Immobilized Metal Ion Research Articles

Bioinertization of NanoLC/MS/MS Systems by Depleting Metal Ions From the Mobile Phases for Phosphoproteomics

We have successfully developed a bioinertized nanoflow LC/MS/MS (nanoLC/MS/MS) system for the highly sensitive analysis of phosphopeptides by depleting metal ions from the mobile phase. We found that not only direct contact of phosphopeptides with metal components, but also indirect contact with nanoLC pumps through the mobile phase causes significant losses during the recovery of phosphopeptides. Moreover, electrospray ionization was adversely affected by the mobile phase containing multiple metal ions as well as by the sample solvents contaminated with metal ions used in immobilized metal ion affinity chromatography for phosphopeptide enrichment. To solve these problems, metal ions were depleted by inserting an online metal ion removal device containing metal-chelating membranes between the gradient mixer and the autosampler. As a result, the peak areas of the identified phosphopeptides increased an average of 9.9-fold overall and 77-fold for multiply phosphorylated peptides with the insertion of the online metal ion removal system. This strategy would be applicable to the highly sensitive analysis of other phosphorylated biomolecules by microscale-LC/MS/MS.

Expression and purification of MERS-CoV envelope protein, an essential viroporin, using the baculovirus expression system.

The causative agent of Middle East Respiratory Syndrome (MERS) is a zoonotic Coronavirus (MERS-CoV) identified in Saudi Arabia in 2012. The envelope (E) protein of MERS-CoV is a small viral protein which plays several essential roles during virus replication. To facilitate study of the structure and function of the E protein, recombinant MERS-CoV E protein was expressed using the baculovirus expression system. A recombinant E open reading frame including an 8-histidine tag at the amino terminus was designed and cloned into a baculovirus transfer vector. Following construction of a recombinant virus insect cells were infected and the expression of the E protein assessed by SDS-PAGE and Western blotting. Recombinant E protein, tagged at the N-terminus with a polyhistidine sequence, with a molecular mass of 10.18 kD was identified by Western blotting with an anti-His antibody. Following large scale infection E protein was released by detergent mediated lysis of infected cells and purified by Immobilized Metal Ion Affinity Chromatography (IMAC). Purified full length recombinant MERS-CoV E protein can be isolated by IMAC and is suitable for further functional, biophysical or immunological studies.

Immobilizing Isatin-Schiff Base Complexes in NH2-UiO-66 for Highly Photocatalytic CO2 Reduction

Reducing carbon dioxide (CO2) via photocatalysis to carbon-based materials is a challenging process due to the complexity of the process and the production of several byproducts. Therefore, it is crucial to develop photocatalysts with high yield and selectivity for CO2 reduction. Herein, we present a strategy for creating high-performance catalysts by immobilizing Ni, Co, and Cu ions in NH2-UiO-66 by covalently linking the isatin-Schiff base metal complexes (IS) to the micropores. The as-synthesized NH2-UiO-66/IS-complex (66-IS-M, M = Ni, Co, Cu) photocatalysts exhibit special photocatalytic activity for the reduction of CO2 because of the immobilized metal ions serving as active sites during the reaction. Among them, 66-IS-Ni has a maximum CO generation rate of 1350 μmol g–1 h–1 and a CO selectivity of 87%, both of which are significantly higher than those of previously reported metal–organic framework (MOF)-based photocatalysts. Experimental characterizations and density functional theory (DFT) calculations reveal that the effective charge separation and lowered free energy of CO2 reduction on 66-IS-Ni promote CO2 conversion. This study presents a universal strategy for the uniform dispersion of molecular catalysts in amino-functionalized MOF for efficient photocatalytic applications.

Complex Formation in the Modified Xerogel Phase: Study and Application in Analysis

The review summarizes the author’s results concerning the complex formation in the phase of modified xerogels synthesized by the sol–gel process for application as sensor materials. The results of studying the complex formation (pH ranges of reactions, light absorption maxima and compositions of complexes, and equilibrium constants) are presented and discussed for 87 systems of two types, that is, immobilized ligand (organic analytical reagent)–metal ion and immobilized metal ion–organic or inorganic compound. The approach proposed by the author to describe the complex formation in the modified xerogel phase is presented. The results of applications of modified xerogels as sensor materials are considered, including their use for determining metal ions; organic compounds; hydrogen peroxide; chloride, fluoride, and oxalate ions in food and hygienic products, pharmaceuticals, biological fluids, and environmental media.

Post-synthesis of a titanium-rich magnetic COF nanocomposite with flexible branched polymers for efficient enrichment of phosphopeptides from human saliva and serum.

A Ti4+-functionalized magnetic covalent organic framework material with flexible branched polymers (mCOF@ε-PL@THBA-Ti4+) built via an immobilized metal ion affinity chromatography (IMAC) enrichment strategy was proposed through post-synthesis modification. Hydrophilic ε-poly-L-lysine (ε-PL) rich in amino active groups was first introduced in the fabrication of the phosphopeptide enrichment material to increase the hydrophilicity while providing more functional modification pathways of the material. 2,3,4-Trihydroxy-benzaldehyde (THBA) provides abundant binding sites for the immobilization of numerous Ti4+, which is advantageous for the subsequent efficient phosphopeptide enrichment. The magnetic nanocomposite exhibited outstanding performance of phosphopeptide enrichment with good selectivity (1 : 5000), a low detection limit (2 fmol), and relatively high loading capacity (66.7 mg g-1). What's more, after treatment with mCOF@ε-PL@THBA-Ti4+, 16 endogenous phosphopeptides from fresh saliva of healthy people were recognized by MALDI-TOF MS, and 50 phosphopeptides belonging to 35 phosphoproteins from the serum of uremia patients were detected by nano-LC-MS/MS. Proteomics data analysis for the differential protein selection between uremia and normal controls was conducted using R software, and four down-regulated and three up-regulated proteins were obtained. The results suggested that the prepared material has potential applications in biomarker discovery.

Hydrophilic magnetic host-guest Ti-phenolic networks: a promising material for the highly sensitive enrichment of glycopeptides and phosphopeptides.

Protein glycosylation and phosphorylation are the two most important post-translational modifications, which play a vital role in physiological and pathological processes. Before the comprehensive characterization of glycoproteome/phosphoproteome through the mass spectrometry (MS) technique, it is necessary to perform a highly specific enrichment procedure because glycoproteins/phosphoproteins inherently occur in low abundances. Herein, we have reported a novel magnetic β-cyclodextrin-based host-guest Ti-phenolic network material, focusing on the simultaneous enrichment of glycopeptides/phosphopeptides via hydrophilic interaction chromatography and immobilized metal ion chromatography. Ti ions and glutathione-derived adamantine were introduced by metal-phenolic interactions and host-guest interactions. The material possesses biocompatibility, good hydrophilicity, strong magnetic response, metal chelation effect, and demonstrates an excellent enrichment ability towards glycopeptides/phosphopeptides. Combined with MS detection, high sensitivity (0.035/0.01 fmol for IgG/β-casein) and good reusability (6 times) were achieved. In addition, its outstanding specificity was validated in quantities as low as 500 : 1 : 1 for BSA : IgG : β-casein (m/m/m). Benefiting from these merits, the adsorbent material was successfully used for the simultaneous enrichment of phosphopeptides/glycopeptides from human serum and HeLa cell lysate and can be expected to exhibit great applicability for precious and small amounts of biosamples in the study of glycoproteomics/phosphoproteomics.

Phosphoproteomic Analysis of the Jejunum Tissue Response to Colostrum and Milk Feeding in Dairy Calves during the Passive Immunity Period.

Improvements in the feeding of calves are of increasing importance for the development of the dairy industry. While colostrum is essential for the health of newborn calves, knowledge of protein phosphorylation alterations in neonatal calves that are fed colostrum or mature milk is lacking. Here, mid-jejunum tissue samples were collected from calves that received colostrum or milk. Subsequently, the jejunum phosphoproteome was analyzed using a phosphopeptide enrichment method, i.e., titanium immobilized metal ion affinity chromatography, coupled with liquid chromatography-tandem mass spectrometry. A total of 2093 phosphopeptides carrying unique 1851 phosphorylation sites corresponding to 1180 phosphoproteins were identified. Of the 1180 phosphoproteins, 314 phosphorylation sites on 241 proteins were differentially expressed between the groups. Gene ontology analysis indicated that the phosphoproteins were strongly associated with developmental and macromolecule metabolic processes, signal transduction, and responses to stimuli and insulin. Pathway analysis showed that the spliceosome, Hippo, insulin, and neurotrophin signaling pathways were enriched. These results reveal the expression pattern and changes in the function of phosphoproteins in bovine jejunum tissues under different feeding conditions and provide further insights into the crucial role of colostrum feeding during the early stages of life.

A phase separation process induced by pH change for purification of His-tagged protein at low salt concentration

Protein purification is critical for protein research and enzyme manufacture, and efficient purification methods with low cost have a high priority. In this study, the difference in solubility between His-tagged protein and nontagged protein led to a liquid-solid phase separation at neutral pH and low salt concentration and purified His-tagged protein to some extent. The investigation of impurity in the precipitate indicated that solvation behavior varied from several oligo-amino acid tagged proteins. Among them, proteins with oligo-Lys, oligo-Trp and oligo-Arg obviously precipitated at low salt concentration and weak acidic pH, which is similar to that for the precipitation of His-tagged protein. Further study suggested that proteins with oligo-Lys, oligo-Trp and oligo-Arg remained a precipitate state while His-tagged protein dissolved at a slightly higher pH value. Based on these finding, a method was developed for the purification of His-tagged protein via phase separation induced by pH (PSIP) at low salt concentration. Though the yield and purity of protein purified by PSIP were slightly inferior to that by immobilized metal ion affinity chromatography (IMAC), PSIP was a high-throughput and low-cost method for purification of His-tagged proteins. Beyond the purification process, the various solvation behaviors of proteins with different oligo-amino acid also provide a reference for solvation research of protein.

Metal-Chelating Peptides Separation Using Immobilized Metal Ion Affinity Chromatography: Experimental Methodology and Simulation

Metal-Chelating Peptides (MCPs), obtained from protein hydrolysates, present various applications in the field of nutrition, pharmacy, cosmetic etc. The separation of MCPs from hydrolysates mixture is challenging, yet, techniques based on peptide-metal ion interactions such as Immobilized Metal Ion Affinity Chromatography (IMAC) seem to be efficient. However, separation processes are time consuming and expensive, therefore separation prediction using chromatography modelling and simulation should be necessary. Meanwhile, the obtention of sorption isotherm for chromatography modelling is a crucial step. Thus, Surface Plasmon Resonance (SPR), a biosensor method efficient to screen MCPs in hydrolysates and with similarities to IMAC might be a good option to acquire sorption isotherm. This review highlights IMAC experimental methodology to separate MCPs and how, IMAC chromatography can be modelled using transport dispersive model and input data obtained from SPR for peptides separation simulation.

Immobilization of Recombinant Endoglucanase (CelA) from Clostridium thermocellum on Modified Regenerated Cellulose Membrane

Cellulases are being widely employed in lignocellulosic biorefineries for the sustainable production of value-added bioproducts. However, the high production cost, sensitivity, and non-reusability of free cellulase enzymes impede their commercial applications. Enzyme immobilization seems to be a potential approach to address the aforesaid complications. The current study aims at the production of recombinant endoglucanase (CelA) originated from the cellulosome of Clostridium thermocellum in Escherichia coli (E. coli), followed by immobilization using modified regenerated cellulose (RC) membranes. The surface modification of RC membranes was performed in two different ways: one to generate the immobilized metal ion affinity membranes RC-EPI-IDA-Co2+ (IMAMs) for coordination coupling and another to develop aldehyde functional group membranes RC-EPI-DA-GA (AMs) for covalent bonding. For the preparation of IMAMs, cobalt ions expressed the highest affinity effect compared to other metal ions. Both enzyme-immobilized membranes exhibited better thermal stability and maintained an improved relative activity at higher temperatures (50–90 °C). In the storage analysis, 80% relative activity was retained after 15 days at 4 °C. Furthermore, the IMAM- and AM-immobilized CelA retained 63% and 53% relative activity, respectively, after being reused five times. As to the purification effect during immobilization, IMAMs showed a better purification fold of 3.19 than AMs. The IMAMs also displayed better kinetic parameters, with a higher Vmax of 15.57 U mg−1 and a lower Km of 36.14 mg mL−1, than those of AMs. The IMAMs were regenerated via treatment with stripping buffer and reloaded with enzymes and displayed almost 100% activity, the same as free enzymes, up to 5 cycles of regeneration.

A Ti/Nb-functionalized COF material based on IMAC strategy for efficient separation of phosphopeptides and phosphorylated exosomes

In this work, on the basis of an immobilized metal ion affinity chromatography enrichment strategy, a new kind of covalent organic framework (COF) material for enrichment of phosphorylated peptides and exosomes was successfully prepared in a facile method, and Ti4+ and Nb5+ were used as dual-functional ions (denoted as COF-S-S-COOH-Ti4+/Nb5+). With the advantage of unbiased enrichment towards phosphopeptides, COF-S-S-COOH-Ti4+/Nb5+ shows ultra-high selectivity (maximum molar ratio of β-casein: BSA is 1:20,000) and low limit of detection (0.2 fmol). In addition, the material has an excellent phosphopeptide loading capacity (100μg/mg) and reusability (at least seven times). Furthermore, applying the material to the actual sample, 4 phosphopeptides were selectively extracted from the serum of renal carcinoma patients. At the same time, exosomes with an intact structure in the serum of renal carcinoma patients were successfully isolated rapidly using this strategy. All experiments have shown that COF-S-S-COOH-Ti4+/Nb5+ exhibits exciting potential in practical applications.

The acidic amino acid-rich C-terminal domain of VanabinX enhances reductase activity, attaining 1.3- to 1.7-fold vanadium reduction

Ascidians accumulate extremely high levels of vanadium (V) in their blood cells. Several V-related proteins, including V-binding proteins (vanabins), have been isolated from V-accumulating ascidians. In this study, to obtain a deeper understanding of vanabins, we performed de novo transcriptome analysis of blood cells from a V-rich ascidian, Ascidia sydneiensis samea, and constructed a database containing 8532 predicted proteins. We found a novel vanabin with a unique acidic amino acid–rich C-terminal domain, designated VanabinX, in the database and studied it in detail. Reverse-transcription polymerase chain reaction analysis revealed that VanabinX was detected in all adult tissues examined, and was most prominent in blood cells and muscle tissue. We prepared recombinant proteins and performed immobilized metal ion affinity chromatography and a NADPH-coupled V(V)-reductase assay. VanabinX bound to metal ions, with increasing affinity for Cu(II) > Zn(II) > Co(II), but not to V(IV). VanabinX reduced V(V) to V(IV) at a rate of 0.170 μM per micoromolar protein within 30 min. The C-terminal acidic domain enhanced the reduction of V(V) by Vanabin2 to 1.3-fold and of VanabinX itself to 1.7-fold in trans mode. In summary, we constructed a protein database containing 8532 predicted proteins expressed in blood cells; among them, we discovered a novel vanabin, VanabinX, which enhances V reduction by vanabins.

Selective Acylation of Proteins at Gly and Lys in His Tags.

The chemical modification of proteins is of great importance in chemical biology, biotechnology, and for the production of modified biopharmaceuticals, as it enables introduction of fluorophores, biotin, half-life extending moieties, and more. We have developed two methods that use poly-His sequences to direct the highly selective acylation of proteins, either at the N-terminus or at a specific Lys residue. For the former, we used an N-terminal Gly-His6 segment (Gly-His tag) that directed acylation of the N-terminal Nα -amine with 4-methoxyphenyl esters, resulting in stable conjugates. Next, we developed the peptide sequences Hisn -Lys-Hism (Lys-His tags) that direct the acylation of the designated Lys Nϵ -amine under mild conditions and with high selectivity over native Lys residues. Both the Gly-His and Lys-His tags maintain the capacity for immobilized metal ion affinity chromatography. We have demonstrated the robustness of these methods by attaching different moieties such as azides, fluorophores, and biotin to different proteins, including antibodies.

Temoneira-1 β-lactamase is not a metalloenzyme, but its native metal ion binding sites allow for purification by immobilized metal ion affinity chromatography

β-lactamases protect bacteria from β-lactam antibiotics. Temoneira (TEM) is a class A serine β-lactamase and its coding sequence is designed into DNA vectors, such as pET-21a (+), to provide antibiotic resistance. TEM-1 β-lactamase was overexpressed efficiently from this vector upon inducing protein expression by IPTG in BL21(DE3) cells. Immobilized metal ion affinity chromatography (IMAC) was used based on the three native putative metal ion binding sites of TEM-1 β-lactamase, each consisting of a pair of histidine sidechains. Elution was achieved at low concentrations of imidazole (∼15–200 mM). Two steps of IMAC and a subsequent anion exchange purification produced highly pure TEM-1 β-lactamase with a yield of 1.9 mg/g of wet bacterial pellet weight. Mass spectrometry revealed that the mature form of β-lactamase (without the signal sequence) was obtained. The secondary structure composition, calculated from the circular dichroism spectrum, showed that the target protein was folded similar to the published crystal structure. Ni(II) binding to the enzyme was also investigated. Increasing amounts of Ni(II) ions had only a small effect on the protein structure. Mass spectrometry detected up to three bound metal ions at 10:1 Ni(II):protein molar ratio, but the major peak was assigned to the monometallated β-lactamase indicating the presence of a paramount metal ion binding site formed by the H151/H156 pair.

Isolation and Characterization of Homologically Expressed Methanol Dehydrogenase from Methylorubrum extorquens AM1 for the Development of Bioelectrocatalytical Systems.

(Ca2+)-dependent pyrroloquinolinequinone (PQQ)-dependent methanol dehydrogenase (MDH) (EC: 1.1.2.7) is one of the key enzymes of primary C1-compound metabolism in methylotrophy. PQQ-MDH is a promising catalyst for electrochemical biosensors and biofuel cells. However, the large-scale use of PQQ-MDH in bioelectrocatalysis is not possible due to the low yield of the native enzyme. Homologously overexpressed MDH was obtained from methylotrophic bacterium Methylorubrum extorquens AM1 by cloning the gene of only one subunit, mxaF. The His-tagged enzyme was easily purified by immobilized metal ion affinity chromatography (36% yield). A multimeric form (α6β6) of recombinant PQQ-MDH possessing enzymatic activity (0.54 U/mg) and high stability was demonstrated for the first time. pH-optimum of the purified protein was about 9–10; the enzyme was activated by ammonium ions. It had the highest affinity toward methanol (KM = 0.36 mM). The recombinant MDH was used for the fabrication of an amperometric biosensor. Its linear range for methanol concentrations was 0.002–0.1 mM, the detection limit was 0.7 µM. The properties of the invented biosensor are competitive to the analogs, meaning that this enzyme is a promising catalyst for industrial methanol biosensors. The developed simplified technology for PQQ-MDH production opens up new opportunities for the development of bioelectrocatalytic systems.

Efficient Enrichment of Global Phosphopeptides Using Magnetic Tannic Acid – Titanium(IV)/Zirconium(IV) Functionalized Spheres as a Novel Sorbent for Immobilized Metal Ion Affinity Chromatography (IMAC)

Inspired by the enrichment preferences of metal ions for phosphopeptides, a layered assembly method was adopted to introduce hydrophilic polyhydroxy ligands tannic acid (TA) to prepare magnetic bimetal ion-functionalized nanomaterials denoted by Fe3O4@TA@Ti4+/Zr4+ to provide the comprehensive enrichment of phosphopeptides. Benefiting from the excellent ion immobilization ability and unbiasedness for phosphopeptide enrichment, Fe3O4@TA@Ti4+/Zr4+ shows excellent properties for the enrichment and identification of global phosphopeptides. The results show that the material possesses high efficiency, outstanding selectivity (the maximum molar ratio β-casein to bovine serum albumin is 1:1000), a low detection limit of 0.4 fmol/μL, and the ability to be recycled at least 10 times. In addition, excellent performance was also demonstrated in the analysis of skimmed milk and human saliva: 13 and 31 phosphopeptides were successfully captured. The results demonstrate that the material has high potential for practical application in phosphoproteomics.

Epitaxial Growth of Guanidyl-Functionalized Magnetic Metal-Organic Frameworks with Multiaffinity Sites for Selective Capture of Global Phosphopeptides.

The flexible and controlled synthesis of metal-organic framework (MOF)-derived hybrid nanostructures is of great significance in fine tuning of their enrichment performance in large-scale and in-depth phosphoproteome analysis. Herein, a magnetic guanidyl-functionalized MOF hybrid coating with multiaffinity sites, denoted as Fe3O4@G-ZIF-8, was fast fabricated via a one-pot epitaxial growth strategy for the first time and applied for selective and highly efficient enrichment of global phosphopeptides. The intrinsic unsaturated metal sites of ZIF-8 endow the surface-mounted MOF coatings with immobilized metal ion affinity chromatography interaction with multiphosphorylated peptides. The oriented anchoring of bifunctional guanidineacetic acid on the magnetic MOF nanospheres provides additional affinity sites (guanidyl groups) for specific recognition of phosphopeptides by "salt bridge" interaction, as well as active site carboxyl groups for the coordination with the metal ions. The as-prepared Fe3O4@G-ZIF-8 exhibits large surface area (382.5 m2 g-1), good superparamagnetic property (41.6 emu g-1) and stability, and size-exclusion effect (1.73 nm), which can serve as a specific adsorbent for global phosphopeptide analysis with satisfactory selectivity, great detection sensitivity (1 fmol), and rapid magnetic separation. Moreover, the successful application of Fe3O4@G-ZIF-8 for selective capture of both multi- and mono-phosphopeptides from human saliva and serum demonstrated the great potential of magnetic surface-mounted MOF coatings in effective identification of low-abundance phosphopeptides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry from complicated biological matrices.

An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen

Developing affordable and easily manufactured SARS-CoV-2 vaccines will be essential to achieve worldwide vaccine coverage and long-term control of the COVID-19 pandemic. Here the development is reported of a vaccine based on the SARS-CoV-2 receptor-binding domain (RBD), produced in the yeast Pichia pastoris. The RBD was modified by adding flexible N- and C-terminal amino acid extensions that modulate protein/protein interactions and facilitate protein purification. A fed-batch methanol fermentation with a yeast extract-based culture medium in a 50 L fermenter and an immobilized metal ion affinity chromatography-based downstream purification process yielded 30–40 mg/L of RBD. Correct folding of the purified protein was demonstrated by mass spectrometry, circular dichroism, and determinations of binding affinity to the angiotensin-converting enzyme 2 (ACE2) receptor. The RBD antigen also exhibited high reactivity with sera from convalescent individuals and Pfizer-BioNTech or Sputnik V vaccinees. Immunization of mice and non-human primates with 50 µg of the recombinant RBD adjuvanted with alum induced high levels of binding antibodies as assessed by ELISA with RBD produced in HEK293T cells, and which inhibited RBD binding to ACE2 and neutralized infection of VeroE6 cells by SARS-CoV-2. Additionally, the RBD protein stimulated IFNγ, IL-2, IL-6, IL-4 and TNFα secretion in splenocytes and lung CD3+-enriched cells of immunized mice. The data suggest that the RBD recombinant protein produced in yeast P. pastoris is suitable as a vaccine candidate against COVID-19.

Affinity of phenolic compounds for transition metal ions immobilized on cation-exchange columns

Immobilized metal ion affinity chromatography (IMAC) is useful in purification of histidine-tagged or histidine-rich proteins and peptides from a variety of hosts. However, phenolic compounds including polyphenols interfere with IMAC due to their high affinities for the transition metals immobilized on the column resins, which hampers the purification of proteins from plant-based host systems. In contrast to extensive knowledge of the mechanism of the interactions between phenolic compounds and transition metal ions in solution, an understanding of the interactions on the columns, where transition metal ions are immobilized on the resins, remains elusive. This study systematically investigated the affinity of phenolic compounds for transition metal ions by varying the number and position of phenolic hydroxyl groups (OH groups) and using different transition metals—Fe(II), Cu(II) and Ni(II)—on various IMACs, in which the columns were fabricated by equilibrating the cation-exchange column with transition metal solutions. It was found that the more OH groups the aromatic compounds have, the higher the affinity for transition metal ions; in particular, methyl gallate and pyrogallol were permanently bound to the IMAC column, which reflected coordinate bond formation with the transition metal ions. Importantly, the phenolic compounds showed no obvious affinity for the Ni(II)-IMAC column, in contrast to the Fe(II)- and Cu(II)-IMAC columns, whereas imidazole and histidine-tagged proteins showed evident binding to the Ni(II)-IMAC column. Ni(II)-IMAC should thus be especially effective in isolating histidine-tagged and histidine-rich species from phenolic compound-containing systems. These results indicate that the affinity between phenolic compounds and transition metal ions on the column is consistent with the results in solution. They also provide a comprehensive view for devising strategies to improve IMAC purification of target proteins and peptides from samples containing phenolic compounds.

Preparation of Core‐Shell Structured Magnetic Superhydrophilic Extractant for Enrichment of Phosphopeptides

A core-shell structured magnetic superhydrophilic Fe3O4@C@TiO2 microspheres are successfully prepared. The as-prepared Fe3O4@C@TiO2 magnetic microspheres possess fast magnetic response, superhydrophilic and high affinity. The magnetic superhydrophilic Fe3O4@C@TiO2 microspheres are employed for selective enrichment phosphopeptides and exhibit excellent enrichment performance such as high selectivity, low detection and excellent reusability. Sixteen phosphopeptides and four endogenous phosphopeptides can be identified from non-fat milk and human serum by Fe3O4@C@TiO2 magnetic microspheres, indicating Fe3O4@C@TiO2 will be a powerful and helpful IMAC material in understanding and analyzing essential biological phosphorylation processes in phosphorylated proteomics research.