The proteome can be defined as the complete set of global protein expression by an organism at any given time. It is this gene expression rather than genome itself that is responsible for most of the reactions taking place within a cell. Tools such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectroscopy (MS) are often utilized for the analysis and identification of the proteins that constitute the proteome. This thesis provides a general overview of proteomics and describes the underlying processes, mechanisms and technologies associated with modern proteomic analysis with specific emphasis on the use of electrospray ionization time of flight mass spectrometry. The use of MS/MS analysis of ESI-QTOF data for the identification of proteins is also explained in detail. Other chromatographic approaches such as high performance liquid chromatography (HPLC), where separation is based on the polarity of the mobile phase, and gel filtration chromatography systems, where separation is based on the molecular size, are also described in detail. The thesis work focuses on identifying the proteomic signature in the soil bacterium Pseudomonas putida using these analytical tools, particularly LC/ESI-Q-TOF MS (Electrospray Ionization Time of Flight Mass Spectrometer). Protein digest analysis was done with lactoperoxidase (LPO), lysozyme, and ribonuclease on HPLC. Whole protein and tryptic peptides were analyzed on the system. Peaks corresponding to the whole proteins as well as the peptides generated from the tryptic digestion proteins were observed. The goal of the entire project is to isolate proteins from the bacteria Pseudomonas Putida strain KT2440 using biochemical techniques to first separate them using 2-D PAGE, subsequently performing in-gel tryptic digest, and finally identifying individual protein spots on the gel using LC / ESI-Q-TOF MS and protein databases.

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Note: imported from RIT’s Digital Media Library running on DSpace to RIT Scholar Works in December 2013. Physical copy available through RIT's The Wallace Library at: QP551 .P36 2007


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