This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Protein deposition as amyloid is the basis of diseases that, overall, have an enormous social and medical impact. More than 21 different proteins are known to be causative agents. In the systemic forms, amyloid deposition is associated with dysfunction of vital organs, and molecular typing of the deposits is necessary for diagnosis and treatment. The traditional diagnostic approach is multidisciplinary, but sometimes fails to identify the correct type. A proteomic approach could help in the diagnosis, through the direct molecular characterization of fibrillar proteins in tissues, and cast insights into the mechanisms of tissue damage. We are working to characterize amyloid deposits in abdominal subcutaneous fat from patients with systemic amyloidosis using 2D-PAGE followed by MALDI-TOF MS and peptide mass fingerprinting. Fat tissue samples were obtained from individuals affected by various forms of systemic amyloidoses. Samples from unaffected volunteers were used as normal controls. Protein was extracted from fat tissue by homogenization directly in IEF buffer followed by ultracentrifugation to clear debris and delipidate samples. Samples were then subjected to 2D-PAGE analysis and Coomassie or silver staining. Protein spots were imaged and quantitated using PDQuest' software. Spots indicating differentially expressed proteins are being excised and subjected to in-gel digestion by trypsin or another protease. Peptides are eluted, de-salted and analyzed by MALDI-TOF MS or by LC-MS and MS/MS. Spectra are analyzed with MoverZ (M/Z') or MassLynx' and ProteinLynx' software, and peptide mass fingerprinting analysis utilizes MASCOT and/or BUPID. A rapid methodology to prepare sample for high-grade 2D-PAGE analysis from fat tissue has been developed. Fat tissues from unaffected (non-amyloid) volunteers were used to generate 2D reference maps for comparison with those generated from patients affected by systemic amyloidosis. Preliminary results arising from the comparison of these maps indicate that significant differences exist. The observations surrounding these differences fall into three categories. (1) New spots appear in the region of the gel consistent with the location where an amyloidogenic protein would typically be found. (2) There is apparent upregulation of some proteins in 2D maps from patient samples compared to those originating from unaffected volunteers. (3) New spots are observed in patient maps that are absent in the non-amyloid maps, suggesting the appearance of novel proteins. These new spots often appear in trains suggesting the presence of modified forms of the same protein. Identifation of the proteins responsible for these differences is being accomplished using in-gel protease digestion and a variety of MS techniques. The use of 2D-PAGE as a tool to highlight the differences between diseased and normal states is well known. Our proteomic approach to the analysis of fat tissues from patients for whom amyloid disease is suspected that should be able to provide a reliable diagnosis. This approach is practical and feasible, given that fat aspirates of potential amyloid patients are routinely acquired for histological analysis. Ongoing analyses may provide identification of new aspects of the disease mechanisms, including the involvement of novel proteins and protein post-translational modifications.
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