Isolation Of Phosphopeptides Research Articles

Facile preparation of porphyrin-based porous organic polymers for specific enrichment and isolation of phosphopeptides and phosphorylated exosomes

Exosomes, which can be used to investigate various disease processes, are novel disease markers that have been extensively studied in recent years. In this work, zirconium-rich porphyrin-based porous organic polymers (Imi-Pops-Zr) were synthesized by a facile and low-cost strategy for specific enrichment and isolation of phosphorylated peptides and exosomes. The proposed material demonstrates a low detection limit (0.5 fmol), a high selectivity (bovine serum albumin (BSA): β-casein = 1000:1), and a loading capability of 100 mg/g for phosphopeptides. For complex practical samples, after enrichment with Imi-Pops-Zr, 4 characteristic phosphopeptides from human serum, 20 and 12 phosphopeptides from human saliva and defatted milk were detected, respectively. Besides, 74 phosphorylated peptides with 67 phosphorylation sites belonging to 61 phosphoproteins and 67 phosphorylated peptides with 63 phosphorylation sites belonging to 65 phosphoproteins were detected from the serum of normal controls and uremic patients, respectively. Biological processes, cellular components and molecular functions revealed that interleukin-6, tumor necrosis factor, high density lipoprotein and proteases binding may be associated with uremia. Furthermore, Imi-Pops-Zr was successfully used to enrich and isolate exosomes from human serum. The experimental results show that Imi-Pops-Zr has promising application in the specific enrichment of phosphorylated peptides and exosomes in complex bio-samples.

Magnetic guanidyl-functionalized covalent organic framework composite: a platform for specific capture and isolation of phosphopeptides and exosomes.

A guanidine-functionalized (GF) covalent organic framework (COF) nanocomposite has been developed by a post-synthetic approach for specific capture and separation of phosphopeptides and exosomes. The abundant binding sites on COF can immobilize a large number of gold nanoparticles (AuNPs), which can be used to react with amino groups to graft polyethyleneimine (PEI). Finally, Fe3O4@COF@Au@PEI-GF is obtained through the reaction of PEI and guanidyl group for phosphopeptides and exosomes detection. This composite shows a low detection limit (0.02 fmol), size exclusion effect (β-casein digests:Albumin from bovine serum protein = 1:10,000), good reusability (10 cycles), and high selectivity (β-casein digests:Albumin from bovine serum digests = 1:10,000). For complex biological sample, 4 phosphopeptides can be successfully identified from human serum. Furthermore, for the first time, we used guanidyl-functionalized probe to capture exosomes in human serum, providing a new method for enriching exosomes. The above experiments showed that Fe3O4@COF@Au@PEI-GF not only effectively enrich phosphopeptides and remove macromolecular proteins, but also successfully separate and capture exosomes. This demonstrates the great potential of this composite for the specific enrichment of phosphopeptides and isolation of exosomes.

Titanium(IV) immobilized affinity chromatography facilitated phosphoproteomics analysis of salivary extracellular vesicles for lung cancer.

Extracellular vesicles (EVs) play critical roles in intercellular communications, which contain valuable biomarkers for the detection of cancers. Phosphoproteomics analysis of human saliva EVs (sEVs) can help to discover lung cancer-related candidates. Due to the low abundance of phosphoproteins in sEVs, an efficient, reproducible, and cost-effective strategy is required for their enrichment. Here, we compared the latest phosphopeptide techniques, including TiO2, ZrO2, CaTiO3, and Ti4+-IMAC (immobilized metal affinity chromatography) methods, for phosphopeptide isolation. Our data demonstrated that Ti4+-IMAC was the superior one. By using the optimized Ti4+-IMAC approach, we identified more than 500 sEV phosphopeptides. Quantitative proteomics was employed to comprehensively decipher the sEV phosphoproteome of the normal group (n = 6) and lung cancer group (n = 6). Accordingly, 524 and 333 phosphopeptides were enriched, respectively, which corresponded to 439 and 282 phosphoproteins. In total, 857 unique sEV phosphopeptides corresponding to 721 phosphoproteins were revealed. Among 493 identified phosphosites, 37 were upregulated (> 1.5) and 217 were downregulated (< 0.66) in the cancer group. Our data collectively demonstrated that Ti4+-IMAC is an efficient and reproducible technology for comprehensive analysis of sEV phosphoproteome. Differentially expressed sEV phosphoproteins and phosphosites might be used for the detection of lung cancer non-invasively.

Application of SILAC Labeling in Phosphoproteomics Analysis.

The analysis of disease-related changes in the phosphorylation status of cellular signal transduction networks is of major interest to biomedical researchers. Mass spectrometry-based proteomics allows the analysis of phosphorylation in a global manner. However, several technical challenges need to be addressed when the phosphorylation of proteins is analyzed. Low-abundant phosphopeptides need to be enriched before analysis, thereby introducing additional steps in sample preparation. Consequently, the applied quantification strategies should be robust towards elaborate sampling handling, rendering label-based quantification strategies the methods of choice in many experiments. Here, we present a protocol for SILAC labeling and the subsequent isolation of phosphopeptides using TiO2 affinity chromatography. We outline the corresponding LC-MS/MS analysis and the essential steps of data processing.

Highly selective enrichment of phosphopeptides using poly(dibenzo-18-crown-6) as a solid-phase extraction material.

A poly(dibenzo-18-crown-6) was used as a new solid-phase extraction material for the selective enrichment of phosphopeptides. Isolation of phosphopeptides was achieved based on specific ionic interactions between poly(dibenzo-18-crown-6) and the phosphate group of phosphopeptides. Thus, a method was developed and optimized, including loading, washing and elution steps, for the selective enrichment of phosphopeptides. To assess this potential, tryptic digest of three proteins (α- casein, β-casein and ovalbumin) was applied on poly(dibenzo-18-crown-6). The nonspecific products were removed by centrifugation and washing. The spectrometric analysis was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Highly selective enrichment of both mono- and multiphosphorylated peptides was achieved using poly(dibenzo-18-crown-6) as solid-phase extraction material with minimum interference from nonspecific compounds. Furthermore, evaluation of the efficiency of the poly(dibenzo-18-crown-6) was performed by applying the digest of egg white. Finally, quantum mechanical calculations were performed to calculate the binding energies to predict the affinity between poly(dibenzo-18-crown-6) and various ligands. The newly identified solid-phase extraction material was found to be a highly efficient tool for phosphopeptide recovery from tryptic digest of proteins.

Facile liquid-phase deposition synthesis of titania-coated magnetic sporopollenin for the selective capture of phosphopeptides.

Titania-grafted magnetic sporopollenin is synthesized by the liquid-phase deposition (LPD) technique, characterized by SEM, EDX, and nitrogen adsorption porosimetry, and used for the selective enrichment of phosphorylated peptides. The material is low cost because of easier availability of pollens and has rich surface chemistry which enables strong attachment of titania onto magnetic sporopollenin. The material shows higher selectivity of 1:1000 with β-casein spiked in BSA. Higher sensitivity of 10fmol is recorded for phosphopeptides from standard β-casein digest. Twenty phosphorylated peptides are enriched from milk digest and four endogenous phosphopeptides from diluted human serum. The magnetic property of titania-coated magnetic sporopollenin facilitates the fast and effective isolation of phosphopeptides from complex mixtures through external magnet. The material is finally applied to tryptic digest of rat brain cell lysate for phosphopeptide enrichment where 2718 phosphopeptides are identified by using LC-MS/MS with C18 column. Titania-coated magnetic sporopollenin captures both mono-phosphorylated (2489) and multi-phosphorylated peptides (229) due to strong affinity of TiO2 with phosphates. TiO2-coated magnetic material also shows better enrichment efficiency in comparison to commercial TiO2. Graphical abstract.

A sensitive and selective phosphopeptide enrichment strategy by combining polyoxometalates and cysteamine hydrochloride-modified chitosan through layer-by-layer assembly

Highly efficient enrichment of phosphopeptides in low abundance prior to mass spectrometry analysis is essential in phosphopeptidomics analysis. Herein, an affinity probe possessing both interactions of polyoxometalates (POMs) and hydrophilic interaction chromatography (HILIC) was facilely prepared. POMs and cysteamine hydrochloride-modified chitosan (CYECS) were assembled onto magnetic nanoparticles (MNPs) via a layer-by-layer approach. The developed MNPs-(POM/CYECS) material owned merits of large metal oxide surfaces, positive charge, and hydrophilicity as well as superparamagnetism. By combining with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), the prepared material was utilized for effective enrichment of phosphopeptides. High selectivity (mass ratio of β-casein and bovine serum albumin digests of 1:5000) and low detection limit (0.02 fmol) toward phosphopeptides were achieved. The recovery of phosphopeptides was measured to be 92.6%. The specific enrichment of phosphopeptides from complex samples (nonfat milk, human saliva, serum, and A549 cell lysate) with MNPs-(POM/CYECS) material further confirmed its bright prospects for isolation of phosphopeptides from biological samples.

Controllable Preparation of CuFeMnO4 Nanospheres as a Novel Multifunctional Affinity Probe for Efficient Adsorption and Selective Enrichment of Low-Abundance Peptides and Phosphopeptides.

A facile solvothermal method for the synthesis of multifunctional magnetic CuFeMnO4 nanospheres affinity probe (NSAP) with controllable morphology and size was developed for the first time. The CuFeMnO4 nanospheres combine the brilliant features of Cu2+, Fe3+, and Mn2+ ions, so their multifunction performances are embodied by strong coordination to carboxyl and amine groups of peptides (Cu2+ and Fe3+), special affinity to phosphate groups of phosphopeptides (Fe3+ and Mn2+), and high magnetic responsiveness in a magnetic field. Their potential as an affinity probe was evaluated for highly effective enrichment, rapid magnetic separation of low-abundance peptides (neutral condition), and effective selective capture of phosphopeptides (acid condition) from various complex biosamples. Notably, CuFeMnO4 NSAP was explored for highly selective capture and isolation of phosphopeptides from A549 cells after exposure to ZnO nanoparticles for different times. Consequently, we put forward a new nanospinel ferrite-based protocol here to analyze and identify the phosphoproteins/phosphopeptides involved in cellular signaling pathways in response to exogenous stimulation.

Spinel-type manganese ferrite (MnFe2O4) microspheres: A novel affinity probe for selective and fast enrichment of phosphopeptides

The spinel-type magnetic manganese ferrite (MnFe2O4) microspheres synthesized by simple solvothermal method were used as a novel adsorbent for selective enrichment and effective isolation of phosphopeptides. The uniform MnFe2O4 magnetic affinity microspheres (MAMSs) had a narrow particle size distribution between 250 and 260nm, and displayed superparamagnetism with a saturation magnetization value of 67.0emu/g. Comprehensively, the possible formation mechanism of MnFe2O4 microspheres with ferric and manganous sources as dual precursors was elucidated by comparison with those of Fe3O4 nanoparticles and MnOOH nanosheets respectively with either ferric or manganous source as single precursor. It was suggested that the spherical or sheet nanostructures could be achieved via secondary recrystallization or Ostwald ripening. The MnFe2O4 MAMSs probe exhibited excellent dispersibility in aqueous solution, and rapid magnetic separation within 15s, as well as good reusability. More importantly, MnFe2O4 was highly selective for phosphopeptides because of the strong coordination interaction between metal ions (Fe3+ and Mn2+) and phosphate groups of phosphopeptdies. This high specificity was demonstrated by effectively enriching phosphopeptides from digest mixture of β-casein and bovine serum albumin (BSA) with high content of non-phosphopeptides, and embodied further in phosphopeptides enrichment from non-fat milk digests and human serum. Consequently, the prepared MnFe2O4 affinity materials are expected to possess great potential in phosphoproteome research.

Immobilized metal affinity chromatography optimized for the analysis of extracellular phosphorylation.

Phosphorylation is the most widely studied posttranslational modification. Its role within the cell has been the focus of numerous large-scale studies. Recently there is growing evidence on the biological significance of extracellular phosphorylation. The analysis of these phosphopeptides is complicated by the abundance of glycosylation in the extracellular space, since glycopeptides are also enriched by the methods used for phosphopeptide isolation. Thus, we optimized IMAC for phosphorylation analysis of secreted proteins, specifically in human serum. Selectivity and efficiency of different enrichment conditions used in earlier large-scale phosphoproteomic studies were evaluated. We found that minimizing hydrophilic interactions in the enrichment allowed selective phosphopeptide isolation. Using a two-step IMAC enrichment protocol under these conditions led to the identification of ∼100 phosphorylation sites from the tryptic digest of as little as 40μL human serum.

Isolation and Characteristics of Lactic Acid Bacteria Producing Casein Phosphopeptides from Kimchi

Isolation and Characteristics of Lactic Acid Bacteria Producing Casein Phosphopeptides from Kimchi

Immobilized metal ion affinity chromatography ZipTip pipette tip with polydopamine modification and Ti4+ immobilization for selective enrichment and isolation of phosphopeptides

As an effective tool in protein analysis, mass spectroscopy (MS) has been widely used in identifying protein phosphorylation and phosphorylation sites. Because of the low abundance of phosphopeptides in protein digestion, isolation and enrichment of phosphopeptides prior to MS analysis is important for efficient phosphopeptides identification. In this work, we initially immobilized titanium ions on polydopamine (PDA)-modified ZipTip pipette tips (denoted as IMAC ZipTip pipette tip) for simple and quick enrichment of phosphopeptides. The preparation process of the novel ZipTip pipette tips is fast and economic since it only contains two simple steps both with mild conditions. The ability of modified ZipTip pipette tips for identifying phosphopeptides in complex biological samples was investigated. The unique ZipTip pipette tip not only exhibited superior ability in selectively capturing phosphopeptides from large amount of non-phosphopeptides, but also remarkably shortened the MS preparation and analysis time, making it an easy-to-use and efficient tool in phosphoproteome research.

Preparation of on-plate immobilized metal ion affinity chromatography platform via dopamine chemistry for highly selective isolation of phosphopeptides with matrix assisted laser desorption/ionization mass spectrometry analysis

In this study, a novel on-plate IMAC technique was developed for highly selective enrichment and isolation of phosphopeptides with high-throughput MALDI-TOF-MS analysis. At first, a MALDI plate was coated with polydopamine (PDA), and then Ti4+ was immobilized on the PDA-coated plate. The obtained IMAC plate was successfully applied to the highly selective enrichment and isolation of phosphopeptides in protein digests and human serum. Because of no loss of samples, the on-plate IMAC platform exhibits excellent selectivity and sensitivity in the selective enrichment and isolation of phosphopeptides, which provides a potential technique for high selectivity in the detection of low-abundance phosphopeptides in biological samples.

Titania composite microspheres endowed with a size-exclusive effect toward the highly specific revelation of phosphopeptidome.

The efficient isolation of low-abundance phosphopeptides from complicated biological samples containing a significant quantity of nonphosphopeptides and proteins is essential for phosphopeptidome research but remains a great challenge. In this Article, magnetic composite microspheres comprising a magnetic colloidal nanocrystal cluster core and a mesoporous titania shell with an average pore diameter of 3.4 nm were modified by directly coating an amorphous titania shell onto the magnetite core, followed by converting the amorphous titania shell into a crystalline structure via a hydrothermal process at 80 °C. The as-prepared magnetic mesoporous titania microspheres possess a remarkable specific surface area that is as high as 603.5 m2/g, which is an appropriate pore size with a narrow size distribution and a high magnetic responsiveness. These outstanding features imply that the composite microspheres exhibit extraordinary performance in phosphopeptidome research, including high specificity toward phosphopeptides, an excellent size-exclusion effect against phosphoproteins, exceptional enrichment capacity, and efficient separation from mixtures. Encouraged by the experimental results, we employed this method to investigate the phosphopeptidome of snake venom for the first time. A total of 35 phosphopeptides was identified from the snake venom from the family Viperidae, accounting for 75% of the total identified peptides. This result represents the largest data set of the phosphopeptidome in snake venom from the family Viperidae.

Влияние сульфата железа на биосинтез внеклеточных метаболитов пропионовокислыми бактериями

In the work, activated cultures of propionic acid bacteria were found to exhibit high antimutagenic activity and adhesion properties, synthesize considerable amount of corrinoids and heme-containing enzymes. Increase of iron concentration in the medium was shown to intensify synthesis of extracellular metabolites promoting adaptation of the culture to the metal. Optimal technological parameters for isolation of casein phosphopeptides were determined. Ability of phosphopeptides to efficiently solubilize divalent iron was confirmed. Relationship between iron concentration and extent of solubilization was established. Iron chelated with casein phosphopeptides was noted to stay in divalent form for prolonged period.

Facile synthesis of magnetic mesoporous titania and its application in selective and rapid enrichment of phosphopeptides

In this paper, a novel magnetic mesoporous titania composite (MNPs/mTiO2) was facilely prepared by anchoring magnetic nanoparticles (MNPs) onto the external surface of mesoporous titania (mTiO2) åsub-microparticles with the use of ethylenebis(nitrilodimethylene)tetraphosphonic acid (EDTMPA) as “bridge” molecule. The usefulness of MNPs/mTiO2 as adsorbent for the selective isolation and enrichment of phosphopeptides with subsequent analysis by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) was studied. The results showed that the MNPs/mTiO2 composite could be used in the highly efficient and rapid enrichment of phosphopeptides.

A new type of metal chelate affinity chromatography using trivalent lanthanide ions for phosphopeptide enrichment

In this study, a new type of immobilized metal-ion affinity chromatography (IMAC) resin for the isolation of phosphopeptides was synthesized which is based on the specific interaction between phosphate groups and chelated lanthanide metal ions. In this regard trivalent lanthanum, holmium and erbium ions were chelated to a highly porous phosphonate polymer which was prepared by radical polymerization of vinylphosphonic acid (VPA) and divinylbenzene (DVB). The developed method was evaluated with peptide mixtures from digested standard proteins (α-casein, β-casein and ovalbumin) as well as with bovine milk, egg white and a spiked HeLa cell lysate. Compared to the commonly used TiO2 approach, the presented method showed higher selectivity for phosphorylated peptides. This can be explained by the strong preference of trivalent lanthanide ions for phosphates with which they form very tight ionic bonds. Mono- and multiply phosphorylated peptides could be enriched and released in a single basic elution step, while non-phosphorylated peptides remained on the resin. Ab initio quantum mechanical energy minimizations of model complexes for polymer-ion-ligand interactions provided geometries, binding energies and charges which are discussed in conjunction with the observed experimental properties, leading to the most satisfying agreement. The presented lanthanide-IMAC resins represent promising affinity materials for the selective isolation of phosphopeptides from biological samples.

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

Grapevine (Vitis vinifera) is an economically important fruit crop that is subject to many types of insect and pathogen attack. To better elucidate the plant response to Lobesia botrana pathogen infection, we initiated a global comparative proteomic study monitoring steady-state protein expression as well as changes in N-glycosylation, phosphorylation, and Lys-acetylation in control and infected mesocarp and exocarp from V. vinifera cv Italia. A multi-parallel, large-scale proteomic approach employing iTRAQ labeling prior to three peptide enrichment techniques followed by tandem mass spectrometry led to the identification of a total of 3059 proteins, 1135 phosphorylation sites, 323 N-linked glycosylation sites and 138 Lys-acetylation sites. Of these, we could identify changes in abundance of 899 proteins. The occupancy of 110 phosphorylation sites, 10 N-glycosylation sites and 20 Lys-acetylation sites differentially changed during L. botrana infection. Sequence consensus analysis for phosphorylation sites showed eight significant motifs, two of which containing up-regulated phosphopeptides (X-G-S-X and S-X-X-D) and two containing down-regulated phosphopeptides (R-X-X-S and S-D-X-E) in response to pathogen infection. Topographical distribution of phosphorylation sites within primary sequences reveal preferential phosphorylation at both the N- and C termini, and a clear preference for C-terminal phosphorylation in response to pathogen infection suggesting induction of region-specific kinase(s). Lys-acetylation analysis confirmed the consensus X-K-Y-X motif previously detected in mammals and revealed the importance of this modification in plant defense. The importance of N-linked protein glycosylation in plant response to biotic stimulus was evident by an up-regulated glycopeptide belonging to the disease resistance response protein 206. This study represents a substantial step toward the understanding of protein and PTMs-mediated plant-pathogen interaction shedding light on the mechanisms underlying the grape infection.

A novel strategy for phosphopeptide enrichment using lanthanide phosphate co-precipitation

Reversible phosphorylation of proteins is a common theme in the regulation of important cellular functions such as growth, metabolism, and differentiation. The comprehensive understanding of biological processes requires the characterization of protein phosphorylation at the molecular level. Although, the number of cellular phosphoproteins is relatively high, the phosphorylated residues themselves are generally of low abundance due to the sub-stoichiometric nature. However, low abundance of phosphopeptides and low degree of phosphorylation typically necessitates isolation and concentration of phosphopeptides prior to mass spectrometric analysis. In this study, we used trivalent lanthanide ions (LaCl(3), CeCl(3), EuCl(3), TbCl(3), HoCl(3), ErCl(3), and TmCl(3)) for phosphopeptide enrichment and cleaning-up. Due to their low solubility product, lanthanide ions form stable complexes with the phosphate groups of phosphopeptides and precipitate out of solution. In a further step, non-phosphorylated compounds can easily be removed by simple centrifugation and washing before mass spectrometric analysis using Matrix-assisted laser desorption/ionisation-time of flight. The precipitation method was applied for the isolation of phosphopeptides from standard proteins such as ovalbumin, α-casein, and β-casein. High enrichment of phosphopeptides could also be achieved for real samples such as fresh milk and egg white. The technology presented here represents an excellent and highly selective tool for phosphopeptide recovery; it is easily applicable and shows several advantages as compared with standard approaches such as TiO(2) or IMAC.

Phosphopeptide enrichment and fractionation by using Click OEG-CD matrix

Reversible protein phosphorylation regulates many significant cellular processes. Identification of phosphorylation sites is vital to elucidate their biofunctions. To aid in phosphoproteome characterization, several selective enrichment methods have been developed, including immobilized metal-ion affinity chromatography (IMAC) and titanium dioxide (TiO2). However, the high pH elution step applied in these two methods, which tends to degrade phosphopeptides. In order to improve phosphopeptide enrichment efficiency, a hydrophilic interaction liquid chromatography (HILIC) based material, cyclodextrin (CD) bonded silica (Click OEG-CD), was synthesized in our group and applied to phosphopeptide enrichment and fractionation. Taking tryptic digest of standard protein α-casein as model sample, the performance of Click OEG-CD in phosphopeptide isolation was investigated. It was found that both the acetonitrile and salt concentrations in mobile phase influence the phosphopeptide enrichment selectivity of the matrix. Under optimized enrichment condition, Click OEG-CD has a similar phosphopeptide enrichment selectivity as commercial TiO2. Meanwhile, phosphopeptides with same charges could be fractionated according to hydrophilicity difference on Click OEG-CD under acetonitrile gradient. Different selectivity is proved with Click OEG-CD from conventional SAX. We demonstrate that Click OEG-CD is an efficient matrix in phosphopeptide enrichment and fractionation.