Proteomic analysis of ubiquitinated proteins employing mass spectrometry-based techniques

Post-translational modification of proteins via the covalent attachment of Ubiquitin plays an important role in the regulation of protein stability and function in eukaryotic cells. In the second chapter of my thesis, a novel method for identifying ubiquitinated proteins from a complex biological sample, such as a whole cell lysate, using a combination of immunoaffinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is described. The applicability of this approach is demonstrated by the identification of seventy ubiquitinated proteins from the human MCF-7 breast cancer cell line after treatment with the proteasome inhibitor MG132. In the third chapter of my thesis, an application of a microfluidic protein processing device, termed the Proteomic Reactor, for enzymatic digestion of proteins for subsequent LC-MS/MS analysis is described. Use of the Proteomic Reactor following immunoaffinity purification enabled the identification of numerous low abundance ubiquitinated proteins from the human 1299 lung cancer cell line expressing reduced amounts of the ubiquitin-dependent chaperone known as the Valosin-Containing Protein. In the fourth chapter of my thesis, a method for determining peptide ion score cutoffs that permit the confident identification of ubiquitinated proteins by MS/MS is described. Experiments involving the analysis of gel bands containing multi-Ubiquitin chains with quadrupole time-of-flight and quadrupole ion trap mass spectrometers revealed that standard ion score cutoffs used for database searching after MS/MS analysis were not sufficiently stringent. False positive and false negative rates were also found to vary significantly depending on the Cutoff scores used and that appropriate cutoffs could only be determined following a systematic evaluation of false positive rates. When standard ion score cutoffs were used for the analysis of complex mixtures of ubiquitinated proteins, unacceptably high false positive rates were observed. Finally, false positive rates for ubiquitinated proteins were found to be affected by the size of the protein database that is searched. In chapters five and six of my thesis, two biological applications employing the methods I developed for identifying ubiquitinated proteins are described. The first application involves the identification of specific lysine residues within the membrane-anchored transcription factor Mga2p that are targets of the E3 ubiquitin ligase Rsp5p in Saccharomyces cerevisiae. Ubiquitination of these lysines were found to mediate mobilization of the Mga2p transcription factor complex and ist subsequent disassembly. The second application involves the identification of proteins that are ubiquitinated upon mitotic exit in Xenopus laevis embryos. Sequence analysis of these proteins revealed that the majority of poly-ubiquitination occurs through lysine-11 chain polymerization, which is an atypical form of ubiquitination