CE-based sample quality assessment prior to 2-D gel electrophoresis: towards the standardization of gel-based proteomics.

2-DE remains one of the most commonly used separation techniques for complex protein mixtures. This article describes a new approach to 2-DE sample assessment using SDS capillary gel electrophoresis (in Beckman Coulter sieving medium) combined with multi-pixel detection. The performance of this platform was investigated using protein samples prepared for 2-DE. The capability to characterize 2-DE sample was tested and the results show that the repeatability of peak migration time and intensity are better than 2% RSD. The system gives good resolution, accurate molecular mass assignment, as well as absolute and relative quantification of proteins. Notably, this study also demonstrates the use of this platform to screen the quality of simple and complex 2-DE samples. Implementation of this technique in the proteomics workflow will not only improve the success rate of 2-DE, but will also enable sample verification before 2-DE and allow the relative quantification of proteins. The validation of differential protein expression is also demonstrated using the combined information of relative molecular mass and relative quantification. It is the first time that a rapid and visual evaluation method is reported for the quality assessment of 2-DE samples.

Development of reagents and assays for the detection of pathogenic Burkholderia species.

Rapid detection of the category B biothreat agents Burkholderia pseudomallei and Burkholderia mallei in acute infections is critical to ensure that appropriate treatment is administered quickly to reduce an otherwise high probability of mortality (ca. 40% for B. pseudomallei). We are developing assays that can be used in clinical laboratories or security applications for the direct detection of surface-localized and secreted macromolecules produced by these organisms. We present our current medium-throughout approach for target selection and production of Burkholderia macromolecules and describe the generation of a Fab molecule targeted to the B. mallei BimA protein. We also present development of prototype assays for detecting Burkholderia species using anti-lipopolysaccharide antibodies.

N255 is a key residue for recognition by a monoclonal antibody which protects against Yersinia pestis infection.

Mab7.3 to Yersinia pestis LcrV antigen (LcrV(Ype)) protected J774A.1 macrophages in vitro from killing by a Yersinia pseudotuberculosis strain expressing LcrV(Ype). Of 4 site-directed mutations in the coiled-coil region (148-169) and 7 mutations in the 225-255 sequence of LcrV(Ype), only the mutation of N255 to D255, abrogated the binding of Mab7.3 and reduced its protective capacity against plague. Since the Mab7.3 epitope in LcrV(Ype) (135-275) encompasses a region (136-180) thought to be exposed on the injectisome, we suggest that Mab7.3 protects by binding to LcrV(Ype) and interfering with protein-protein interactions necessary for type three secretion.