Evaluation of the extracellular proteins in full-scale activated sludges

The proteins present in the extracellular polymeric substances (EPS) of activated sludge flocs were investigated using three cation-associated extraction methods. The subproteomes generated from four full-scale activated sludges were subsequently fractionated by ammonium sulfate precipitation and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that each extraction method led to unique SDS-PAGE protein profiles, which provided strong evidence that the extracted proteins are uniquely associated with specific cations in activated sludge flocs. The comparison of protein profiles across sludges from different treatment plants revealed that extracts obtained using a cation-exchange resin exhibited similar protein banding patterns while sulfide- and base-extracted EPS led to more variable protein profiles. Analysis of several SDS-PAGE bands by liquid chromatography-tandem mass spectrometry of tryptic digests led to the identification of several bacterial proteins as well as sewage-derived polypeptides (human elastase IIIA and keratins). Their putative roles in activated sludges and their association with targeted cations are proposed.     

Cow's milk allergens identification by two-dimensional immunoblotting and mass spectrometry

Cow's milk allergy (CMA) has become a common disease in early childhood, its prevalence ranging from 1.6% to 2.8% among children younger than 2 years of age. The role of different cow's milk protein (CMP) in the pathogenesis of CMA is still controversial. Even if the proteins most frequently and most intensively recognized by immunoglobulin E (IgE) seem to be the most abundant in milk (caseins and beta-lactoglobulin), with an although great variability all milk proteins appear to be potential allergens, even those that are present in trace amounts (i.e., lactoferrin, IgG, and BSA). In this work proteomics techniques have been applied for CMP allergens analysis. Allergens have been identified by immunoblotting following resolution of CMP components by two-dimensional electrophoresis. Sera from 20 milk-allergic subjects, as proven by oral provocation test, CAP-RAST and skin prick test, have been used for cow's milk major allergen identification. Cow's milk proteins and their isoforms were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. In our group of patients, the prevalence of CMP allergens, i.e., the total number of subjects sensitized to CMP divided by the total number of the subjects enrolled in the study, was: 55% alpha(s1)-casein, 90% alpha(s2)-casein, 15% beta-casein, 50% kappa-casein, 45% beta-lactoglobulin, 45% BSA, 95% IgG-heavy chain, 50% lactoferrin, and 0% alpha-lactalbumin.     

Glutathione improves the cold resistance of Lactobacillus sanfranciscensis by physiological regulation

The microenvironmental manipulation of glutathione (GSH) on improving cold resistance of Lactobacillus sanfranciscensis DSM 20451^T was investigated in this study. It was proved that GSH relieves the metabolic disorder of cells under cold stress, and prevents the decreased activities of related key enzymes such as pyruvate kinase (PK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) upon cold challenges. Higher intracellular ATP level was also found in cells with GSH under cold stress. Moreover, cells with imported GSH had significantly higher intracellular than the control during cold treatment. In addition, proteomics analysis showed more exciting findings that the protective function of GSH under cold stress was related to metabolic regulation and the multi-control against induced cross-stresses. These results broaden the knowledge about the physiological function of GSH, and suggest a practicable approach to improve the cold resistance of L. sanfranciscensis, a starter culture for sourdough, by the addition of GSH.     

Identification of small troponin T peptides generated in dry-cured ham

Troponin T (TnT) is a myofibrillar protein present in striated muscle that develops important structural and regulatory functions. This protein is easily degraded during post-mortem ageing but details of the TnT hydrolysis occurring during much longer processes, e.g. the dry-cured processing, as well as the specific sequences of the generated fragments, still remain undetermined. In the present study, the isolation and identification of 27 peptides, generated from TnT during dry-cured processing, are reported for the first time. This study has been developed using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF) and it evidences the extensive degradation of TnT occurring during the processing of dry-cured ham. Some of the cleavage sites, corresponding to these sequenced fragments, confirm the sites reported by other authors both in vitro and during post-mortem ageing of meat.     

The Impact of Activity-Based Protein Profiling in Malaria Drug Discovery

Activity-based protein profiling (ABPP) is an approach used at the interface of chemical biology and proteomics that uses small molecular probes to provide dynamic fingerprints of enzymatic activity in complex proteomes. Malaria is a disease caused by Plasmodium parasites with a significant death burden and for which new therapies are actively being sought. Here, we compile the main achievements from ABPP studies in malaria and highlight the probes used and the different downstream platforms for data analysis. ABPP has excelled at studying Plasmodium cysteine proteases and serine hydrolase families, the targeting of the proteasome and metabolic pathways, and in the deconvolution of targets and mechanisms of known antimalarials. Despite the major impact in the field, many antimalarials and enzymatic families in Plasmodium remain to be studied, which suggests ABPP will be an evergreen technique in the field.