Operate and maintain laboratory utilizing expertise in protein biochemistry and state-of-art tools for mass spectroscopy, organic and solid phase synthesis of peptides/proteins and liquid chromatographic techniques. The primary focus of the lab is the analysis, synthesis and characterization of proteins and peptides. This research covers primarily four collaborative areas. 1. In collaboration with NICHD, we are focusing on the development of low femtomole mass spectrometric methods sufficient for identification of phosphorylation sites in CTS and Ang II-stimulated AT1. Protein kinase levels in AT1 containing cells with and without Ang II stimulation have been studied that permit selection of certain protein kinases for further studies using the CTS and its mutants as substrates. Ultimately, protein kinases that show positive results will be selected for transfection into AT1 containing cells to qualitate the phosphorylation patterns and to investigate the role of phosphorylation in the activation of the receptor. It is only recently that common methods for analyzing O-phosphorylation sites have been achieved by mass spectrometry. However tandem MS identification of the location of phosphorylated amino acids in the peptide sequence is complicated by not only intrinsic characterization of low ionization efficiency and suppression of the phosphate moiety but also the loss of the phosphate moiety from both Ser and Thr residues during the collision induced dissociation (CID) necessary in tandem mass spectrometry. To address these problems, we and others have developed reaction conditions for the b-elimination/Michael addition to convert the phosphate moiety on Ser and Thr residues to CID stable derivatives to allow definitive determination of the phosphorylation site in the peptide. Along the same lines we will also apply similar techniques to the study and identification of the phosphorylation sites found on the UL3 protein from herpesvirus in a joint effort with OVRR/DCGT. 2. In collaboration with CBER/OBRR/DH we will utilize mass spectrometry in conjunction with chemical cross linking methodologies to study the structural properties of chemically cross-linked alpha 1-protease inhibitor (alpha 1-PI) polymer. It has been known for some time that liver disease in alpha 1- P1 deficiency is accompanied by the deposition of alpha 1- PI aggregates. The nature of the polymers remains the subject of debate. 3. In collaboration with CBER/OBRR/DH we are utilizing mass spectrometry together with other biochemical tools to study and evaluate blood substitutes of regulatory significance. 4. In collaboration with CBER/OD/FBR we are utilizing mass spectrometry as a tool to sequence and identify conserved protein sequences from the Limulus ameobycte proteome. Limulus lysate continues to be a primary regulatory concern for its sensitivity to endotoxin. Even though the horseshoe crab predates many dinosaurs very little is known of it cellular proteome. Not long ago, it was discovered that the carapace of the horseshoe crab may possibly possess antibiotic agents. The protein limulin for example has been identified as having potent cell-killing abilities. The group of proteins known as petraxins of which CRP is one of them was found in Limulus and could be an ideal model for study in more complex organisms. 4. In collaboration with CBER/OBRR/DETTD we continue to evaluate synthetic peptides that are based on the HIV Tat protein as possible vaccine candidates. In the past we have focused on and identified the functional domains of Tat and have also studied the immunogenicity of various multiple peptide conjugates with emphasis on possible therapeutic vaccine use. In addition our studies are also now focused on the metal binding region of this protein. The metal binding complexes are being studied with the aid of mass spectrometry.
