We have successfully integrated MSe acquisitions into our proteomics experiments and have closely worked with bioinformatics group to validate our results on several projects. 1. Method Development. The use of label-free approaches for quantitative proteomics studies is being implemented. This methodology involves data-independent analyses (MSe) and is being evaluated to determine if this MS approach is better for protein quantitation studies than data dependent (DDA) methods. In addition, an online 2D LC method suitable for the identification of proteins from complex samples is being optimized. MSe can rapidly acquire accurate mass precursor and fragment ion information while simultaneously obtaining fairly accurate quantitative profiles from every detectable component in the sample, in theory. A comparison of the results obtained from depleted serum (depletion of 14 most abundant proteins) followed by six different treatments is being evaluated. 2. Proteomic Studies Involving Myositis. A differential proteomics project comparing the quantitation of proteins from sera of healthy individuals to individuals diagnosed with a rheumatic disease is underway. This work is in collaboration with F. Miller (EAG) as part of the EAGs study of families with twins or siblings discordant for systemic rheumatic disorders. The goal of this project is to determine whether a protein(s) can be identified that would allow for the early diagnosis, prognosis, and treatment of these diseases. Sera samples have been depleted and processed and are awaiting LC/MSe analysis. 3. Proteomic Studies of LCM Tissue. A protein expression study of LCM tissue is underway. Additionally, FFPE kidney tissue has been evaluated (formalin-fixed paraffin-embedded) for protein extraction. Initial experiments using FFPE kidney tissue of rat compared extraction efficacies of proteins from FFPE tissues using home-made and published protocols. The best results were obtained when protein was extracted using DTT and a nonlabile detergent. Compatibility with mass spectrometry and sample size requirements are under investigation. 4. Proteomic Studies following Environmental Exposure. A quantitative serum proteomics study of serum from healthy individuals following time spent in an isolation chamber under conditions similar to a space-like life support system was designed to address potential long-term effects of space exploration. Plasma samples were taken at six different time points throughout a 103 day isolation period from six individuals. The samples were depleted of the 14 most abundant proteins, digested, and analyzed by LC/MSe. A manuscript reporting these results is being finalized. 5. Proteomic Studies of COPD Serum. A quantitative serum proteomics pilot study of serum from healthy individuals versus individuals with varying degrees of COPD is underway. ITIH4 (inter-alpha-trypsin inhibitor heavy chain H4) is perhaps a biomarker. Using 2D LC/MSe the potential for ITIH4 as a biomarker is being investigated. 6. Proteomic Studies of Oviduct Tissue. A quantitative proteomics study of proteins from oviduct tissue from wild-type and knock- out mice from oviduct tissue was conducted. Protein samples from each group of mice were pooled, digested, and analyzed using mass spectrometry. 7. Proteomic Studies of Yeast. A quantitative proteomics study of proteins from schizosaccharomyces pombe was conducted. A comparison of extraction buffers was performed and the mass spectrometry results that we obtained were evaluated. The results indicated that the best extraction buffer yielded about 500 proteins that could be quantitated.

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