High throughput mass spectrometry for microbial identification.

摘要

Bacteria cause significant morbidity and mortality throughout the world, including deadly diseases such as tuberculosis, meningitis, cholera, and pneumonia. Timely and accurate bacterial identification is critical in areas such as clinical diagnostics, environmental monitoring, food safety, water and air quality assessment, and identification of biological threat agents2. At present, there is an established need for high throughput, sensitive, selective, and rapid methods for the detection of pathogenic bacteria, as existing methods, while nominally effective, have failed to sufficiently reduce the massive impact of bacterial contamination and infection. The work presented in this thesis focuses on addressing this need and augmenting conventional microorganism research through development of mass spectrometry (MS)-based proteomic applications. MS, a well established tool for addressing biological problems, offers a broad range of laboratory procedures that can be used for taxonomic classification and identification of microorganisms3, 4. These methods provide a powerful complement to many of the widely used molecular biology approaches and play critical functions in various fields of science. While implementation of modern biomolecule-identifying instrumentation, such as MS, has long been postulated to have a role in the microbiology laboratory, it has yet to be accepted on a large scale. Described in this document are MS methods that erect strong foundations on which new bacterial diagnostics may be based. A general introduction on key aspects of this work is presented in Chapter 1, where different approaches for detection of pathogenic bacteria are reviewed, and an overview regarding MS and microbial identification is provided. Chapter 2 presents the first implementation of microbial identification via rapid, open air Direct Analysis in Real Time MS (DART MS) to generate ions directly from microbial samples, including the disease-causing bacteria, Coxiella burnetii, Streptococcus pyogenes, and Escherichia coli. Chapter 3 expands on whole cell C. burnetii MS analysis and presents a rapid differentiation method to the strain-level for C. burnetii using mass profiling/fingerprinting matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and multivariate pattern recognition. Chapter 4 presents a unique “top-down” proteomics approach using 15N-labeled bacteriophage amplification coupled with MALDI-TOF MS as a detector for the rapid and selective identification of Staphylococcus aureus. Chapter 5 extends the idea of using isotopically labeled bacteriophage amplification by implementing a “bottom-up” proteomics approach that not only identifies S. aureus in a sample, but also quantifies the bacterial concentration in the sample using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) as a detector. In conclusion, Chapter 6, summarizes and contextualizes the work presented in this dissertation, and outlines how future research can build upon the experimentation detailed in this document.