Development of proteomic characterization and speciation techniques utilizing tryptic peptides with MALDI-TOF MS and LC-ESI MS-MS.

摘要

The characterization of microbes which can be opportunists and pathogens (e.g., methicillin resistant Staphylococcus aureus (MRSA)) is important in understanding and potentially treating diseases caused by various bacterial species. Common genera found in the human skin micro-biome include Micrococcus and Staphylococcus, but there only a limited number of tests to differentiate these genera and/or species. My research reflects methods development from distinguishing one closely related genera from another and then expanded to species identification. Tryptic peptides were analyzed by MALDI TOF MS and the mass profiles compared with those of a reference strain in both genus and species identification. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor tu provided the highest confidence in staphylococcal speciation. Ultimately, refinement in speed and accuracy of analysis was accomplished with LC ESI MS-MS for peptide analysis on whole proteomes and data analysis also employing a customized X!tandem database allowing for successful identification and differentiation of the 11 Staphylococcus species commonly found on humans (genomes gathered from UNIPROT). The methodological approach described can be utilized for bacterial identification across multiple species, creating more accurate methods for identification of species that may be pathogenic to humans.