- MeSH
- antigeny fungální diagnostické užití krev MeSH
- beta-glukany diagnostické užití krev MeSH
- biologické markery * krev MeSH
- bronchoalveolární lavážní tekutina cytologie mikrobiologie MeSH
- chromatografie afinitní metody přístrojové vybavení MeSH
- fungální polysacharidy krev MeSH
- invazivní plicní aspergilóza * diagnóza MeSH
- lidé MeSH
- mannany diagnostické užití krev MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
Synthetic heptapeptides containing D-amino acid residues and differing in the content of L-phenylalanine and L-tyrosine residues and their position (Val-D-Leu-Pro-Tyr-Phe-Val-D-Leu, Val-D-Leu-Pro-Tyr-Tyr-Val-D-Leu, Val-D-Leu-Pro-Phe-Tyr-Val-D-Leu) were immobilized to two types of carriers: glyoxal-activated magnetic agarose particles and CNBr-activated Sepharose. In both cases, peptides were immobilized via their terminal amino group. Immobilized peptides were used for the study of binding properties of two gastric aspartic proteases (porcine pepsin A and rat pepsin C). Porcine pepsin A was adsorbed to all studied peptide-modified magnetic carriers, while rat pepsin C interacted with immobilized ligands only slightly. Similar results were obtained in affinity chromatographic experiments using heptapeptides immobilized to Sepharose.
- MeSH
- chromatografie afinitní přístrojové vybavení metody MeSH
- kinetika MeSH
- krysa rodu rattus MeSH
- magnetismus MeSH
- pepsin A chemie MeSH
- peptidy chemie MeSH
- potkani Wistar MeSH
- prasata MeSH
- sefarosa chemie MeSH
- vazba proteinů MeSH
- žaludeční sliznice chemie enzymologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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
The interactions of pepsin with immobilized trivalent metal ions and the participation of the enzyme phosphate group in this process were investigated using high performance immobilized metal affinity chromatography. Two different sorbents were used: the newly prepared one, consisting of Ga(3+ )chelate of (6-amino-1-hydroxyhexane-1,1-diyl) bis(phosphonic acid) covalently bound to a methacrylate support (BP-Ga(3+)), and the commercial one, containing immobilized Fe(3+ )ions (POROS MC20-Fe(3+)). The comparison of the behavior of porcine pepsin A and its partially dephosphorylated form on both sorbents showed that both forms of pepsin were adsorbed under the same conditions. To eliminate the participation of free carboxyl groups in pepsin adsorption, both enzyme forms were modified by amidation or esterification. Native enzyme and its partially dephosphorylated form both with modified carboxyl groups differed in their interaction with immobilized Ga(3+ )and Fe(3+). Phosphorylated pepsin molecules with esterified carboxyl groups were adsorbed on both sorbents while nonphosphorylated ones with esterified carboxyl groups were not adsorbed.
- MeSH
- adsorpce MeSH
- chelátory chemie MeSH
- chromatografie afinitní metody přístrojové vybavení MeSH
- financování organizované MeSH
- fosforylace MeSH
- gadolinium chemie MeSH
- ionty MeSH
- kovy analýza MeSH
- lidé MeSH
- pepsin A analýza chemie MeSH
- polymery chemie MeSH
- prasata MeSH
- železo chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG)-Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach.
- MeSH
- bioreaktory MeSH
- časové faktory MeSH
- chromatografie afinitní metody přístrojové vybavení MeSH
- enzymy imobilizované chemie MeSH
- financování organizované MeSH
- lidé MeSH
- ligandy MeSH
- magnetismus MeSH
- metaloendopeptidasy chemie MeSH
- nanočástice chemie MeSH
- neurotensin analýza MeSH
- oxid křemičitý MeSH
- peptidy analýza chemie MeSH
- proteomika MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- serinové endopeptidasy chemie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- trypsin chemie izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- MeSH
- chemické techniky analytické metody normy MeSH
- chemie fyzikální metody normy MeSH
- chromatografie afinitní metody přístrojové vybavení využití MeSH
- chromatografie kapalinová metody využití MeSH
- klinické laboratorní techniky využití MeSH
- ligandy MeSH
- organické látky chemie izolace a purifikace normy MeSH
- pufry MeSH
Three stationary phases have been prepared for affinity liquid chromatography isolation and separation of porcine and human pepsin. The phases contain 3,5-diiodo-L-tyrosine (DIT) bound to the supports HEMA BIO VS, HEMA BIO E and EPOXY TOYOPEARL. These phases have been tested on a model sample of porcine pepsin A and applied to human pepsin. Fractions have been collected and the chymase activity determined in selected analyses. For affinity CE, capillaries have been prepared by modifying the wall with 3-aminopropyltriethoxysilane, followed either by direct binding of DIT, or by binding L-tyrosine that was subsequently iodated. The dissociation constant K(d) has been determined for the pepsin-DIT complex from the changes in the electrophoretic mobilities.
- MeSH
- chromatografie afinitní metody přístrojové vybavení MeSH
- dijodtyrosin chemie MeSH
- elektroforéza kapilární metody přístrojové vybavení MeSH
- finanční podpora výzkumu jako téma MeSH
- lidé MeSH
- oxid křemičitý chemie MeSH
- pepsin A izolace a purifikace MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
