Oil bodies, lipid-storage organelles, are stabilized by a number of specific proteins. These proteins are very hydrophobic, which complicates their identification by "classical" proteomic protocols using trypsin digestion. Due to the lack of trypsin cleavage sites, the achievable protein coverage is limited or even insufficient for reliable protein identification. To identify such proteins and to enhance their coverage, we introduced a modified method comprising standard three-step procedure (SDS-PAGE, in-gel digestion, and LC-MS/MS analysis). In this method, chymotrypsin, single or in combination with trypsin, was used, which enabled to obtain proteolytic peptides from the hydrophobic regions and to identify new oil bodies' proteins. Our method can be easily applied to identification of other hydrophobic proteins.

• MeSH
• Arabidopsis metabolismus   MeSH
• chromatografie kapalinová MeSH
• chymotrypsin metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• hmotnostní spektrometrie MeSH
• hydrofobní a hydrofilní interakce * MeSH
• lipidy * MeSH
• organely metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• proteomika metody   MeSH
• trypsin metabolismus   MeSH
• Publikační typ
• časopisecké články 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.

• MeSH
• cytochromy c chemie   MeSH
• elektroforéza kapilární metody   MeSH
• inzulin chemie   MeSH
• kaseiny chemie   MeSH
• peptidové fragmenty chemie   MeSH
• proteiny chemie   MeSH
• proteomika metody   MeSH
• trypsin chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300Å pores, packed in 2.1×30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using Nα-Benzoyl-l-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37°C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented.

• MeSH
• chromatografie kapalinová * přístrojové vybavení   metody   MeSH
• enzymy imobilizované * chemie   metabolismus   MeSH
• trypsin * chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

Plant seed oil bodies, subcellular lipoprotein inclusions providing storage reserves, are composed of a neutral lipid core surrounded by a phospholipid monolayer with several integrated proteins that play a significant role in stabilization of the particles and probably also in lipid mobilization. Oil bodies' proteins are generally very hydrophobic, due to the long uncharged sequences anchoring them into the lipid core, which makes them extremely difficult to handle and to digest successfully. Although oil bodies have been intensively studied during last decades, not all their proteins have been identified yet. To overcome the problems connected with their identification, a method based on SDS-PAGE, in-gel digestion and LC-MS/MS analysis was used. Digestion was carried out with trypsin and chymotrypsin, single or in combination, which increased significantly the number of identified peptides, namely the hydrophobic ones. Thanks to this methodology it was possible to achieve an extensive coverage of proteins studied, to analyze their N-terminal modifications and moreover, to detect four new oil bodies' protein isoforms, which demonstrates the complexity of oil bodies' protein composition.

• MeSH
• Arabidopsis chemie   metabolismus   MeSH
• chromatografie kapalinová MeSH
• chymotrypsin chemie   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• hmotnostní spektrometrie MeSH
• molekulární sekvence - údaje MeSH
• oleje rostlin chemie   MeSH
• peptidové fragmenty analýza   chemie   MeSH
• protein - isoformy chemie   klasifikace   izolace a purifikace   MeSH
• proteiny huseníčku chemie   izolace a purifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza proteinů MeSH
• semena rostlinná chemie   metabolismus   MeSH
• trypsin chemie   MeSH
• vakuoly chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Trypsin is the protease of choice for protein sample digestion in proteomics. The most typical active forms are the single-chain β-trypsin and the two-chain α-trypsin, which is produced by a limited autolysis of β-trypsin. An additional intra-chain split leads to pseudotrypsin (ψ-trypsin) with three chains interconnected by disulfide bonds, which can be isolated from the autolyzate by ion-exchange chromatography. Based on experimental data with artificial substrates, peptides, and protein standards, ψ-trypsin shows altered kinetic properties, thermodynamic stability and cleavage site preference (and partly also cleavage specificity) compared to the above-mentioned proteoforms. In our laboratory, we have analyzed the performance of bovine ψ-trypsin in the digestion of protein samples with a different complexity. It cleaves predominantly at the characteristic trypsin cleavage sites. However, in a comparison with common tryptic digestion, non-specific cleavages occur more frequently (mostly after the aromatic residues of Tyr and Phe) and more missed cleavages are generated. Because of the preferential cleavages after the basic residues and more developed side specificity, which is not expected to occur for the major trypsin forms (but may appear anyway because of their autolysis), ψ-trypsin produces valuable information, which is complementary in part to data based on a strictly specific trypsin digestion and thus can be unnoticed following common proteomics protocols.

• MeSH
• autolýza MeSH
• kinetika MeSH
• protein - isoformy chemie   metabolismus   MeSH
• proteolýza MeSH
• skot MeSH
• stabilita enzymů MeSH
• trypsin chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

• Konspekt
• Lékařské vědy. Lékařství
• NLK Obory
• gastroenterologie
• fyziologie
• patologie

• MeSH
• chymotrypsin analýza   MeSH
• gnotobiologické modely MeSH
• infekce vyvolané Escherichia coli MeSH
• psi MeSH
• střevní sliznice cytologie   enzymologie   mikrobiologie   MeSH
• trávení analýza   MeSH
• trypsin analýza   MeSH
• Check Tag
• psi MeSH

For the understanding of pathological states of bone tissues in oral surgery, it would be desirable to have the possibility to simulate these processes on bone cell models in vitro. These cultures, similarly to bone tissues, contain numerous proteins entrapped in the insoluble matrix. The major goal of this study was to verify whether a method based on direct in-matrix protein digestion could be suitable for the discrimination between different induced pathological states of bone cell models cultivated in vitro. Using in-sample specific protein digestion with trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released peptides, 446 proteins (in average per sample) were identified in a bone cell in vitro model with induced cancer, 440 proteins were found in a model with induced inflammation, 451 proteins were detected in control in vitro culture, and 491 proteins were distinguished in samples of vestibular laminas of maxillary bone tissues originating from six different patients. Subsequent partial least squares - discrimination analysis of obtained liquid chromatography-tandem mass spectrometry data was able to discriminate among in vitro cultures with induced cancer, with induced inflammation, and control cultivation. Thus, the direct in-sample protein digestion by trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released specific peptide fragments from the insoluble matrix and mathematical analysis of the mass spectrometry data seems to be a promising tool for the routine proteomic characterization of in vitro human bone models with induced different pathological states.

• MeSH
• chromatografie kapalinová metody   MeSH
• lidé MeSH
• peptidy analýza   MeSH
• proteiny chemie   MeSH
• proteolýza MeSH
• proteomika metody   MeSH
• stomatochirurgické výkony * MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• trypsin chemie   MeSH
• zánět MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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

The major objective of proteomics is to identify and examine the large numbers of proteins extracted from complex biological systems. This is generally achieved by combining various techniques of protein separation with a mass spectrometric analysis of proteins that are digested enzymatically. Recently, several alternatives to this standard protocol have been developed for efficient and fast protein digestion. One option is the use of modified trypsin instead of native trypsin for the in-gel digestion of proteins. Microwave, ultrasonic-assisted protein enzymatic digestion and proteolysis accelerated by infrared radiation are other suitable alternatives. The application of the variable performance of the fast enzymatic digestion of proteins by using different techniques is reported here. The advantage of these methods is to have the ability to detect proteins in a shorter span of time. For example, using alternative protein digestion takes only minutes, in contrast to the several hours required by conventional methods. To demonstrate the suitability of this fast procedure, the digestion of carbonic anhydrase, bovine serum albumin, lysozyme and proteins extracted from plants (Hordeum vulgare, Arabidopsis thaliana) were used. Considering that the required reaction time for the conventional method is much longer, these applied methodic approaches tend to give in-gel digestion a much higher efficiency rating. This study examines the fast, efficient and low-cost proteolytic strategies for the digestion process, and for protein identification based on the use of ultrasound and infrared technology. In addition, comparisons of the applied techniques were studied. Several differences were found, suggesting the potential use of proteolysis accelerated by infrared radiation.

• MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• molekulární sekvence - údaje MeSH
• peptidové fragmenty analýza   chemie   metabolismus   MeSH
• proteiny analýza   chemie   metabolismus   MeSH
• proteomika metody   MeSH
• rostlinné proteiny MeSH
• sekvence aminokyselin MeSH
• skot MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• trypsin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH