Yüksel Güzel

Selective enrichment of phosphopeptides by a metal–organic framework

A metal–organic framework, consisting of Er(III) linked together by 1,4-phenylenediacetate, was synthesised by a one-pot reaction and successfully used as an affinity material for the selective capturing of phosphopeptides. An optimised protocol for loading, washing and elution was developed and the eluents were analysed via matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. A standard protein digest (α-casein, β-casein and ovalbumin) as well as digested egg white proteins were used to test the efficiency and selectivity of the presented approach. 14 phosphopeptides could be recovered from the peptide mixture and in the case of digested egg-white, four phosphorylated peptides which could be assigned to ovalbumin were isolated. Ab initio calculations based on the affinities of various ligands to the material have provided reasonable explanations of the observed experimental properties.

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

AbstractReversible 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 (LaCl3, CeCl3, EuCl3, TbCl3, HoCl3, ErCl3, and TmCl3) 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 TiO2 or IMAC. FigureHighly selective co-precipitation of phosphopeptides by trivalent lanthanide ions

Solid-phase extraction of galloyl- and caffeoylquinic acids from natural sources (Galphimia glauca and Arnicae flos) using pure zirconium silicate and bismuth citrate powders as sorbents inside micro spin columns

Galloyl- and caffeoylquinic acids are among the most important pharmacological active groups of natural compounds. This study describes a pre-step in isolation of some selected representatives of these groups from biological samples. A selective solid-phase extraction (SPE) method for these compounds may help assign classes and isomer designations within complex mixtures. Pure zirconium silicate and bismuth citrate powders (325 mesh) were employed as two new sorbents for optimized SPE of phenolic acids. These sorbents possess electrostatic interaction sites which accounts for additional interactions for carbon acid moieties as compared to hydrophilic and hydrophobic sorbents alone. Based on this principle, a selective SPE method for 1,3,4,5-tetragalloylquinic acid (an anti-HIV and anti-asthamatic agent) as a starting compound was developed and then deployed upon other phenolic acids with success. The recoveries and selectivities of both sorbents were compared to most commonly applied and commercially available sorbents by using high performance liquid chromatography. The nature of interaction between the carrier sorbent and the acidic target molecules was investigated by studying hydrophilic (silica), hydrophobic (C18), mixed-mode (ionic and hydrophobic: Oasis(®) MAX) and predominantly electrostatic (zirconium silicate) materials. The newly developed zirconium silicate and bismuth citrate stationary phases revealed promising results for the selective extraction of galloyl- and caffeoylquinic acids from natural sources. It was observed that zirconium silicate exhibited maximum recovery and selectivity for tetragalloylquinic acid (84%), chlorogenic acid (82%) and dicaffeoylquinic acid (94%) among all the tested sorbents.

Newly Fabricated Magnetic Lanthanide Oxides Core–Shell Nanoparticles in Phosphoproteomics

Metal oxides show high selectivity and sensitivity toward mass spectrometry based enrichment strategies. Phosphopeptides/phosphoproteins enrichment from biological samples is cumbersome because of their low abundance. Phosphopeptides are of interest in enzymes and phosphorylation pathways which lead to the clinical links of a disease. Magnetic core-shell lanthanide oxide nanoparticles (Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3) are fabricated, characterized by SEM, FTIR, and EDX and employed in the enrichment of phosphopeptides. The nanoparticles enrich phosphopeptides from casein variants, nonfat milk, egg yolk, human serum and HeLa cell extract. The materials and enrichment protocols are designed in a way that there are almost no nonspecific bindings. The selectivity is achieved up to 1:8500 using β-casein/BSA mixture and sensitivity down to 1 atto-mole. Batch-to-batch reproducibility is high with the reuse of core-shell nanoparticles up to four cycles. The enrichment followed by MALDI-MS analyses is carried out for the identification of phosphopeptides from serum digest and HeLa cell extract. Characteristic phosphopeptides of phosphoproteins are identified from human serum after the enrichment, which have the diagnostic potential toward prostate cancer. Thus, the lanthanide based magnetic core-shell materials offer a highly selective and sensitive workflow in phosphoproteomics.

Development of erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns for selective enrichment of phosphopeptides

In this study, a novel method for the highly selective enrichment of phosphopeptides using erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns is presented. Erbium phosphate was synthesized by precipitation from boiling phosphoric acid and incubated overnight in erbium chloride solutions. The resulting powder was embedded in a monolithic poly(glycidyl methacrylate/ethylene dimethacrylate) polymer. The monolith was synthesized in a spin column by radical polymerization. Erbium phosphate demonstrated a high affinity and selectivity for phosphopeptides due to the strong interaction of trivalent erbium ions with the phosphate groups of phosphopeptides. The high selectivity and performance of the designed spin columns were demonstrated by successfully enriching phosphopeptides from tryptically digested protein mixtures containing the model phosphoproteins α- and β-casein, bovine milk, and human saliva. By the implementation of several washing steps, unspecific components were removed and the enriched phosphopeptides were effectively eluted from the spin columns under alkaline conditions. The selective performance of the presented method was further demonstrated by the enrichment of two synthetic phosphopeptides, which were spiked in tryptically digested and dephosphorylated HeLa cell lysates at low ratios. Finally, the presented approach was compared to conventional phosphopeptide enrichment by titanium oxide and revealed higher recoveries for the erbium phosphate doped monoliths.

Highly selective enrichment of phosphopeptides using aluminum silicate

This study presents a novel strategy for highly selective enrichment of phosphopeptides using aluminium silicate (mullite) powder. Mullite is a nontoxic and inexpensive material and offers excellent performance for the purification of phosphopeptides from complex samples. The selectivity of the method was investigated by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The method was validated with tryptic peptides from model proteins, bovine milk and human saliva samples. For sample preparation, the digested samples were loaded on self-assembled extraction columns containing mullite as the sorbent. Non-phosphorylated compounds could be easily removed by several washing steps, while phosphorylated peptides were successfully immobilized on the mullite substrate. In the final step, phosphopeptides were eluted from the extraction column under alkaline conditions. To further assess the enrichment efficiency of the presented method, HeLa cell lysates were spiked with two synthetic phosphopeptides at different ratios. The method showed high selectivity and allowed the detection of phosphopeptides at a ratio of 1:1000. In a further study, the performance of the presented approach was compared with that of conventional phosphopeptide enrichment by TiO2 and revealed superior results for aluminium silicate.

Zirconium silicate assisted removal of residual proteins after organic solvent deproteinization of human plasma, enhancing the stability of the LC-ESI-MS response for the bioanalysis of small molecules.

An efficient blood plasma clean-up method was developed, where methanol protein precipitation was applied, followed by zirconium silicate assisted exclusion of residual proteins. A strong binding of zirconium (IV) silicate to the proteins enabled the elimination of remaining proteins after solvent deproteinization through a rapid solid-phase extraction (SPE) procedure. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) was used for monitoring the proteins during clean-up practice applied to human plasma samples. The proteins were quantified by colorimetric detection using the bicinchoninic acid (BCA) assay. The presented analytical strategy resulted in the depletion of >99.6% proteins from human plasma samples. Furthermore, high-performance liquid chromatography hyphenated to diode-array and electrospray ionization mass spectrometric detection (HPLC-DAD/ESI MS) was applied for qualitative and quantitative analysis of the caffeoylquinic acids (CQAs) and their metabolites in human plasma. The procedure demonstrated high recoveries for the standard compounds spiked at different concentrations. Cynarin and chlorogenic acid were recovered in the range of 81-86% and 78-83%, respectively. Caffeic acid was extracted in the excess of 89-92%, while ferulic acid and dihydroxyhydrocinnamic acid showed a recovery of 87-91% and 92-95%, respectively. The method was partially validated in accordance with FDA-Industry Guidelines for Bioanalytical Method Validation (2001). The presented scheme improves the clean-up efficacy of the methanol deproteinization, significantly reduces the matrix effects and provides a great analytical tool for the isolation of small molecules from human plasma.

Development of magnetic ytterbium oxide core–shell particles for selectively trapping phosphopeptides

Protein phosphorylation is one of the most important post-translational modifications and plays a key role in a large number of cellular processes. The progress in studying protein phosphorylation is largely based on the development of novel, selective and sensitive sample preparation tools for mass spectrometry. Here we report for the first time magnetic core–shell particles based on ytterbium for the selective enrichment of phosphopeptides from complex samples including human saliva. The newly fabricated Fe3O4@SiO2@Yb2O3 particles exhibit two unique features, which are specificity and response to a magnetic field. Thus, phosphopeptides are easily enriched on magnetic core–shell particles. In a further step, particles are simply removed from the sample matrix by applying a magnetic field. Electrostatic interactions, stable bidentate ligand chemistry and the high coordination number of ytterbium provide high selectivity, which was demonstrated for a spiked protein standard with a 10 000 fold background. The limit of detection was found to be in the femtomolar range for the applied mass spectrometer. Furthermore, magnetic Fe3O4@SiO2@Yb2O3 particles were compared to classical phosphopeptide enrichment by titanium oxide and revealed higher recoveries for the core–shell particles. The synthesized core–shell particles also allow the enrichment of multiply phosphorylated peptides.

Solid‐phase extraction of plant thionins employing aluminum silicate based extraction columns

Thionins belong to a family of cysteine-rich, low-molecular-weight (∼5 KDa) biologically active proteins in the plant kingdom. They display a broad cellular toxicity against a wide range of organisms and eukaryotic cell lines. Thionins protect plants against different pathogens, including bacteria and fungi. A highly selective solid-phase extraction method for plant thionins is reported deploying aluminum silicate (3:2 mullite) powder as a sorbent in extraction columns. Mullite was shown to considerably improve selectivity compared to a previously described zirconium silicate embedded poly(styrene-co-divinylbenzene) monolithic polymer. Due to the presence of aluminum(III), mullite offers electrostatic interactions for the selective isolation of cysteine-rich proteins. In comparison to zirconium(IV) silicate, aluminum(III) silicate showed reduced interactions towards proteins which resulted into superior washings of unspecific compounds while still retaining cysteine-rich thionins. In the presented study, European mistletoe, wheat and barley samples were subjected to solid-phase extraction analysis for isolation of viscotoxins, purothionins and hordothionins, respectively. Matrix-assisted laser desorption/ionization time of flight mass spectroscopy was used for determining the selectivity of the sorbent toward thionins. The selectively retained thionins were quantified by colorimetric detection using the bicinchoninic acid assay. For peptide mass-fingerprint analysis tryptic digests of eluates were examined.