Cheeses are a group of fermented dairy products that are produced all over the world in various forms and flavours. Milk, especially sheep or goat milk, is still regarded as an expensive raw material in the world, which makes milk and milk products highly attractive as a fraud target. Most often, such fraud includes partial or complete substitution with cheaper sorts of milk (e.g. bovine milk). The aim of this work was to verify the authenticity of 27 cheeses commonly emerging on the Czech food market. The cheeses were distinguished on the basis of milk animal species origin. For this purpose, two mass spectrometry techniques were used: matrix-assisted laser desorption/ionization with time of flight mass spectrometry together with principal component analysis method and liquid chromatography coupled with electrospray ionization and quadrupole time-of-flight mass spectrometry. The results were a partial success, because the cheeses could only be partially distinguished with the first mass spectrometry technique probably because of the influence of some protein additive materials in cheeses. The second technique allowed for collecting higher quality results and thus appears to be highly suitable for the research task.
Hydrophobins are small proteins that play a role in a number of processes during the filamentous fungi growth and development. These proteins are characterized by the self-assembly of their molecules into an amphipathic membrane at hydrophilic-hydrophobic interfaces. Isolation and purification of hydrophobins generally present a challenge in their analysis. Hydrophobin SC3 from Schizophyllum commune was selected as a representative of class I hydrophobins in this work. A novel procedure for selective and effective isolation of hydrophobin SC3 based on solid-phase extraction with polytetrafluoroethylene microparticles loaded in a small self-made microcolumn is reported. The tailored binding of hydrophobins to polytetrafluoroethylene followed by harsh elution conditions resulted in a highly specific isolation of hydrophobin SC3 from the model mixture of ten proteins. The presented isolation protocol can have a positive impact on the analysis and utilization of these proteins including all class I hydrophobins. Hydrophobin SC3 was further subjected to reduction of its highly stable disulfide bonds and to chymotryptic digestion followed by mass spectrometric analysis. The isolation and digestion protocols presented in this work make the analysis of these highly hydrophobic and compact proteins possible.
- MeSH
- albuminy chemie MeSH
- ananasovník chemie MeSH
- bromelainy chemie MeSH
- Canavalia chemie MeSH
- chymotrypsin chemie MeSH
- cytochromy c chemie MeSH
- disulfidy chemie MeSH
- erytrocyty enzymologie MeSH
- extrakce na pevné fázi metody MeSH
- hmotnostní spektrometrie metody MeSH
- karboanhydrasy chemie MeSH
- kaseiny chemie MeSH
- koně MeSH
- konkanavalin A chemie MeSH
- kur domácí MeSH
- lidé MeSH
- mikrosféry * MeSH
- mléko enzymologie MeSH
- myokard metabolismus MeSH
- polytetrafluoroethylen chemie MeSH
- proteomika metody MeSH
- Schizophyllum chemie MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- tandemová hmotnostní spektrometrie MeSH
- thermolysin chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Proteome modifications in a biological fluid can potentially indicate the occurrence of pathologies, even if the identification of a proteome fingerprint correlated to a specific disease represents a very difficult task. When a nanomaterial is introduced into a biological fluid, macromolecules compete to form a protein corona on the nanoparticle surface, and depending on the specific proteome, different patterns of proteins will form the final protein corona shell depending on their affinity for the nanoparticle surface. Novel surface active maghemite nanoparticles (SAMNs) display a remarkable selectivity toward protein corona formation, and they are able to concentrate proteins and peptides presenting high affinities for their surface even if they are present in very low amounts. Thus, SAMNs may confer visibility to hidden biomarkers correlated to the occurrence of a pathology. In the present report, SAMNs were introduced into milk samples from healthy cows and from animals affected by mastitis, and the selectively bound protein corona shell was easily analyzed and quantified by gel electrophoresis and characterized by mass spectrometry. Upon incubation in mastitic milk, SAMNs were able to selectively bind αs2-casein fragments containing the FALPQYLK sequence, as part of the larger casocidin-1 peptide with strong antibacterial activity, which were not present in healthy samples. Thus, SAMNs can be used as a future candidate for the rapid diagnosis of mastitis in bovine milk. The present report proposes protein competition for SAMN protein corona formation as a means of mirroring proteome modifications. Thus, the selected protein shell on the nanoparticles results in a fingerprint of the specific pathology.
- MeSH
- biologické markery chemie metabolismus MeSH
- hmotnostní spektrometrie metody MeSH
- kaseiny chemie metabolismus MeSH
- kovové nanočástice chemie MeSH
- mastitida diagnóza metabolismus MeSH
- mléčné bílkoviny chemie metabolismus MeSH
- mléko chemie metabolismus MeSH
- povrchové vlastnosti MeSH
- proteinová korona chemie metabolismus MeSH
- proteom chemie metabolismus MeSH
- proteomika metody MeSH
- reprodukovatelnost výsledků MeSH
- sekvence aminokyselin MeSH
- senzitivita a specificita MeSH
- skot MeSH
- železité sloučeniny chemie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hlavní mléčné proteiny − kaseiny a syrovátkové proteiny − jsou vysoce kvalitní bílkoviny, jejichž podání vyvolává inzulínovou sekreci, aniž by vedlo k vzestupu postprandiální glykémie. Předpokládá se, že inzulínovou sekreci zvyšují přímým účinkem některých zejména větvených aminokyselin na β-buňku Langerhansových ostrůvků pankreatu spolu s aktivací inkretinových hormonů. Pro rozdílné složení, trávení a vstřebávání aminokyselin dosahují syrovátkové proteiny ve srovnání s kaseiny vyššího inzulinotropního efektu.
Major milk proteins − casein and whey proteins − are high-quality proteins with unique insulinotropic property that do not increase postprandial blood glucose upon administration. It is believed that these proteins increase insulin secretion by both a direct effect of particulary branched chain amino acids on β-cell of islets of Langerhans in pancreas and also by activation of incretin hormones. However, due to a different amino acid profile and digestion, whey proteins achieve higher insulinotropic effect in comparison with casein proteins.
- Klíčová slova
- zdraví jedinci,
- MeSH
- beta-buňky fyziologie MeSH
- fyziologie výživy fyziologie MeSH
- inkretiny fyziologie MeSH
- inzulin lidský sekrece MeSH
- inzulin * sekrece MeSH
- kaseiny * farmakologie chemie MeSH
- klinické zkoušky jako téma statistika a číselné údaje MeSH
- krevní glukóza fyziologie MeSH
- lidé MeSH
- mléčné bílkoviny * farmakologie chemie MeSH
- postprandiální období fyziologie MeSH
- větvené aminokyseliny fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy 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
Magnetic bead cellulose activated with divinyl sulfone was used for the immobilization of Staphylococcus aureus endoproteinase Glu-C (EC 3.4.21.19). The immobilized proteinase was characterized by increased thermostability, by decreased self-cleavage activity, and a possibility of repeated use. The prepared immobilized enzyme was applied for the proteolytic cleavage of α-casein and BSA under different conditions (different composition of buffers, different pH, and different time of digestion). The possibilities of the direct use of enzyme reaction products for MALDI TOF MS analysis were shown.
- MeSH
- bakteriální proteiny chemie MeSH
- celulosa chemie MeSH
- enzymy imobilizované chemie MeSH
- kaseiny chemie MeSH
- koncentrace vodíkových iontů MeSH
- magnetismus MeSH
- proteomika přístrojové vybavení metody MeSH
- serinové endopeptidasy chemie MeSH
- sérový albumin hovězí chemie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- stabilita enzymů MeSH
- Staphylococcus aureus enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.
- MeSH
- design vybavení MeSH
- isoelektrická fokusace přístrojové vybavení metody MeSH
- kaseiny chemie MeSH
- peptidové fragmenty analýza chemie izolace a purifikace MeSH
- peptidy analýza izolace a purifikace MeSH
- proteiny analýza izolace a purifikace MeSH
- skot MeSH
- trypsin chemie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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
In this work, electrophoretically mediated microanalysis (EMMA) was applied to the in-capillary tryptic digestion of proteins for proteomic purposes. Compared with classical in-solution tryptic digestion or the trypsin reactor commonly used for this purpose, the EMMA-based method is rapid, can be automated and requires only a small amount of trypsin preparation. Moreover, the protein digestion and the analysis of the resulting peptides are integrated into one procedure. A combination of the EMMA methodology with a partial filling technique was used in this study, since the pH optimum of the trypsin reaction differs strongly from the best pH for the CZE separation of peptides. In this set-up, a part of the capillary is filled with the best buffer for the tryptic digestion (50 mM Tris-HCl buffer, pH 8.5) whereas the rest of the capillary is filled with the BGE optimal for peptide separation (0.1 M phosphate buffer, pH 2.5). As the proteins differ in their isoelectric points, a sandwich type of injection was used. The analysed protein is thus injected between two trypsin zones, which ensures their mixing and digestion. The analysis of one protein comprising both the digestion and the peptide separation is then completed in 1 h using a commercial instrument for CE with no modifications.
