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Liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOF MS) utilizing electrospray ionization was employed to monitor protein expression in Escherichia coli and Shigella organisms. Automated charge state deconvolution, spectral subtraction and spectral mirroring were used to reveal subtle differences in the LC/MS data. Reproducible intact protein biomarkers were discovered based on their unique mass, retention time and relative intensity. These markers were implemented to differentiate closely related strain types, (e.g. two distinct isolates of E. coli O157:H7) and to correctly identify unknown pathogens. Notable, was the distinction of multiple serotypes of enterohaemorrhagic E. coli which cannot be distinguished by clinical manifestation alone. Additionally, speciation of Shigella was achieved, a task for which no commercial real-time polymerase chain reaction (PCR) primers exist. This method was subsequently applied to two pathogenic Clostridium species: C. difficile and C. perfringens. Due to the increased difficulty during lysis, two new lysis protocols were developed, and each extracted a distinct set of proteins (by both mass and retention time). Extracts from both lysis procedures were utilized to discover biomarkers useful for identification and characterization at the species and strain levels. These biomarkers were successfully implemented to identify unknowns during a blind study and would enhance serological and genetic approaches by serving as new targets for detection. Two sets of the C. perfringens isolates that were deemed 100% similar by the gold standard for strain differentiation, pulsed-field gel electrophoresis (PFGE), were distinguished using LC/MS, demonstrating the high specificity of this approach. The final part of this work demonstrated the application of ultra performance liquid chromatography (UPLC) to this project to improve the throughput of the method. Given that numerous small molecule applications of UPLC have been published, efforts were made to examine the potential of UPLC to enhance the separation of intact proteins. Beginning with typically employed conditions, column temperature and organic solvent were optimized followed by an HPLC vs. UPLC comparison. When applied to a mixture of ten protein standards, the optimized UPLC method yielded improved chromatographic resolution, enhanced sensitivity, and a three-fold increase in throughput. Application of this method to cell lysate analysis demonstrated no compromise in chromatographic or mass spectral data quality; a reduction in run time from 75 minutes to 25 minutes was achieved.

The analysis of large intact proteins and complex biological samples containing drug molecules is a common complicated task for many scientists. However, due to the importance of these molecules, there is a growing interest in pharmaceutical and medicinal research to develop rapid, highly sensitive and efficient analytical techniques. The advantages of capillary electrophoresis (CE) in combination with mass spectrometry (MS) provide a powerful analytical tool. However, further improvement and development of these techniques are required to extend their utility and to meet the challenges of selected analytes. Thus, the scope of this thesis deals with the development of novel analytical methods to achieve efficient and high performance analysis of peptides, intact proteins, digests of complex samples and basic pharmaceutical drug compounds in biological matrices. Implementation of CE for routine analysis of proteins and complex samples is constrained by the partial adsorption to the capillary wall. Consequently, the use of surface modified capillaries is required to control the surface properties and prevent analyte adsorption. In this thesis, analyte adsorption was successfully prevented using tailored covalent cationic (M7C4I) and electrostatic cationic (PVPy-Me) coatings. Rapid and efficient separations of peptides, proteins and digests of complex samples such as cerebrospinal fluids were obtained with these coatings. The M7C4I coating showed a distinct ability to handle large intact proteins with a molecular size of over 0.5 MDa. The highest peak efficiencies and surprisingly high peak stacking effects were obtained by adding salts to the protein samples. The effect of salt additives on peak efficiencies of intact proteins was further demonstrated and compared using different surface modified capillaries. Additionally, rapid CE-ESI-MS quantification of pharmaceutical drug molecules in human plasma was performed after a SCX-SPE sample preparation method using the M7C4I coating. In conclusion, the results presented in this thesis show the strong potential of CE in combination with MS using electrospray ionization (ESI) for the analysis of peptides and large intact proteins and the applicability for clinical monitoring of the levels of pharmaceutical drug molecules in human plasma with high sensitivity and efficiency. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 734