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
- Názvy látek
- proteinové hydrolyzáty MeSH
- trypsin 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
- Názvy látek
- aldolasa MeSH
- biologické markery MeSH
- fosfoglycerátkinasa MeSH
- fosfopyruváthydratasa MeSH
- glycerolfosfátdehydrogenasa MeSH
- izoenzymy MeSH
- peptidy MeSH
Separation of peptides and proteins by capillary zone electrophoresis suffers from the interaction of these solutes with the capillary wall which results in the formation of broad peaks and low resolution. To minimize the protein/peptide-capillary wall interaction we tried to use Pluronic F127, a triblock copolymer of the general formula (polyethylene oxide)(x)(polypropylene oxide)(y)(polyethylene oxide)(z) when x=106, y=70 and z=106 which can be considered a surfactant capable of self-association both into isotropic and anisotropic gels. The analytes studied were enzymatic digests (obtained by trypsin or pepsin treatment) of insoluble matrix proteins from avian eggshell. The best separations were obtained by a system exploiting 10% Pluronic F127 in 20 mmol/l phosphate buffer, pH 2.5. Electrophoretic peptide profiles obtained were very complex owing to the complicated nature of the samples (the exact composition of the proteinous insoluble part of the eggshell is still unknown). The separation in phosphate buffer only offered complex maps of incompletely resolved peaks. The use of Pluronic F127 distinctly improved the separation with a considerably better resolution regarding both the number of peaks obtained and the quality of the separation.
- MeSH
- elektroforéza kapilární MeSH
- hydrolýza MeSH
- pepsin A chemie MeSH
- peptidové mapování metody MeSH
- poloxamer chemie MeSH
- povrchově aktivní látky MeSH
- pufry MeSH
- trypsin chemie MeSH
- vaječné proteiny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- pepsin A MeSH
- poloxamer MeSH
- povrchově aktivní látky MeSH
- pufry MeSH
- trypsin MeSH
- vaječné proteiny MeSH
An overview of the recent developments in the applications of high-performance capillary electromigration methods, namely zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography, to analysis, preparation, and physicochemical characterization of peptides is presented. New approaches to the theoretical description and experimental verification of the electromigration behavior of peptides and the methodological aspects of capillary electroseparations of peptides, such as rational selection of separation conditions, sample treatment, and suppression of adsorption, are discussed, and new developments in individual separation modes and new designs of detection systems applied to peptide separations are shown. Several types of applications of capillary electromigration methods to peptide analysis are presented: quality control and purity tests, determination in biomatrices, monitoring of physical and chemical changes and enzymatic conversions, amino acid and sequence analysis, and peptide mapping. The examples of micropreparative peptide separations are given and capabilities of capillary electromigration techniques to provide important physicochemical characteristics of peptides are demonstrated.
- MeSH
- chromatografie MeSH
- elektroforéza kapilární MeSH
- hmotnostní spektrometrie MeSH
- isoelektrická fokusace MeSH
- lidé MeSH
- peptidové mapování metody MeSH
- peptidy analýza MeSH
- spektrofotometrie ultrafialová MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- peptidy MeSH
