The BioAnalytical Chemistry Core B will provide expert bio-analytical chemistry support to the three projects of this Program Project Grant, 'Innate Responses to Microbial Infection'. These areas include mass spectrometry, protein chemistry &proteomics (Part I), and carbohydrate &lipid chemistries (Part II). In Part I, a comparative analysis of proteins, including discovery-based and quantitative mass spectrometry based proteomic methods, will be carried out on human neutrophil preparations, capsule-like material (CLM). and various traget proteins of interest. For Project 1 (Apicella). we will examine CLM perparations for the presence of a putative lipoprotein anchor and. if present, its identity and lipid modifications will be elucidated. As part of Project 2 (Nauseef). we will target human cell types that are critical to the reguation or dysregulation of the innate immune response and identify changes in protein composition and expression contained in detergent-resistant membrane microdomains or lipid rafts. For these experiments, various proteomic approaches will be used, including non-lableing and ITRAQ labeling strategies. Lastly, in Project 2 and 3 (Nauseef and Allen), a targeted examination of posttranslational modifications will be carried out with an emphasis on phosphorylation for several key host proteins invloved in immune regulation, i.e., p47phox, CaMKII and PKCa. For these experiment, a multireaction monitoring approach using stable isotope-labeled peptides will be employed for targeted quantitation. In Part II, we will use our expertise in mass spectrometry and carbohydrate and lipid chemistries for the structural characterization of host plasma phospholipids and capsule-like material (CLM). With Dr. Apicella (Project 1), we will carry out an exhaustive analysis of CLM preparations to identify its polysaccharide structure and its membrane anchor, likely a diacylglycerolphosphate-linked moiety. For Project 2 (Nauseef). an examination of the changes in phospholipid composition in recruited neutrophils will be undertaken. All experiments will be carried out in the Buck Institute's proteomics and mass spectrometry facility. This facility, of which Dr. Gibson is Director, is capable of both high-throughput and targeted analysis of complex mixtures of proteins, as well as detailed analysis of complex of carbohydrates and lipids. Dr. Gibson will also work with the NMR facility at lowa. who will acquire 1 and 2D NMR data from CLM preparations.
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