In this work, two mixed-mode columns from a different manufacturers and one marketed as a reversed-phase column were characterized and compared in the terms of their interaction abilities, retentivity, peak symmetry, and applicability for peptide separation. All the tested columns contain octadecyl ligand and positively charged modifier, i.e. pyridyl group for the reversed-phase column XSelect CSH C18, quaternary alkylamine for mixed-mode column Atlantis PREMIER BEH C18 AX, and permanently charged moiety (details not available from the manufacturer) for mixed-mode column Luna Omega PS C18. For detailed characterization and comparison of their interaction potential, several approaches were used. First, a simple Walters test was performed to estimate hydrophobic and silanophilic interactions of the tested columns. The highest values of both parameters were observed for column Atlantis PREMIER BEH C18 AX. To investigate the effect of pH and buffer concentration on retention, mobile phases composed of acetonitrile and buffer (ammonium formate, pH 3.0; ammonium acetate pH 4.7 and pH 6.9) in various concentrations (5mM; 10mM; 15mM and 20mM) were used. The analysis of permanently charged compounds was used to describe the electrostatic interaction abilities of the stationary phases. The most significant contribution of electrostatic interactions to the retention was observed for Atlantis PREMIER BEH C18 AX column in the mobile phase with buffer of pH 3.0. A set of ten dipeptides, three pentapeptides and one octapeptide was used to investigate the effects of pH and buffer concentration on retention and peak symmetry. Each of the tested columns provides the optimal peak shape under different buffer pH and concentration. The gradient separation of the 14 tested peptides was used to verify the application potential of the tested columns for peptide separation. The best separation was achieved within 4 minutes on column Atlantis PREMIER BEH C18 AX.
The review presents a comprehensive survey of recent developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, micropreparation, and physicochemical and biochemical characterization of peptides since 2017 up to about the middle of 2019. Progress in the study of electromigration properties of peptides and in the methodology of their analysis (sample preseparation, preconcentration and derivatization, adsorption suppression, EOF control, and detection) are described. Advances in CE and CEC methods are demonstrated and their applications in the following areas are presented: qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. In addition, micropreparative separations and determinations of important physicochemical characteristics of peptides by CE and CEC methods are reported.
Continuous flow electrophoretic separation with continuous sample loading provides the advantage of processing volumes of any sizes, as well as the benefit of a real-time monitoring and optimization of the separation process. In addition, the spatial separation of the sample enables collecting multiple separated components simultaneously and in a continuous manner. The separation is usually performed in mild buffers without organic solvents and detergents (sample biological activity is retained) and it is carried out without usage of a solid support in the separation space preventing the interaction of the sample with it (high sample recovery). The method is used for the separation of proteins/peptides in proteomic applications, and its great applicability is to the separation of the cells, cellular organelles, vesicles, membrane fragments, and DNA. This review focuses on the electrophoretic separation performed in a continuous flow and it describes various electrophoretic modes and instrumental setups. Recent developments in methodology and instrumentation, the integration with other techniques, and the application to the biological sample analysis are discussed as well.
Peptide spectral libraries enable targeted identification and quantitation of low-abundance proteins in a complex plant proteome. Here we describe parallel protein and peptide fractionation techniques to improve plant proteome coverage and facilitate construction of spectral libraries.
Cyanobacteria represent a bacterial phyllum characteristic by the ability to photosynthesize. They are potentially applicable for the production of useful compounds but may also cause poisoning or at least health problems as they can produce cyanotoxins. The introduction of a fast methodology is important not only for fundamental taxonomic purposes, but also for reliable identifications in biological studies. In this work, we have used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact cells to study Chroococcidiopsis strains. A library of the obtained reference mass spectra containing characteristic peptide/protein profiles was examined by software tools to characterize similarities and differences applicable for diagnostics and taxonomy. Both a similarity tree and heat map constructed from the mass spectrometric data proved consistent with 16S rRNA sequencing results. We show as novelty that a binary matrix combining ferulic and sinapinic acids performs well in acquiring reproducible mass spectra of cyanobacteria. Using the matrix solvent, a protein extraction from cells was done. After polyacrylamide gel electrophoresis, the separated protein fractions were in-gel digested and the resulting peptides analyzed by liquid chromatography coupled with tandem mass spectrometry. For the first time, photosystem protein components, phycobilisome proteins, electron transport proteins, nitrogen-metabolism and nucleic acids binding-proteins, cytochromes plus other enzymes and various uncharacterized proteins could be assigned to characteristic peaks in the mass spectrometric profiles and some of them suggested as markers in addition to 30S and 50S ribosomal proteins known from previous studies employing intact cell mass spectrometry of microorganisms.
- MeSH
- bakteriální proteiny analýza izolace a purifikace MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fylogeneze MeSH
- peptidy analýza izolace a purifikace MeSH
- RNA ribozomální 16S genetika MeSH
- sinice chemie klasifikace genetika MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The review brings a comprehensive overview of recent developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) to analysis, microscale isolation, purification, and physicochemical and biochemical characterization of peptides in the years 2015, 2016, and ca. up to the middle of 2017. Advances in the investigation of electromigration properties of peptides and in the methodology of their analysis (sample preseparation, preconcentration and derivatization, adsorption suppression and EOF control, and detection) are described. New developments in particular CE and CEC methods are presented and several types of their applications to peptide analysis are reported: qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. Some micropreparative peptide separations are shown and capabilities of CE and CEC methods to provide important physicochemical characteristics of peptides are demonstrated.
- MeSH
- aminokyseliny chemie MeSH
- chemická frakcionace MeSH
- elektroforéza kapilární metody MeSH
- lidé MeSH
- mikročipová analýza metody MeSH
- peptidové mapování MeSH
- peptidy analýza izolace a purifikace MeSH
- stereoizomerie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Eight beetle species of the superfamily Scarabaeoidea were investigated with respect to peptides belonging to the adipokinetic hormone (AKH) family in their neurohemal organs, the corpora cardiaca (CC). The following beetle families are represented: Scarabaeidae, Lucanidae, and Geotrupidae. AKH peptides were identified through a heterospecific trehalose-mobilizing bioassay and by sequence analyses, using liquid chromatography coupled to positive electrospray mass spectrometry (LC-ESI-MS) and analysis of the tandem MS2 spectra obtained by collision-induced dissociation. All the beetle species have octapeptide AKHs; some have two AKHs, while others have only one. Novel AKH members were found in Euoniticellus intermedius and Circellium bacchus (family Scarabaeidae), as well as in Dorcus parallelipipedus (family Lucanidae). Two species of the family Geotrupidae and two species of the Scarabaeidae subfamily Cetoniinae contain one known AKH peptide, Melme-CC, while E. intermedius produces a novel peptide code named Euoin-AKH: pEINFTTGWamide. Two AKH peptides were each identified in CC of C. bacchus and D. parallelipipedus: the novel Cirba-AKH: pEFNFSAGWamide and the known peptide, Scade-CC-I in the former, and the novel Dorpa-AKH: pEVNYSPVW amide and the known peptide, Melme-CC in the latter. Kheper bonelli (subfamily Scarabaeinae) also has two AKHs, the known Scade-CC-I and Scade-CC-II. All the novel peptides were synthesized and the amino acid sequence assignments were unequivocally confirmed by co-elution of the synthetic peptides with their natural equivalent, and identical MS parameters of the two forms. The novel synthetic peptides are all active in inducing hypertrehalosemia in cockroaches.
- MeSH
- brouci chemie genetika MeSH
- chromatografie kapalinová MeSH
- hmotnostní spektrometrie MeSH
- hmyzí hormony chemie genetika izolace a purifikace MeSH
- kyselina pyrrolidonkarboxylová analogy a deriváty chemie izolace a purifikace MeSH
- oligopeptidy chemie genetika izolace a purifikace MeSH
- peptidy chemie genetika izolace a purifikace MeSH
- sekvence aminokyselin genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The applicability and predictive properties of the linear solvent strength model and two nonlinear retention-time models, i.e., the quadratic model and the Neue model, were assessed for the separation of small molecules (phenol derivatives), peptides, and intact proteins. Retention-time measurements were conducted in isocratic mode and gradient mode applying different gradient times and elution-strength combinations. The quadratic model provided the most accurate retention-factor predictions for small molecules (average absolute prediction error of 1.5%) and peptides separations (with a prediction error of 2.3%). An advantage of the Neue model is that it can provide accurate predictions based on only three gradient scouting runs, making tedious isocratic retention-time measurements obsolete. For peptides, the use of gradient scouting runs in combination with the Neue model resulted in better prediction errors (<2.2%) compared to the use of isocratic runs. The applicability of the quadratic model is limited due to a complex combination of error and exponential functions. For protein separations, only a small elution window could be applied, which is due to the strong effect of the content of organic modifier on retention. Hence, the linear retention-time behavior of intact proteins is well described by the linear solvent strength model. Prediction errors using gradient scouting runs were significantly lower (2.2%) than when using isocratic scouting runs (3.2%).
- MeSH
- časové faktory MeSH
- chromatografie s reverzní fází * MeSH
- fenoly chemie izolace a purifikace MeSH
- molekulární modely MeSH
- molekulová hmotnost MeSH
- peptidy chemie izolace a purifikace MeSH
- proteiny chemie izolace a purifikace MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A group of peptides from the salivary gland of the tick Hyalomma marginatum rufipes, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, Haemaphysalis longicornis. We have evaluated the anti-serine protease activity of one of these H. marginatum peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic.
- MeSH
- agregace trombocytů účinky léků MeSH
- antikoagulancia chemie izolace a purifikace farmakologie MeSH
- hemokoagulace účinky léků MeSH
- klíšťata chemie MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- peptidy chemie izolace a purifikace farmakologie MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- trombin antagonisté a inhibitory metabolismus MeSH
- trombóza farmakoterapie MeSH
- vyšetření krevní srážlivosti MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
There is still a considerable need for development of new tools and methods detecting specific viral proteins for the diagnosis and pathogenesis study of the Yellow fever virus (YFV). This study aimed to develop and characterize polyclonal peptide antisera for detection of YFV-C and -NS1 proteins. The antisera were used further to investigate NS1 protein expression during YFV infection in mammalian cells. YFV target proteins were detected by all antisera in western blot and immunofluorescence assays. No cross-reactivity was observed with Dengue virus, West Nile virus, Tick-borne encephalitis virus and Japanese encephalitis virus. Nuclear localization of the YFV-C protein was demonstrated for the first time. Experiments investigating NS1 expression suggested a potential use of the YFV-NS1 antisera for development of diagnostic approaches targeting the secreted form of the NS1 protein. The antisera described in this study offer new possibilities for use in YFV research and for the development of novel diagnostic tests.
- MeSH
- antigeny virové analýza imunologie metabolismus MeSH
- buněčné jádro chemie virologie MeSH
- Cercopithecus aethiops MeSH
- diagnostické testy rutinní metody MeSH
- fluorescenční protilátková technika MeSH
- králíci MeSH
- morčata MeSH
- peptidy izolace a purifikace metabolismus MeSH
- protilátky virové imunologie izolace a purifikace metabolismus MeSH
- senzitivita a specificita MeSH
- Vero buňky MeSH
- virologie metody MeSH
- virové nestrukturální proteiny analýza imunologie metabolismus MeSH
- virové plášťové proteiny analýza imunologie metabolismus MeSH
- virus žluté zimnice imunologie izolace a purifikace MeSH
- western blotting MeSH
- zkřížené reakce MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- morčata MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
