Research Project
6934914
DESCRIPTION (provided by applicant): Proteomic analysis of laser capture microdissection (LCM) procured specimens is severely constrained by sample amounts ranging from 1,000-100,000 cells, corresponding to a total protein content of 0.1-10 microgram. Current proteome technologies, including two-dimensional (2D) polyacrylamide gel electrophoresis and shotgun-based multidimensional liquid chromatography separations, require large cellular samples that are orders of magnitude greater than those obtained during a clinical biopsy. Thus, this project aims to develop and demonstrate a capillary gel electrophoresis (CGE)-based multidimensional separation platform, capable of performing comprehensive and ultrasensitive studies of protein profiles within LCM procured specimens. A key feature of the proposed proteome technology is the simplification of many of the common sample handling steps, as the tissue sample acquired through LCM is directly processed and applied to the inlet end of the CGE capillary through electrokinetic injection and stacking of SDS-protein complexes. This feature allows the quantitative use of limited protein samples by combining analyte concentration, protein/peptide separations, and in situ proteolytic digestion in an integrated platform while eliminating analyte loss and dilution to achieve comprehensive and ultrasensitive proteomic studies. Several performance factors in the proposed multidimensional separation platform equipped with ESI-qTOF MS, including the protein concentration coefficient of at least 500-fold contributed by electrokinetic stacking, the dynamic range (the ratio of high abundance to low abundance proteins) of 100,000: 1 or higher, and the overall peak capacity of more than 70,000, will be evaluated using yeast cell lysates containing model proteins such as ribonuclease A, casein, and green fluorescence protein of known concentrations. The analytical capabilities of the proposed proteome technology will be demonstrated using LCM procured tissue specimens obtained from Professor Wittliff's laboratory at the University of Louisville. The respective scientific milestones include the use of 1 microgram or less total protein loading for enabling the identification of at least 1,000 proteins from tissue specimens with greater than 80% reproducibility in identified proteins among replicates.
