Due to the increased threat of terrorism there is currently a need to develop sensitive and specific methods for the detection and identification of biological threat agents. Here, the utility of the bottom-up tandem mass spectrometry (MS/MS) approach was investigated for the detection and identification of biological threat agents based on the analysis of unique protein biomarkers. Specifically, alternative methods for bacterial cell lysis and sample clean-up were investigated in order to address the challenges associated with the analysis of small sample quantities likely to be encountered in biological threat agent detection and identification. Guanidine hydrochloride was shown to be an effective reagent for achieving cell lysis at small scales, even for as little as 1 mg of starting cellular material. The number of proteins identified following guanidine hydrochloride lysis was found to be comparable with the number of proteins typically identified following large-scale lysis by sonication. This technique also demonstrated minimal bias in terms of the types of proteins which were identified. For sample clean-up prior to MS/MS analysis, on-line desalting was found to be an effective technique. The 2D linear quadrupole ion trap was also shown to significantly outperform the traditional 3D quadrupole ion trap, with over 2, 000 proteins confidently identified following lysis of 1 mg of starting cellular material. Finally, the small-scale bottom-up MS/MS approach was successful in identifying proteins derived from a complex microbial mixture.Perhaps one of the greatest challenges in analyzing limited quantities of starting cellular material is bacterial cell lysis. ... French press, on the other hand, passes cells through a narrow valve under high pressure (typically 10, 000-20, 000 psi). ... techniques which could be utilized including enzymatic- [40-43] and detergent- based methods, as well as the combined use of solvents and detergents [44, 45].
|Title||:||Development of a Small-scale Single-tube Proteomics Approach for the Analysis of Protein Biomarkers from Biological Threat Agents|
|Publisher||:||ProQuest - 2008|