Hana Konečná

Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis

High concentrations of cytokinins (CKs) in the cultivation medium can induce partial photomorphogenesis in dark-grown Arabidopsis seedlings. However, no significant increases in endogenous CK levels have been found in de-etiolated mutants, suggesting that either parallel pathways are involved in the light and CK responses, or changes in the sensitivity to CKs occur during photomorphogenesis. Here it is shown that even modest increases in endogenous CK levels induced by transgenic expression of the CK biosynthetic gene, ipt, can lead to many typical features of light-induced de-etiolation, including inhibition of hypocotyl elongation and partial cotyledon opening. In addition, significant changes in expression of 37 proteins (mostly related to chloroplast biogenesis, a major element of light-induced photomorphogenesis) were detected by image and mass spectrometric analysis of two-dimensionally separated proteins. The identified chloroplast proteins were all up-regulated in response to increased CKs, and more than half are up-regulated at the transcript level during light-induced photomorphogenesis according to previously published transcriptomic data. Four of the up-regulated chloroplast proteins identified here have also been shown to be up-regulated during light-induced photomorphogenesis in previous proteomic analyses. In contrast, all differentially regulated mitochondrial proteins (the second largest group of differentially expressed proteins) were down-regulated. Changes in the levels of several tubulins are consistent with the observed morphological alterations. Further, 10 out of the 37 differentially expressed proteins detected have not been linked to either photomorphogenesis or CK action in light-grown Arabidopsis seedlings in previously published transcriptomic or proteomic analyses.

Exploration of Beer Proteome Using OFFGEL Prefractionation in Combination with Two-Dimensional Gel Electrophoresis with Narrow pH Range Gradients

Two-dimensional gel electrophoresis in combination with mass spectrometry has already been applied successfully to study beer proteome. Due to the abundance of protein Z in beer samples, prefractionation techniques might help to improve beer proteome coverage. Proteins from four lager beers of different origins were separated by two-dimensional electrophoresis (2-DE) followed by tandem mass spectrometric analysis. Initially 52 proteins mostly from Hordeum vulgare (22 proteins) and Saccharomyces species (25 proteins) were identified. Preparative isoelectric focusing by OFFGEL Fractionator was applied prior to 2-DE to improve its resolution power. As a result of this combined approach, a total of 70 beer proteins from Hordeum vulgare (30 proteins), from Saccharomyces species (31 proteins), and from other sources (9 proteins) were identified. Of these, 37 proteins have not been previously reported in beer samples.

Proteomic profiling of human embryonic stem cell-derived microvesicles reveals a risk of transfer of proteins of bovine and mouse origin

BACKGROUND AIMS Microvesicles (MV) shed from the plasma membrane of eukaryotic cells, including human embryonic stem cells (hESC), contain proteins, lipids and RNA and serve as mediators of cell-to-cell communication. However, they may also contain immunogenic membrane domains and infectious particles acquired from xenogenic components of the culture milieu. Therefore, MV represent a potential risk for clinical application of cell therapy. METHODS We tested the ability of hESC and their most commonly used feeder cells, mouse embryonic fibroblasts (MEF), to produce MV. We found that hESC are potent producers of MV, whereas mitotically inactivated MEF do not produce any detectable MV. We therefore employed a combined proteomic approach to identify the molecules that constitute the major components of MV from hESC maintained in a standard culture setting with xenogenic feeder cells. RESULTS In purified MV fractions, we identified a total of 22 proteins, including five unique protein species that are known to be highly expressed in invasive cancers and participate in cellular activation, metastasis and inhibition of apoptosis. Moreover, we found that hESC-derived MV contained the immunogenic agents apolipoprotein and transferrin, a source of Neu5Gc, as well as mouse retroviral Gag protein. CONCLUSIONS These findings indicate that MV represent a mechanism by which hESC communicate; however, they also serve as potential carriers of immunogenic and pathogenic compounds acquired from environment. Our results highlight a potential danger regarding the use of hESC that have previously been exposed to animal proteins and cells.

A combined approach for the study of histone deacetylase inhibitors

Overexpression of histone deacetylases (HDACs), with consequent hypoacetylation of histones, is reportedly associated with transcriptional repression of tumour suppressor genes. Thus, inhibition of HDACs has emerged as a promising strategy in cancer therapy. In order to monitor the effects of potential HDAC inhibitors, a multi-level approach consisting of preliminary screening (measurement of HDAC activity and semi-quantitative evaluation of histone H4 modification profile by MALDI-TOF MS) and detailed analysis of histone modification forms (using 2-D AUT/AU PAGE and LC-ESI-IT MS) has been used in this study. The data obtained provide a global insight into the effects of HDAC inhibitors on the histone acetylation status that participates in gene transcription control. Using two example inhibitors, valproic acid sodium salt and entinostat, we show that similar levels of HDAC inhibition induced by different agents can lead to distinct rates of histone hyperacetylation, suggesting that except for the direct inhibition of HDACs, additional molecular mechanisms amplifying the response are likely to be involved in the inhibitory process. The approach used in our study makes it possible not only to follow the dynamics of individual histone modification forms, but also of their combined occurrence in the N-terminal fragment.

Deep coverage of the beer proteome

We adopted an approach based on peptide immobilized pH gradient-isoelectric focusing (IPG-IEF) separation, coupled with LC-MS/MS, in order to maximize coverage of the beer proteome. A lager beer brewed using traditional Czech technology was degassed, desalted and digested. Tryptic peptides were separated by isoelectric focusing on an immobilized pH gradient strip and, after separation, the gel strip was divided into seven equally sized parts. Peptides extracted from gel fractions were analyzed by LC-MS/MS. This approach resulted in a three-fold increase in the number of proteins identified (over 1700) when compared to analysis of unfractionated beer processed by a filter-aided sample preparation (FASP). Over 1900 protein groups (PGs) in total were identified by both approaches. SIGNIFICANCE The study significantly extends knowledge about the beer proteome and demonstrates its complexity. Detailed knowledge of the protein content, especially gluten proteins, will enhance the evaluation of potential health risks related to beer consumption (coeliac disease) and will contribute to improving beer quality.

Preventing the mixed detergent micelle effect in two-dimensional electrophoresis on Tris-Tricine gels

Application of Tris‐N‐[Tris(hydroxymethyl)methyl]glycine gels for 2DE is hampered by formation of mixed CHAPS–SDS micelles resulting in typical swirling pattern in the low mass range, which makes reliable quantitative and qualitative gel evaluation impossible. Modification of 2DE strip equilibration procedure prevented the direct interaction between both detergents during equilibration process, thus substantially improving gel separation.

P136 Two-dimensional molecular profiling of multiple myeloma

Multiple myeloma (MM) is a B-cell malignancy characterized by proliferation and accumulation of B lymphocytes and plasma cells, secreting monoclonal immunoglobulins, in the bone marrow and, less frequently, at extramedullary sites. Proteome analysis of clinical samples aims at characterizing disease-specific changes in the protein expression profile of an affected cell. Such changes could serve as potential diagnostic markers. Two-dimensional polyacrylamide gel electrophoresis (2-DE) is widely used in comparative studies of protein expression levels. The potential of this method is strongly dependent on good sample preparation, in order to obtain proteomic maps with good reproducibility and resolution. Aims. The aim of this study was to optimize two-dimensional electrophoresis (2-DE) conditions for separation of human myeloma proteins. Methods. We have compared two different solubilization buffers, we also evaluated protein precipitation with ethanol (EtOH) and optimized 2-DE conditions for human myeloma proteins. Myeloma cell line ARH-77 was used to in the pilot study. Proteins from myeloma cell line ARH-77 were separated by 2-DE using a 17 cm pH 3-10 nonlinear immobilized pH gradient strips in the first dimension. Separation in the second-dimension was carried out on the Protean II Cell (Bio-Rad) using a 12% SDS-polyacrylamide gel. Separated proteins were stained with ProteoSilver Plus (Sigma). Spot detection, quantification, and alignment were performed with the PDQuest software (version 7.3.0., Bio-Rad). Results. Two concentrations of the detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) in the solubilization buffer were tested, 2% and 4%. Higher concentration of CHAPS showed a greater ability to solubilize proteins. The most efficient solubilization was obtained with lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 60 mM DTT (dithiothreitol), 0.8% carrier ampholytes and 0.003% BPB (bromphenol blue). About 1448 spots were visualized in the gel using this lysis buffer. Many proteins (206 spots) were lost after the EtOH precipitation, though. Conclusions. Initial extraction and solubilization are key factors of proteomic analysis. The successful solubilization and 2-DE separation of proteins will be valuable tool for further study of the mechanisms of therapy resistance in purified plasma cells of patients with MM and further study of the biomarkers for prediction of progression of MGUS to overt myeloma.