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.

• Klíčová slova
• Enrichment, Forcespinning, Nanofibers, Phosphopeptides,
• MeSH
• fosfopeptidy izolace a purifikace   MeSH
• nanovlákna * MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• titan chemie   MeSH
• zirkonium chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfopeptidy MeSH
• titan MeSH
• titanium dioxide MeSH Prohlížeč
• zirconium oxide MeSH Prohlížeč
• zirkonium MeSH

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
• Názvy látek
• ferric oxide MeSH Prohlížeč
• fosfopeptidy MeSH
• hydroxyapatit MeSH
• kaseiny MeSH
• syntetické pryskyřice MeSH
• železité sloučeniny MeSH

We report substantial in-situ enrichment of phosphopeptides in peptide mixtures using titanium and zirconium dioxide-coated matrix assisted laser desorption-ionization (MALDI) plates prepared by recently reported ambient ion landing deposition technique. The technique was able to modify four common materials currently used for MALDI targets (stainless steel, aluminum, indium-tin oxide glass and polymeric anchor chip). The structure of the deposited dioxide was investigated by electron microscopy, and different surfaces were compared and discussed in this study. Two standard proteins were used to test the enrichment capabilities of modified MALDI plates: casein and in-vitro phosphorylated trehalase. The enrichment of casein tryptic digest resulted in identification of 20 phosphopeptides (including miscleavages). Trehalase was used as a suitable model of larger protein that provided more complex peptide mixture after the trypsin digestion. All four possible phosphorylation sites in trehalase were identified and up to seven phosphopetides were found (including methionine oxidations and miscleavages). Two different mass spectrometers, MALDI-Fourier transform ion cyclotron resonance (FTICR) and MALDI-time of flight, were used to detect the phosphopeptides from modified MALDI plates after the enrichment procedure. It was observed that the desorption-ionization phenomena on the modified surfaces are not critically influenced by the parameters of the different MALDI ion sources (e.g. different pressure, different extraction voltages), and thus the presence of dioxide layer on the standard MALDI plate does not significantly interfere with the main MALDI processes. The detection of phosphopeptides after the enrichment could be done by both instruments. Desorption electrospray ionization coupled to the FTICR was also tested, but, unlike MALDI, it did not provide satisfactory results.

• MeSH
• chemické modely MeSH
• fosfopeptidy chemie   izolace a purifikace   MeSH
• fosforylace MeSH
• kaseiny chemie   MeSH
• molekulární sekvence - údaje MeSH
• peptidové fragmenty chemie   izolace a purifikace   MeSH
• sekvence aminokyselin MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice přístrojové vybavení   metody   MeSH
• trehalasa chemie   MeSH
• trypsin chemie   MeSH
• zirkonium chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfopeptidy MeSH
• kaseiny MeSH
• peptidové fragmenty MeSH
• trehalasa MeSH
• trypsin MeSH
• zirconium oxide MeSH Prohlížeč
• zirkonium 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.

• Klíčová slova
• Enrichment, Iron oxide nanoparticles, Monolithic column, Phosphopeptides,
• 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
• Názvy látek
• ferric oxide MeSH Prohlížeč
• fosfopeptidy MeSH
• kaseiny MeSH
• polymery MeSH
• železité sloučeniny 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
• Názvy látek
• fosfopeptidy MeSH
• iminodiacetic acid MeSH Prohlížeč
• iminokyseliny MeSH
• polyhydroxyethylmethakrylát MeSH