Dana Flodrová

Study of quantitative changes of cereal allergenic proteins after food processing

BACKGROUND Within last few years, the occurrence of food allergens and corresponding food allergies has been increasing, therefore research into the individual allergens is required. In the present work, the effect of cereal processing on the amounts of allergenic proteins is studied by modern proteomic-based approaches. The most important wheat and barley allergens are low-molecular-weight (LMW) proteins. Therefore we investigated the relative quantitative changes of these proteins after food technological processing, namely wheat couscous production and barley malting. RESULTS A comparative study using mass spectrometry in connection with the technique of isobaric tag for relative and absolute quantification (iTRAQ) revealed that the amount of wheat allergenic LMW proteins decreased significantly during couscous production (approximately to 5-26% of their initial content in wheat flour). After barley malting, the amounts of the majority of LMW proteins decreased as well, although to a lesser extent than in the case of wheat/couscous. The level of two allergens even slightly increased. CONCLUSION Suggested proteomic strategy proved as universal and sensitive method for fast and reliable identification of various cereal allergens and monitoring of their quantitative changes during food processing. Such information is important for consumers who suffer from allergies.

Proteomic analysis of changes in the protein composition of MCF-7 human breast cancer cells induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination.

Retinoic acid (all-trans and 9-cis) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a commercially manufactured kit RIPA and separated on two dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with both retinoic acid isomers. We found significant differences in occurrence of proteins probably affecting the cell migration process in tumour cells. Heat shock protein 27, ribonucleoprotein SmD3, and cofilin-1 were significantly upregulated after treatment with combination of individual retinoic acid isomers. On the other hand, AP-5 complex subunit beta-1 shows the different response. Thus, the results might help to find the answer to important medical questions on (i) the identification of signaling pathways affected by retinoic acid isomers or (ii) how the observed proteomic pattern might reflect the effectiveness of retinoic acids treatment.

A comparative study of protein patterns of human estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cell lines.

In the present study, we analyzed the cell lysates of human tumour cell lines representing two major clinically different types of breast cancer. Our main goal was to show the differences between them on proteomic level. Gel electrophoresis followed by MALDI-TOF MS analysis was used for proteins determination. Exactly 98 proteins were unequivocally identified and 60 of them were expressed differentially between MDA-MB-231 and MCF-7 cell lines. Among the proteins reported here, some well-known breast cancer markers (e.g., annexin A1, annexin A2 and vimentin) were identified in the MDA-MB-231 cell line and thus we were able to distinguish both cell lines sufficiently.

Incorporation of β-(1,6)-linked glucooligosaccharides (pustulooligosaccharides) into plant cell wall structures

Protein extract of germinating nasturtium (Tropaeolum majus) seeds containing xyloglucan endotransglycosylase (xyloglucan xyloglucosyl transferase, EC 2.4.1.207, abbreviated XET) exhibited the heterotransglycosylating activity with donor/acceptor substrate pair xyloglucan/sulphorhodamine labelled pustulooligosaccharides (XG/PUOS-SR) in a dot blot assay. The heterotransglycosylating activity was confirmed by the substrate-product changes during transglycosylation by HPLC size-exclusion chromatography. Another donor substrate capable of being coupled with PUOS-SR was cellulose, probably owing to its structural similarity to xyloglucan. Surprisingly, microscopic comparison of the incorporation of the labelled xyloglucan nonasaccharide XGO9-SR (specific substrate for XET) and PUOS-SR into the cell wall structures clearly showed differences in their binding to specific cell structures: the primary cell wall and the plasma membrane. These findings indicate the existence in nasturtium of XETs with different localisation, substrate specificity and, probably, function.

APPLICATION OF MONOLITHIC AFFINITY HPLC COLUMN FOR RAPID DETERMINATION OF MALT GLYCOPROTEINS

Of all known protein post-translational modifications, glycosylation is the most common and complex one, which plays an important role in many biological processes and shows great diversity. For identification and detailed analysis of glycoproteins, separation from complex samples is required. Lectin affinity chromatography has been frequently used for purification and enrichment of glycoproteins or glycopeptides. The most well characterized and widely used is concanavalin A (ConA), binding specifically to α-mannosyl and α-glucosyl residues. It is known that the protein as well as glycoprotein composition of barley malt is fundamental with regard to beer production. For this reason, water soluble glycoproteins were investigated. The aim of this study was to optimize separation and enrichment of individual modified proteins on a monolithic ConA affinity HPLC column. This chromatographic strategy demonstrates the potential of affinity chromatography on this type of column, providing higher reproducibility, efficiency, and faster performance in comparison with the previously used manually filled columns. Most importantly, the ConA column used is resistant to bleeding. Furthermore, ConA-bound proteins were separated on SDS-PAGE and then chymotryptic digested and identified using MALDI-TOF/TOF MS. Our proteomic analysis allowed successful determination of several putative glycoproteins present in barley malt.

Vliv pivovarského procesu na profil proteinů ječmene.

DAGMAR BENKOVSKÁ, DANA FLODROVÁ, VRATISLAV PSOTA, JANETTE BOBÁĽOVÁ 1 Ústav analytické chemie AV ČR, v. v. i., Veveří 97, 602 00 Brno / Institute of Analytical Chemistry of the ASCR, v. v. i., Veveří 97, 602 00 Brno, Czech Republic 2 Vysoké učení technické v Brně, Fakulta chemická, Purkyňova 118, 612 00 Brno / Brno University of Technology, Faculty of Chemistry, Purkyňova 118, 612 00 Brno, Czech Republic 3 Výzkumný ústav pivovarský a sladařský, a. s., Mostecká 7, 614 00 Brno / Research Institute of Brewing and Malting, PLC, Mostecká 7, 614 00 Brno, Czech Republic

Consequences of the natural retinoid/retinoid X receptor ligands action in human breast cancer MDA-MB-231 cell line: Focus on functional proteomics

The main intention of this study was the investigation of impact of natural biologically active ligands of nuclear retinoid/retinoid X receptors (all-trans and 9-cis retinoic acid) on proteomic pattern in human estrogen receptor negative breast cancer cell line MDA-MB-231. For this purpose, proteomic strategies based on bottom-up method were applied. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and their characterization was achieved by MALDI-TOF/TOF. By employing PDQuest™ software, we identified more than 50 proteins affected by retinoic acid isomers. For more information, 9 proteins which are associated with tumor process were selected. We determined that derivatives of retinoic acid led to significantly reduced level of proteins belonging to metabolic pathway (e.g. glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase 2) or to other cellular processes as apoptosis, regulation of transcription process or epithelial-mesenchymal transition (e.g. annexins, nucleoside diphosphate kinase B, vimentin). On the other hand all-trans retinoic acid treatment indicates up-regulated effect for heterogeneous nuclear ribonucleoprotein A2/B1.

Oligogalacturonate hydrolase with unique substrate preference from the pulp of parsley roots

The main form of pectate hydrolases in the cell wall of parsley roots showed a unique substrate preference of a plant exopolygalacturonase because it clearly preferred the substrates with degree of polymerization about 10. This form was separated from the others, purified and characterized. Enzyme exhibited sharp pH optimum corresponding to pH 4.7, molecular mass 53.5 kDa, and isoelectric point 5.3. It was stable at 50°C in 2-h assay and had optimum of temperature at 60°C (activation energy being 37.0 kJ/mol). The interaction with concanavalin A indicated the glycosylation of enzyme. Substrates were cleaved from the non-reducing end.