Janette Bobalova

2D-HPLC and MALDI-TOF/TOF analysis of barley proteins glycated during brewing.

The barley proteins have been the subject of interests of many research groups dealing with barley grains, malt and beer. The proteins which remain intact after harsh malting conditions influence the quality and flavor of beer. The characteristic feature of the proteins present in malt and beer is their extensive modification with carbohydrates, mainly glucose that comes from the starch degradation during technological processes. The degree of the protein glycation has an effect on the quality of malt and beer and on the properties of the beer foam. A combination of two-dimensional high performance liquid chromatography (2D-HPLC) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS) was used for the analysis of the protein extracts that were reduced, alkylated, and degraded enzymatically without prior protein separation. This so-called shot-gun approach enabled us to determine glycation sites in one third of the proteins identified in the study and to propose potential glycation markers for fast and efficient monitoring during malting.

Influence of different proteomic protocols on degree of high-coverage identification of nonspecific lipid transfer protein 1 modified during malting

Both top‐down (combining protein separation with MS analysis of intact proteins) and bottom‐up (MS analysis of digested proteins) proteomic approaches were used for detailed characterization of nonspecific lipid transfer protein from barley malt. The aim was obtaining high‐coverage of the primary structure of the proteins and the determination of PTMs such as lipid adduction and glycation. Here we present an influence of 15 proteomic protocols (differing in applied separation technique, enzyme and digestion procedure) on the extent of the coverage of the protein primary structure. The most successful protocols were in‐gel digestion with trypsin of alkylated protein and in‐solution digestions with trypsin or trypsin/chymotrypsin mixture of the nonalkylated protein. Totally, full sequence coverage based on the PMF and 85% sequence coverage based on the peptide fragmentation including PTMs was obtained.

Monitoring of barley starch amylolysis by gravitational field flow fractionation and MALDI-TOF MS

BACKGROUNDnIn barley, starch occurs in the form of granules with bimodal size distribution. Enzymatic hydrolysis of the starch granule is one of the most important reactions occurring during malting and mashing. Previous studies revealed the discrepancies in the assumption that barley varieties with better malting qualities should have a higher A/B (large/small starch granules) ratio. This led us to focus our attention on detailed analysis of two barley varieties, Jersey and Tolar, both with high malting quality but significantly differing in A/B (1.28 and 0.66, respectively), were chosen for more detailed analysis in the actual work. In this study, the capacity of gravitational field flow fractionation (GFFF) to monitor amylolysis of the starch granules was investigated.nnnRESULTSnIsolated starch granules from these two barley cultivars were treated with amylases. The changes in starch granule size and bimodal distribution were studied by GFFF. Simultaneously, free sugars released during enzymatic digestion were observed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The changes in the fractogram and in the mass spectra reflect a correlation with the progress of enzymatic hydrolysis.nnnCONCLUSIONnThe results show the interest in utilization of GFFF as a simple and cheap method for monitoring changes in the distribution of the starch granule size during amylolysis.

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.

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.

Tributyltin-resistant Methanothermobacter thermautotrophicus mutant with mutational substitutions in the A1A0-ATP synthase operon.

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to tributyltin chloride (TBT) was isolated. TBT, the inhibitor of the A(0) domain of A(1)A(0)-ATP synthase, inhibits methanogenesis in the wild-type cells; however, the TBT-resistant mutant exhibited methanogenesis even in the presence of 800 microM TBT. ATP synthesis driven by methanogenic electron transport was markedly diminished in the mutant strain. While TBT profoundly inhibited ATP synthesis driven by methanogenic electron transport in the wild type, only a slight inhibition was observed in the mutant strain. These results suggested a modification in the ATP-synthesizing system of the mutant strain. The sequence of the complete A(1)A(0)-ATP synthase operon (Mth952-Mth961) in the wild-type and mutant strains was determined and compared. Three mutations leading to amino acid substitutions in two A(1)A(0)-ATP synthase subunits were identified - Val(338)Ala in subunit A and Leu(252)Ile and Ser(293)Ala in subunit B. Moreover, this study revealed the differential expression of several proteins that may contribute to TBT resistance. The results imply that change of TBT sensitivities of TBT-resistant mutant is due to mutational substitutions in the A(1)A(0)-ATP synthase operon.