Identification and evaluation of small changes in β-amyloid peptide (Aβ) levels in cerebrospinal fluid is of crucial importance for early detection of Alzheimer's disease. Microfluidic detection methods enable effective preconcentration of Aβ using magnetic microparticles coated with Aβ antibodies. Poly(glycidyl methacrylate) microspheres are coated with α-amino-ω-methoxy-PEG5000 /α-amino-ω-Boc-NH-PEG5000 Boc groups deprotected and NH2 succinylated to introduce carboxyl groups. Capillary electrophoresis with laser-induced fluorescence detection confirms the efficient capture of Aβ 1-40 peptides on the microspheres with immobilized monoclonal anti-Aβ 6E10. The capture specificity is confirmed by comparing Aβ 1-40 levels on the anti-IgG-immobilized particles used as a control.

• Klíčová slova
• CE-LIF detection, functionalization, magnetic, microspheres, β-amyloid peptides,
• MeSH
• adsorpce MeSH
• amyloidní beta-protein izolace a purifikace   MeSH
• chromatografie afinitní MeSH
• elektroforéza kapilární MeSH
• imunoglobulin G metabolismus   MeSH
• kyseliny karboxylové chemie   MeSH
• kyseliny polymethakrylové chemie   MeSH
• magnetické jevy MeSH
• mikrosféry * MeSH
• mikroskopie atomárních sil MeSH
• peptidové fragmenty izolace a purifikace   MeSH
• polyethylenglykoly chemie   MeSH
• skot MeSH
• termogravimetrie MeSH
• velikost částic MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amyloid beta-protein (1-40) MeSH Prohlížeč
• amyloidní beta-protein MeSH
• imunoglobulin G MeSH
• kyseliny karboxylové MeSH
• kyseliny polymethakrylové MeSH
• peptidové fragmenty MeSH
• polyethylenglykoly MeSH
• polyglycidyl methacrylate MeSH Prohlížeč

Protein or peptide sample losses could accompany all steps of the proteomic analysis workflow. We focused on suppression of sample adsorptive losses during sample storage in autosampler vials. We examined suppression capabilities of six different sample injection solutions and seven types of autosampler vial surfaces using a model sample (tryptic digest of six proteins, 1 fmol per protein). While the vial material did not play an essential role, the choice of appropriate composition of sample injection solution reduced adsorptive losses substantially. The combination of a polypropylene vial and solution of poly(ethylene glycol) (PEG) (0.001%) or a mixture of high concentrated urea and thiourea (6 M and 1 M) as injection solutions (both acidified with formic acid (FA) (0.1%)) provided the best results in terms of number of significantly identified peptides (p < 0.05). These conclusions were confirmed by analyses of a real sample with intermediate complexity (in-gel digest from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)). Addition of PEG into the real sample solution proved to prevent higher losses, concerning mainly hydrophobic peptides, during up to 48 h storage in the autosampler in comparison with a formic acid solution and even with a solution of highly concentrated urea and thiourea. Using PEG for several months was not accompanied by any adverse effect to the liquid chromatography system.

• MeSH
• adsorpce MeSH
• chromatografie kapalinová normy   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• formiáty MeSH
• hydrofobní a hydrofilní interakce MeSH
• močovina MeSH
• odběr biologického vzorku normy   MeSH
• peptidové fragmenty izolace a purifikace   normy   MeSH
• polyethylenglykoly MeSH
• proteiny chemie   MeSH
• průtoková injekční analýza normy   MeSH
• thiomočovina MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• formiáty MeSH
• formic acid MeSH Prohlížeč
• močovina MeSH
• peptidové fragmenty MeSH
• polyethylenglykoly MeSH
• proteiny MeSH
• thiomočovina MeSH

We report substantial in-situ enrichment of phosphopeptides in peptide mixtures using titanium and zirconium dioxide-coated matrix assisted laser desorption-ionization (MALDI) plates prepared by recently reported ambient ion landing deposition technique. The technique was able to modify four common materials currently used for MALDI targets (stainless steel, aluminum, indium-tin oxide glass and polymeric anchor chip). The structure of the deposited dioxide was investigated by electron microscopy, and different surfaces were compared and discussed in this study. Two standard proteins were used to test the enrichment capabilities of modified MALDI plates: casein and in-vitro phosphorylated trehalase. The enrichment of casein tryptic digest resulted in identification of 20 phosphopeptides (including miscleavages). Trehalase was used as a suitable model of larger protein that provided more complex peptide mixture after the trypsin digestion. All four possible phosphorylation sites in trehalase were identified and up to seven phosphopetides were found (including methionine oxidations and miscleavages). Two different mass spectrometers, MALDI-Fourier transform ion cyclotron resonance (FTICR) and MALDI-time of flight, were used to detect the phosphopeptides from modified MALDI plates after the enrichment procedure. It was observed that the desorption-ionization phenomena on the modified surfaces are not critically influenced by the parameters of the different MALDI ion sources (e.g. different pressure, different extraction voltages), and thus the presence of dioxide layer on the standard MALDI plate does not significantly interfere with the main MALDI processes. The detection of phosphopeptides after the enrichment could be done by both instruments. Desorption electrospray ionization coupled to the FTICR was also tested, but, unlike MALDI, it did not provide satisfactory results.

• MeSH
• chemické modely MeSH
• fosfopeptidy chemie   izolace a purifikace   MeSH
• fosforylace MeSH
• kaseiny chemie   MeSH
• molekulární sekvence - údaje MeSH
• peptidové fragmenty chemie   izolace a purifikace   MeSH
• sekvence aminokyselin MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice přístrojové vybavení   metody   MeSH
• trehalasa chemie   MeSH
• trypsin chemie   MeSH
• zirkonium chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfopeptidy MeSH
• kaseiny MeSH
• peptidové fragmenty MeSH
• trehalasa MeSH
• trypsin MeSH
• zirconium oxide MeSH Prohlížeč
• zirkonium MeSH

Rapid changes of protein phosphorylation play a crucial role in the regulation of many cellular processes. Being post-translationally modified, phosphoproteins are often present in quite low abundance and tend to co-exist with their unphosphorylated isoform within the cell. To make their identification more practicable, the use of enrichment protocols is often required. The enrichment strategies can be performed either at the level of phosphoproteins or at the level of phosphopeptides. Both approaches have their advantages and disadvantages. Most enriching strategies are based on chemical modifications, affinity chromatography to capture peptides and proteins containing negatively charged phosphate groups onto a positively charged matrix, or immunoprecipitation by phospho-specific antibodies.In this article, the most up-to-date enrichment techniques are discussed, taking into account their optimization, and highlighting their advantages and disadvantages. Moreover, these methods are compared to each other, revealing their complementary nature in providing comprehensive coverage of the phosphoproteome.

• MeSH
• barvení a značení MeSH
• chromatografie afinitní MeSH
• fosfoproteiny chemie   izolace a purifikace   metabolismus   MeSH
• fosforylace MeSH
• hmotnostní spektrometrie MeSH
• imunoprecipitace MeSH
• peptidové fragmenty chemie   izolace a purifikace   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• proteom chemie   izolace a purifikace   metabolismus   MeSH
• proteomika MeSH
• rostlinné proteiny chemie   izolace a purifikace   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• fosfoproteiny MeSH
• peptidové fragmenty MeSH
• proteom MeSH
• rostlinné proteiny MeSH

A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.

• MeSH
• design vybavení MeSH
• isoelektrická fokusace přístrojové vybavení   metody   MeSH
• kaseiny chemie   MeSH
• peptidové fragmenty analýza   chemie   izolace a purifikace   MeSH
• peptidy analýza   izolace a purifikace   MeSH
• proteiny analýza   izolace a purifikace   MeSH
• skot MeSH
• trypsin chemie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• caseinomacropeptide MeSH Prohlížeč
• kaseiny MeSH
• peptidové fragmenty MeSH
• peptidy MeSH
• proteiny MeSH
• trypsin MeSH

Open-tubular CEC (OT-CEC) with a new stationary phase, salophene-lanthanide-Zn(2+) complex, has been applied to the separation of tryptic peptides of native BSA and BSA glycated by glucose and ribose. Glycation of proteins (non-enzymatic modification by sugars) significantly affects their properties and it is of great importance from a physiological point of view. Separation of tryptic peptides of glycated BSA by CZE was poor because of their strong adsorption to the bare fused silica capillary. An improved separation of tryptic peptides of both native and glycated BSA was achieved by OT-CEC in the fused silica capillary non-covalently coated with salophene-lanthanide-Zn(2+) complex, which suppressed the adsorption of peptides to the capillary and via specific interactions with some (glyco)peptides enhanced selectivity of the separation. Significant differences have been found in OT-CEC analyses of tryptic hydrolysates of native and glycated BSA. In OT-CEC-UV profile of tryptic peptides of native BSA, 44 peaks could be resolved, whereas a reduced number of 38 peaks were observed in the profile of tryptic peptides of glucose-glycated BSA and only 30 peaks were found in the case of ribose-glycated BSA. The developed OT-CEC can be potentially used for monitoring of protein glycation.

• MeSH
• adsorpce MeSH
• elektroforéza kapilární metody   MeSH
• glukosa chemie   MeSH
• lanthanoidy chemie   MeSH
• peptidové fragmenty chemie   izolace a purifikace   MeSH
• salicylany chemie   MeSH
• sérový albumin hovězí chemie   MeSH
• trypsin chemie   MeSH
• zinek chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glukosa MeSH
• lanthanoidy MeSH
• peptidové fragmenty MeSH
• salicylany MeSH
• salophen MeSH Prohlížeč
• sérový albumin hovězí MeSH
• trypsin MeSH
• zinek MeSH

The optimized expression of recombinant Potato virus A coat protein (ACP) carrying two different epitopes from Human papillomavirus type 16 (HPV16) was developed. Epitope derived from minor capsid protein L2 was expressed as N-terminal fusion with ACP while an epitope derived from E7 oncoprotein was fused to its C-terminus. The construct was cloned into Potato X potexvirus (PVX) based vector and transiently expressed in plants using Agrobacterium tumefaciens mediated inoculation. To increase the level of expressed protein the transgenic Nicotiana benthamiana plants expressing Potato virus A HC-Pro gene and transgenic Nicotiana tabacum, cv. Petit Havana SR1 carrying Potato virus A P3 protein gene were tested. Synergistic infection of host plants with PVX carrying the construct and Potato virus Y(O) (PVY(O)) increased the expression of L2ACPE7 in N. tabacum and in transgenic N. benthamiana carrying potyviral HC-Pro gene as compared to control plants infected with L2ACPE7 only.

• MeSH
• genetické vektory * MeSH
• geneticky modifikované rostliny MeSH
• imunoelektronová mikroskopie MeSH
• lidé MeSH
• onkogenní proteiny virové genetika   metabolismus   MeSH
• Papillomavirus E7 - proteiny MeSH
• peptidové fragmenty genetika   izolace a purifikace   metabolismus   MeSH
• Potexvirus genetika   fyziologie   MeSH
• rekombinantní fúzní proteiny biosyntéza   izolace a purifikace   MeSH
• virové plášťové proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• L2 protein, Human papillomavirus type 16 MeSH Prohlížeč
• oncogene protein E7, Human papillomavirus type 16 MeSH Prohlížeč
• onkogenní proteiny virové MeSH
• Papillomavirus E7 - proteiny MeSH
• peptidové fragmenty MeSH
• rekombinantní fúzní proteiny MeSH
• virové plášťové proteiny MeSH

Humanin (HN), Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-IIe-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala, recently discovered in the human brain, is an important neuroprotective peptide. Some derivatives of HN show even higher biological activity, for example [G-14]-HN, where Ser at position 14 is replaced with Gly. As structurally related HN peptide derivatives have similar chemical properties, their separation by CE is difficult. In this work, the electrophoretic behaviour of HN derivatives including [G-14]-HN, a tryptophan HN derivative [W-14]-HN, several other HN derivatives and HN fragments was studied. While phosphate buffer was used as the general BGE, the effects of the buffer concentration and various additives were examined, including sulphate, heptane sulphonate, 2-morpholinoethanesulphonic acid N-[tris(hydroxymethyl)methyl]-2-aminoethane sulphonic acid (TES), sulphated-beta-CD and beta-CD. Separation efficiency of 200,000 theoretical plates was achieved in a BGE of 80 mM phosphate at pH 2.5 where seven out of nine major peaks were partially separated. By investigating the influence of concentration of the interrogated ions on peptides migration, the association between positively charged protonated sites of peptides and various anions was proved. Especially a strong interaction with phosphate, sulphate and sulphonate groups was established. Conditional stability constant of the [Pep(z+), (H(2)PO(4)(-))(n)](z - n) ion associate (n = 1) for [G-14]-HN equals to log K approximately 1.78.

• MeSH
• alkylsulfonany chemie   MeSH
• beta-cyklodextriny chemie   MeSH
• elektroforéza kapilární metody   MeSH
• fosfáty chemie   MeSH
• intracelulární signální peptidy a proteiny chemická syntéza   chemie   izolace a purifikace   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• neuropeptidy chemická syntéza   chemie   izolace a purifikace   MeSH
• peptidové fragmenty chemická syntéza   chemie   izolace a purifikace   MeSH
• pufry MeSH
• roztoky MeSH
• sekvence aminokyselin MeSH
• sírany chemie   MeSH
• substituce aminokyselin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkylsulfonany MeSH
• beta-cyklodextriny MeSH
• fosfáty MeSH
• humanin MeSH Prohlížeč
• intracelulární signální peptidy a proteiny MeSH
• neuropeptidy MeSH
• peptidové fragmenty MeSH
• pufry MeSH
• roztoky MeSH
• sírany MeSH

The method leading to overexpression of the full-length mouse recombinant prion protein (mrPrP 23-231) in the cytoplasm of E. coli as a his-PrP fusion protein and its effective purification using affinity chromatography is described. A typical yield of the method was 8-10 mg his-mrPrP per L of the bacterial culture. The purity of purified protein was > 95 %. The purified his-mrPrP was converted to a soluble form and its folding to alpha-helical and beta-sheet conformations was studied. The properties of differently folded mrPrP were determined by measuring their circular dichroism spectra, partial resistance to cleavage by proteinase K and by centrifugation in sucrose gradient.

• MeSH
• chromatografie afinitní MeSH
• cirkulární dichroismus MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• Escherichia coli genetika   metabolismus   MeSH
• myši MeSH
• oxidace-redukce MeSH
• peptidové fragmenty biosyntéza   chemie   genetika   izolace a purifikace   MeSH
• priony biosyntéza   chemie   genetika   izolace a purifikace   MeSH
• rekombinantní proteiny biosyntéza   chemie   genetika   izolace a purifikace   MeSH
• sbalování proteinů MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• peptidové fragmenty MeSH
• prion protein (23-231) MeSH Prohlížeč
• priony MeSH
• rekombinantní proteiny MeSH

We examined the use of monolithic capillary columns prepared via ring-opening metathesis polymerization (ROMP) for peptide separation in voltage-assisted capillary LC (voltage-assisted CLC). In order to demonstrate their potential for peptide separation, ROMP-derived monoliths with RP properties were prepared. The preparation procedure of monoliths was transferred from ROMP monoliths optimized for CLC. ROMP monoliths were synthesized within the confines of 200 microm id fused-silica capillaries with a length of 37 cm. After optimization of the chromatographic conditions, the separation performance was tested using a well-defined set of artificial peptides as well as two peptidic mixtures resulting from a tryptic digest of BSA as well as a collagenase digest of collagen. ROMP monoliths showed comparable performance to other monolithic separation media in voltage-assisted CLC published so far. Therefore, we conclude that by optimizing the composition of the ROMP monoliths as well as by using the controlled manner of their functionalization, ROMP monoliths bear a great potential in CLC and CEC.

• MeSH
• kapilární elektrochromatografie přístrojové vybavení   metody   MeSH
• kolagen metabolismus   MeSH
• kolagenasy metabolismus   MeSH
• oligopeptidy izolace a purifikace   MeSH
• peptidové fragmenty izolace a purifikace   MeSH
• peptidy izolace a purifikace   MeSH
• polymery chemická syntéza   chemie   MeSH
• sérový albumin hovězí metabolismus   MeSH
• skot MeSH
• trypsin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kolagen MeSH
• kolagenasy MeSH
• oligopeptidy MeSH
• peptidové fragmenty MeSH
• peptidy MeSH
• polymery MeSH
• sérový albumin hovězí MeSH
• trypsin MeSH