic responsivity, which make them a simple and efficient kind of adsorbent for the enrichment of low‐concentration peptides. For bovine serum albumin (BSA, 15 fmol μL^–1) digest, after concentration by Fe₃O₄·nSiO₂·meso‐hybrid‐C8 microspheres, the enrichment performance was evidently better than those obtained by solvent evaporation and C8‐functionalized magnetic particles, and comparable to those obtained by commercial Anchor chip target and ZipTipC18 pipette tip. Such microspheres were further applied in the enrichment of the tryptic digests of rat cerebellum proteins and endogenous peptides of crude human serum, and more peaks with higher signal‐to‐noise (S/N) ratio were obtained than before pre‐concentration. Furthermore, the pre‐concentration reproducibility of magnetic microspheres for biological samples was good, and the limit of detection (LOD) for BSA digests by MALDI‐TOF MS was decreased by at least one order of magnitude compared with that obtained without pre‐concentration. All the above‐mentioned results indicate that the synthesized Fe₃O₄·nSiO₂·meso‐hybrid‐C8 microspheres are promising for the enrichment of low‐concentration peptides from complex biosamples. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

Immobilization of trypsin in polyaniline-coated nano-Fe3O4/carbon nanotube composite for protein digestion

In this report, a four-component nanocomposite, trypsin-immobilized polyaniline-coated Fe₃O₄/carbon nanotube composite, was synthesized for highly efficient protein digestion. Fe₃O₄ was deposited by the chemical coprecipitation of Fe²⁺ and Fe³⁺ in an alkaline solution containing carbon nanotubes (CNTs) to prepare nano-Fe₃O₄/CNT composite. Subsequently, polyaniline (PA) was assembled on the Fe₃O₄/CNT composite by the in situ polymerization of aniline in the presence of trypsin to obtain trypsin-immobilized PA/Fe₃O₄/CNT nanocomposite. The novel 1D superparamagnetic biomaterial has been characterized by TEM, SEM, XRD, and magnetometric analysis. The feasibility and performance of the unique magnetic biomaterial have been demonstrated by the tryptic digestion of bovine serum albumin, myoglobin, and lysozyme within 5 min. The digests were identified by MALDI-TOF MS with sequence coverages that were comparable to those obtained from the conventional in-solution tryptic digestion. The present biocomposite offers considerable promise for protein analysis due to its high magnetic responsivity and excellent dispersibility. It can be easily isolated from the digests with the aid of an external magnetic field. Because the enzyme-immobilized nanocomposite can be prepared by a simple two-step deposition approach at low cost, it may find a wide range of biological applications including proteome research.     

Preparation of core–shell structure Fe3O4@SiO2 superparamagnetic microspheres immoblized with iminodiacetic acid as immobilized metal ion affinity adsorbents for His‐tag protein purification

The core–shell structure Fe₃O₄/SiO₂ magnetic microspheres were prepared by a sol–gel method, and immobiled with iminodiacetic acid (IDA) as metal ion affinity ligands for protein adsorption. The size, morphology, magnetic properties and surface modification of magnetic silica nanospheres were characterized by various modern analytical instruments. It was shown that the magnetic silica nanospheres exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu²⁺, Ni²⁺ and Zn²⁺, were chelated on the Fe₃O₄@SiO₂–IDA magnetic microspheres to adsorb lysozyme. The results indicated that Ni²⁺‐chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, adsorption equilibrium could be achieved within 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe₃O₄@SiO₂–IDA–Ni²⁺ magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His‐tag recombinant Homer 1a from the inclusion extraction expressed in Escherichia coli. The result indicated that the magnetic microspheres showed unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption, and easy handling. This demonstrates that the magnetic silica microspheres can be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample. Copyright © 2015 John Wiley & Sons, Ltd.     

Application of polydopamine-coated nylon capillary-channeled polymer fibers as a stationary phase for mass spectrometric phosphopeptide analysis

Capillary-channeled polymer (C-CP) fibers are demonstrated as a selective stationary phase for phosphopeptide analysis via LC–MS. Taking advantage of the oxidative self-polymerization of dopamine under alkaline conditions, a simple system involving a dilute aqueous solution of 0.2% w/v dopamine hydrochloride in 0.15% w/v TRIS buffer, pH 8.5 was utilized to coat polydopamine onto nylon 6 C-CP fibers. Confirmation of the polydopamine coating on the fibers (nylon-PDA) was made through attenuated total reflection-FTIR (ATR-FTIR) analysis. Imaging using SEM was also performed to examine the morphology and topography of the nylon-PDA. Subsequent loading of Fe³⁺ to the nylon-PDA matrix was confirmed by SEM/energy dispersive X-ray spectroscopy (SEM/EDX). The Fe³⁺-bound nylon-PDA fibers packed in a microbore column format were tested in the off-line preconcentration of phosphopeptides from a 1:100 mixture of β-casein/BSA digests for MALDI-TOF analysis. The packed column was also installed onto an HPLC system as a platform for the online sample clean-up and enrichment of phosphopeptides from a 1:1000 mixture of β-casein/BSA protein digests that were determined by subsequent ESI–MS analysis.     

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。     

Fe3O4@Al2O3 magnetic core-shell microspheres for rapid and highly specific capture of phosphopeptides with mass spectrometry analysis

Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres with phosphopeptides has been developed. With a well-defined core-shell structure, the Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres not only have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy isolation by positioning an external magnetic field. The prepared Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres have been successfully applied to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and ovalbumin. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:50 as well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres over Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC (immobilized metal affinity chromatography) resin, and TiO(2) beads. Finally, the Al(2)O(3) coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.     

Magnetic porous polymer microspheres: Synthesis,characterization and adsorption performance for the removal of phenol

The magnetic poly(ethylene glycol dimethacrylate-n-vinylimidazole) (Fe₃O₄@poly (EGDMA@VIM)) microspheres were prepared by suspension polymerization method using magnetite Fe₃O₄ nano-powder and the porosity, morphology, chemical composition and structure of the magnetic polymer microspheres were characterized. The specific surface area and swelling ratio of the Fe₃O₄@poly(EGDMA@VIM) microspheres were found to be 278.6?m²·g^–1 and 48%, respectively. The Fe₃O₄@poly(EGDMA@VIM) microspheres were used as an adsorbent for phenol removal. The effects of the parameters such as adsorbent dosage, temperature, pH and initial concentration of phenol solutions on the adsorption were investigated. The experimental adsorption equilibrium data obtained were fitted with Langmuir, Freundlich and Dubinin-Radushkevich isotherms and the pseudo-first-order, pseudo-second-order and intra–particle diffusion kinetic models. The adsorption equilibrium data agreed well with the Freundlich isotherm and the pseudo-second-order kinetic model. The maximum capacity of the Fe₃O₄@poly(EGDMA@VIM) microspheres was calculated to be 33.83?mg·g^–1 at 298?K and natural pH from Langmuir isotherm. The Fe₃O₄@poly(EGDMA@VIM) microspheres were found to be reusable for removal of phenol after desorption for several times. The result indicated that the Fe₃O₄@poly(EGDMA@VIM) microspheres are potential candidate for removal of phenol in wastewaters.     

Enrichment of phosphopeptides using bare magnetic particles

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

Study on Fe3O4/polyaniline electromagnetic composite hollow spheres prepared against sulfonated polystyrene colloid template

Fe₃O₄/polyaniline (PANI) composite hollow spheres were prepared by using sulfonated polystyrene (SPS) microspheres as templates. The sulfonic acid groups were applied to induce absorbing Fe₃O₄ nanoparticle, and subsequently, conductive PANI was grown. Finally, the polystyrene cores were selectively dissolved to yield composite hollow microspheres with electromagnetic properties. The analysis results indicated that the adsorption of Fe₃O₄ on template core by electrostatic interaction resulted in magnetic composite microspheres. The conductivity of composite hollow spheres was remarkably increased after polyvinylpyrrolidone modification which favored the growth of PANI on SPS/Fe₃O₄ and enhanced the integrity of hollow microspheres. The saturated magnetization of the composite hollow microspheres was tuned from 2.7 to 9.1 emu/g, and the conductivity was in the range from 10^?2 to 10⁰?S/cm.     

用于检测重金属离子的磁性荧光复合微球

采用静电逐层自组装的方法,首先将PSS和PAH聚电解质交替沉积在CaCO₃中空微球表面,然后将Fe₃O₄磁性纳米粒子与CdSe量子点负载在中空微球表面不同的聚电解质层中,制备出具有磁性和荧光双重功能的复合微球,并将其作为荧光离子探针,研究了其对Cu²⁺和Pb²⁺离子检测的灵敏度、选择性及可行性。结果表明,复合微球显示出良好的磁性和荧光性能,对Cu²⁺和Pb²⁺离子的检测具有较高的灵敏度和选择性。尤为重要的是,可通过磁分离的方法将微球快速地从待测液中回收,从而能够避免量子点对环境造成的二次污染。     

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.     

Immobilized Metal Affinity Chromatography of Phosphorylated Proteins Using High Performance Sorbents

The interactions of two model phosphoproteins (porcine pepsin and ovalbumin) with two different immobilized metal affinity chromatography (IMAC) sorbents containing immobilized Fe³⁺, Ga³⁺, and UO₂ ²⁺ ions have been investigated under various conditions. Both proteins were adsorbed on immobilized uranyl ions under acidic conditions similar to those on immobilized Fe³⁺ and Ga³⁺ ions. The retained proteins could be released either by the presence of phosphate ions in the elution buffer (immobilized Ga³⁺ and Fe³⁺ ions) or by an increased pH (all tested immobilized ions). The IMAC sorbents employed could be used under the conditions of high-performance chromatography and are suitable for the separation and analysis of intact phosphoproteins.