We used a proteomic approach to identify constitutively formed extracellular proteins of Hypocrea atroviridis (Trichoderma atroviride), a known biocontrol agent. The fungus was cultivated on glucose and the secretome was examined by two‐dimensional gel electrophoresis. The two predominant spots were identified by MALDI MS utilizing peptide mass fingerprints and amino acid sequence tags obtained by postsource decay and/or high‐energy collision‐induced dissociation (MS/MS) experiments, and turned out to be the same protein (12 629 Da as determined with MS, pI 5.5–5.7), probably representing the monomer and the dimer. The corresponding gene was subsequently cloned from H. atroviridis and named epl1 (eliciting plant response‐like), because it encodes a protein that exhibits high similarity to the cerato‐platanin family, which comprises proteins such as cerato‐platanin from Ceratocystis fimbriata f. sp. platani and Snodprot1 of Phaeosphaeria nodorum, which have been reported to be involved in plant pathogenesis and elicitation of plant defense responses. Additionally, based on the similarity of the N‐terminus to that of H. atroviridis Epl1, we conclude that a previously identified 18 kDa plant response elicitor isolated from T. virens is an ortholog of epl1. Our results showed that epl1 transcript was present under all growth conditions tested, which included the carbon sources glucose, glycerol, l‐arabinose, d‐xylose, colloidal chitin and cell walls of the plant pathogen Rhizoctonia solani, and also plate confrontation assays with R. solani. Epl1 transcript could even be detected under osmotic stress, and carbon and nitrogen starvation.
Highlights d Distinct transcriptional programs characterize prostate CSC and bulk tumor cells d BRD4 promotes mitochondrial biogenesis and metabolic plasticity in prostate CSCs d Mitochondrial fission enables asymmetric division and prostate CSC self-renewal d BRD4 inhibitors block mitochondrial fission and hinder selfrenewal of prostate CSCs
Knowledge about the extent of total variation experienced between samples from different individuals is of great importance for the design of not only proteomics but every clinical study. This variation defines the smallest statistically significant detectable signal difference when comparing two groups of individuals. We isolated platelets from 20 healthy human volunteers aged 56 -100 years because this age group is most commonly encountered in the clinics. We determined the technical and total variation experienced in a proteome analysis using two-dimensional DIGE with IPGs in the pI ranges 4 -7 and 6 -9. Only spots that were reproducibly detectable in at least 90% of all gels (n ؍ 908) were included in the study. All spots had a similar technical variation with a median coefficient of variation (cv) of about 7%. In contrast, spots showed a more diverse total variation between individuals with a surprisingly low median cv of only 18%. Because most known biomarkers show an effect size in a 1-2-fold range of their cv, any future clinical proteomics study with platelets will require an analytical method that is able to detect such small quantitative differences. In addition, we calculated the minimal number of samples (sample size) needed to detect given protein expression differences with statistical significance. Molecular & Cellular Proteomics 7:193-203, 2008.Biomarkers are used to differentiate between different biological states and to monitor disease progress or the success of medical treatment. To fulfill these tasks, there are not only strict requirements for each biomarker candidate in terms of selectivity and specificity but also for the precision of the analytical process. Therefore knowledge on the extent of variation between the individuals within each group is crucial for the study design as well as the selection of the method of measurement. The total variation experienced between individuals within one group is the result of the biological variation caused by factors like sex, age, genetic background, lifestyle, or health status and the technical variation introduced by the applied sample handling and the method of measurement itself. This study investigated the extent of variation in human blood platelets. Platelets are responsible for the maintenance of vascular integrity and are also involved in inflammation and wound healing (1). They are anucleate cytoplasmic fragments released from megakaryocytes in the bone marrow. During platelet biogenesis, organelles, especially the mitochondria and the ␣-and dense granules, are actively transported into the platelets. Furthermore platelets receive a certain set of mRNAs during their biogenesis from the megakaryocytes and are still capable of protein synthesis and processing (2). Several proteomics studies have focused on different aspects in platelet research using 2D 1 electrophoresis followed by mass spectrometry. These studies provided 2D maps of the total platelet proteome (3, 4), the platelet secretome (5), and the processes during platelet activ...
Our experiments show that it is possible to detect different types of recombinant human erythropoietins (rhEPOs), EPO-a, EPO-b and novel erythropoesis stimulating protein (NESP), based on exact molecular weight (MW) determination by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) applying a high-resolution time-of-flight (TOF) mass analyser in the linear mode. Detection limits for the highly purified, intact glycoproteins were achievable in the low fmol range (25-50 fmol) using a sample preparation method applying a hydrophobic sample support (DropStop 1 ) as MALDI target surface. These results are very promising for the development of highly sensitive detection methods for a direct identification of rhEPO after enrichment from human body fluids. During our investigation we were able to differentiate EPO-a, EPO-b and NESP based on distinct molecular substructures at the protein level by specific enzymatic reactions. MW determination of the intact molecules by high resolving one-dimensional sodium dodecyl sulfate /polyacrylamide gel electrophoresis (1D SDS-PAGE) and isoform separation by planar isoelectric focusing (IEF) was compared with MALDI-MS data. Migration differences between the rhEPOs were observed from gel electrophoresis, whereby MWs of 38 kDa in the case of EPO-a/b and 49 kDa for NESP could be estimated. In contrast, an exact MW determination by MALDI-MS based on internal calibration revealed average MWs of 29.8 AE 0.3 kDa for EPO-a/b and 36.8 AE 0.4 kDa for NESP. IEF separation of the intact rhEPOs revealed the presence of four to eight distinct isoforms in EPO-a and EPO-b, while four isoforms, which appeared in the more acidic area of the gels, were detected by immunostaining in NESP. A direct detection of the different N-or O-glycoform pattern from rhEPOs using MALDI-MS was possible by de-sialylation of the glycan structures and after de-N-glycosylation of the intact molecules. Thereby, the main glycoforms of EPO-a, EPO-b and NESP could be characterised based on their N-glycan composition. A microheterogeneity of the molecules based on the degree of sialylation of the O-glycan was observable directly from the de-N-glycosylated protein.
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