phosphopeptide Dotaz Zobrazit nápovědu
Efficient selective sample enrichment is often a key procedure in protocols for analyses of complex samples. This applies not only to trace sample components but also to species with weak detection response. For example enrichment of phosphopeptides using selective affinity techniques prior to mass spectrometry analysis is necessary to increase detection sensitivity of phosphopeptides from highly complex peptide mixtures. In this work we have developed inorganic nanofibrous materials based on titanium or zirconium dioxides for selective and efficient enrichment of phosphopeptides for MALDI/MS detection. In comparison to the common bead based materials the presented nanofibrous materials exhibit much higher permeability allowing their use not only for batch mode or packed in the column operation, but also in the pipette tip format without the need for high pressure. Both the methods of preparation and characterization of the resulting materials are presented.
We have developed nanoparticle-modified monoliths in pipette tips for selective and efficient enrichment of phosphopeptides. The 5 μL monolithic beds were prepared by UV-initiated polymerization in 200 μL polypropylene pipette tips and either iron oxide or hydroxyapatite nanoparticles were used for monolith modification. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of iron oxide nanoparticles on the pore surface of the monolith was obtained via multivalent electrostatic interactions of citrate ions on the surface of nanoparticles with a quaternary amine functionalized poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith. Hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The nanoparticle-modified monoliths were compared with commercially available titanium dioxide pipette tips. Performance of the developed and commercially available sorbents was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and β-casein digests followed by off-line MALDI/MS analysis.
- MeSH
- chromatografie přístrojové vybavení metody MeSH
- fosfopeptidy chemie izolace a purifikace MeSH
- hydroxyapatit chemie MeSH
- kaseiny chemie MeSH
- nanočástice chemie MeSH
- poréznost MeSH
- syntetické pryskyřice chemická syntéza chemie MeSH
- železité sloučeniny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
A new monolithic capillary column with an iron oxide nanoparticle coating has been developed for selective and efficient enrichment of phosphopeptides. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of nanoparticles was obtained via multivalent electrostatic interactions of citrate ions on the surface of iron oxide nanoparticles with a quaternary amine functionalized poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith. A high dynamic binding capacity of 86 μmol/mL column volume was measured with an adenosine-5'-triphosphate. Performance of the monolithic column was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and β-casein digests and their MALDI/MS characterization in off-line mode.
- MeSH
- chromatografie kapalinová přístrojové vybavení metody MeSH
- fosfopeptidy chemie izolace a purifikace MeSH
- kaseiny chemie MeSH
- nanočástice chemie MeSH
- polymery chemická syntéza chemie MeSH
- železité sloučeniny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Magnetic non-porous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres prepared by the dispersion polymerization and modified with iminodiacetic acid (IDA) were employed for the IMAC separation of phosphopeptides. Fe(3+) and Ga(3+) ions immobilized on IDA-modified magnetic microspheres were used for the enrichment of phosphopeptides from the proteolytic digests of two model proteins differing in their physico-chemical properties and phosphate group content: porcine pepsin A and bovine α-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The phosphopeptides separated from the proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ability of the prepared Fe(3+)- and Ga(3+)-IDA-modified magnetic microspheres to capture phosphopeptides from complex mixtures was shown on an example of bovine milk proteolytic digest.
- MeSH
- adsorpce MeSH
- chromatografie afinitní přístrojové vybavení metody MeSH
- fosfopeptidy analýza izolace a purifikace MeSH
- iminokyseliny chemie MeSH
- magnetismus MeSH
- mikrosféry MeSH
- mléko chemie MeSH
- polyhydroxyethylmethakrylát chemie MeSH
- polymerizace MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.
- MeSH
- acetonitrily chemie MeSH
- chemická frakcionace metody MeSH
- chromatografie kapalinová metody MeSH
- fosfopeptidy chemie MeSH
- fosfoproteiny metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- proteom MeSH
- proteomika MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- titan chemie MeSH
- tlak MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
75, xii, s. ; 23 cm
- MeSH
- fosfopeptidy MeSH
- fosfoproteiny MeSH
- fosfor MeSH
- Publikační typ
- vysokoškolské kvalifikační práce MeSH
- Konspekt
- Chemie. Mineralogické vědy
- NLK Obory
- chemie, klinická chemie
- MeSH
- dietní proteiny metabolismus MeSH
- fosfopeptidy biosyntéza MeSH
- opioidní peptidy biosyntéza MeSH
- peptidy metabolismus MeSH
- Publikační typ
- přehledy MeSH
Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. In contrast to other formats such as StageTips or batch incubations using TiO2or Ti-IMAC beads, Fe-IMAC HPLC columns do not suffer from issues regarding incomplete phosphopeptide binding or elution and enrichment efficiency scales linearly with the amount of starting material. Here, we provide a step-by-step protocol for the entire phosphopeptide enrichment procedure including sample preparation (lysis, digestion, desalting), Fe-IMAC column chromatography (column setup, operation, charging), measurement by LC-MS/MS (nHPLC gradient, MS parameters) and data analysis (MaxQuant). To increase throughput, we have optimized several key steps such as the gradient time of the Fe-IMAC separation (15 min per enrichment), the number of consecutive enrichments possible between two chargings (>20) and the column recharging itself (<1 h). We show that the application of this protocol enables the selective (>90 %) identification of more than 10,000 unique phosphopeptides from 1 mg of HeLa digest within 2 h of measurement time (Q Exactive Plus).
- MeSH
- buněčné linie MeSH
- chromatografie kapalinová MeSH
- fosfopeptidy MeSH
- fosfoproteiny * MeSH
- imidazoly chemie MeSH
- lidé MeSH
- proteom * MeSH
- proteomika metody MeSH
- průběh práce MeSH
- software MeSH
- statistika jako téma MeSH
- tandemová hmotnostní spektrometrie MeSH
- železo chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
14-3-3 proteins are important dimeric scaffolds that regulate the function of hundreds of proteins in a phosphorylation-dependent manner. The SARS-CoV-2 nucleocapsid (N) protein forms a complex with human 14-3-3 proteins upon phosphorylation, which has also been described for other coronaviruses. Here, we report a high-resolution crystal structure of 14-3-3 bound to an N phosphopeptide bearing the phosphoserine 197 in the middle. The structure revealed two copies of the N phosphopeptide bound, each in the central binding groove of each 14-3-3 monomer. A complex network of hydrogen bonds and water bridges between the peptide and 14-3-3 was observed explaining the high affinity of the N protein for 14-3-3 proteins.
- MeSH
- COVID-19 MeSH
- fosfopeptidy chemie MeSH
- fosfoproteiny chemie MeSH
- koronavirové nukleokapsidové proteiny * chemie MeSH
- lidé MeSH
- proteiny 14-3-3 * chemie MeSH
- SARS-CoV-2 * MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
