Hydrogen/deuterium exchange connected with mass spectrometry is increasingly applied for the interrogation of protein conformation, mapping protein dynamics, identification of protein-ligand interaction sites, and allosteric conformation changes. The dynamics of protein changes is determined with the m/z value of deuterated and non-deuterated protein or percentage of deuterium incorporation for the digested peptides. Hydrogen/deuterium exchange data are processed with selected software and finally quaternary structure of protein is visualized showing how different protein and ligand chains hook up with each other. Obtained results can help understand how protein interacts with its ligand and elucidate the role of this complex in a living organism. Utilization of this method is demonstrated by the amyloid-beta peptide aggregation associated with Alzheimer´s disease; determination of the structure toxin-co-regulated pili Vibrio cholerae in connection with its pathogenesis or revelation of binding sites on Mouse double minute 2 homolog complex with small molecule Nutlin-3 which is important for elucidation of drug effects in cancer research.
- MeSH
- alosterické místo účinky léků MeSH
- Alzheimerova nemoc etiologie MeSH
- hmotnostní spektrometrie * metody využití MeSH
- konformace proteinů MeSH
- lidé MeSH
- ligandy MeSH
- mapování interakce mezi proteiny * využití MeSH
- nádory MeSH
- peptidové mapování metody využití MeSH
- simulace molekulární dynamiky využití MeSH
- vazba proteinů * MeSH
- výzkum MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Aortic stenosis is one of the most common heart diseases that occur in developed countries. The disease has many causes; among the most discussed is excessive intake of fluorides. The aim of our work is to clarify the biochemical nature of biomineralizations in cardial tissue as well as to select an appropriate medication and to mitigate the disease. Peptide mapping combined with UHPLC and MS were used in the study of the mineralized cardial tissue. We managed to identify proteins such as alkaline phosphatase, biglycan, mimecan, osteopontin, periostin and proteoglycan, which are probably related to mineralization of the valves.
- MeSH
- aortální stenóza * etiologie MeSH
- chromatografie kapalinová využití MeSH
- fluor škodlivé účinky MeSH
- hmotnostní spektrometrie * metody přístrojové vybavení využití MeSH
- kalcinóza * patofyziologie MeSH
- lidé MeSH
- peptidové mapování * metody trendy využití MeSH
- tandemová hmotnostní spektrometrie využití MeSH
- vitamin K chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Klíčová slova
- diagnostika, fingerprint, agregáty,
- MeSH
- adenokarcinom diagnóza genetika MeSH
- elektroforéza metody statistika a číselné údaje využití MeSH
- financování organizované MeSH
- lidé MeSH
- metalothionein analýza diagnostické užití MeSH
- nádory prostaty diagnóza genetika MeSH
- peptidové mapování metody statistika a číselné údaje využití MeSH
- sérum MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.
- MeSH
- chromatografie afinitní MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- mikrochemie metody MeSH
- peptidové mapování metody MeSH
- proteinové hydrolyzáty chemie MeSH
- proteinurie moč MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- trypsin izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Sturgeon and paddlefish (Acipenseriformes), the source of roe consumed as caviar, are a unique and commercially valuable group of ancient fishes. In this study, comparative proteomics was used to analyze protein profiles of spermatozoa from five sturgeon species and one paddlefish: Siberian sturgeon (Acipenser baerii), sterlet (A. ruthenus), Russian sturgeon (A. gueldenstaedtii), starry sturgeon (A. stellatus), beluga (Huso huso), and Mississippi paddlefish (Polyodon spathula). Protein profiles of spermatozoa were determined by isoelectric focusing and two-dimensional electrophoresis (2-DE) high-resolution gels. The peptides, previously selected by 2-DE analysis as potentially species-specific, were obtained by "in-gel" tryptic digestion, followed by matrix-associated laser desorption/ionization time-of-flight/mass spectrometry (MALDI-TOF/MS). Among the 23 protein spots selected, 14 were identified as isoforms of enolase B present in all species, but with different isoelectric points or molecular mass. Exceptions were A. ruthenus and H. huso, species with a close phylogenetic relationship. Glycerol-3-phosphate dehydrogenase was detected exclusively in P. spathula. Phosphoglycerate kinase was detected only in A. ruthenus and H. huso, and 3 additional proteins (fructose bisphosphate aldolase A-2, glycogen phosphorylase type IV and glyceraldehyde-3-phosphate dehydrogenase) were found exclusively in A. gueldenstaedtii and H. huso. This study points to the application of proteomics for differential characterization and comparative studies of acipenseriform species at the molecular level.
- MeSH
- 2D gelová elektroforéza metody MeSH
- aldolasa genetika metabolismus MeSH
- biologické markery metabolismus MeSH
- druhová specificita MeSH
- fosfoglycerátkinasa genetika metabolismus MeSH
- fosfopyruváthydratasa genetika metabolismus MeSH
- glycerolfosfátdehydrogenasa genetika metabolismus MeSH
- isoelektrická fokusace metody MeSH
- izoenzymy genetika metabolismus MeSH
- peptidové mapování metody MeSH
- peptidy genetika metabolismus MeSH
- proteomika metody MeSH
- ryby klasifikace genetika metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- spermie metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Geografické názvy
- Mississippi MeSH
- Sibiř MeSH
- MeSH
- elektroforéza metody přístrojové vybavení využití MeSH
- enzymy imobilizované analýza MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody přístrojové vybavení MeSH
- peptidové mapování metody přístrojové vybavení využití MeSH
- proteiny analýza MeSH
- trypsin analýza MeSH
- vazba proteinů genetika MeSH
Práce se zabývá možnostmi určení lidského věku na základě poznatku o stereospecifickém štěpení proteinů, obsahujících D-formy kyseliny asparagové. Stereospecifita enzymatického štěpení předpokládá, že po enzymatické hydrolýze vzniknou peptidové štěpy o různé molekulové hmotnosti - tzv. peptidová mapa, podle toho, kolik D-aspartylových zbytků protein obsahuje. V této předběžné studii jsme prokázali u různě starých osob vznik rozdílně velkých štěpů v nekolagenních proteinech lidského dentinu, který byl hydrolyzován proteázou V8 ze Staphylococcus aureus.
The submitted paper deals with possibilities of assessment of human age based on findings of stereospecific breakdown of proteins containing D-forms of aspartic acid.The stereospecificity of enzymatic breakdown assumes that after enzymatic hydrolysis peptide breakdown products with different molecular weights - the so-called peptide map - will be formed, depending how many D-aspartyl residues the protein contains. The authors proved in the submitted preliminary study in subjects of different age the formation of breakdown products of different size in non-collagenous proteins of human dentin which was hydrolyzed by protease V8 fi*om Staphylococcus aureus.
