As one of the most powerful techniques in analytical sciences, mass spectrometry (MS), coupled with the techniques for sample preparation and separation, plays an essential role on the rise of proteomics. In this dissertation, we focused on the development and application of MS and chemical tools for the identification and quantification of several important classes of proteins, including nucleotide-binding proteins, nucleic acid-binding proteins and plasma membrane proteins.
In Chapter two, we described the synthesis and application of a biotin-conjugated chemical probe in probing nucleotide-binding proteins. This activity-based chemical probe can react specifically with the lysine residue at the nucleotide-binding site. The strategy developed for proteomic application is relatively simple, fast, and straightforward. This method is useful in the activity-based functional study and in profiling nucleotide-binding proteins, e.g., kinases, in complex biological samples.
In Chapter three, a strategy for the comprehensive investigation of DNA-binding proteins with in vivo chemical cross-linking and LC-MS/MS was developed. DNA-binding proteins were isolated via the isolation of DNA-protein complexes and released from the complexes by reversing the cross-linking. By using this method, we were able to identify more than one hundred DNA-binding proteins, including proteins involved in transcription, gene regulation, DNA replication and repair.
In Chapter four, we described a strategy, including SILAC, cell-surface biotinylation, affinity purification and LC-MS/MS, for the identification and quantification of cell-surface membrane proteins. Integrins, cell adhesive molecules, CD antigens and receptors, which are essential in tumor development, were quantified in this work. The identification of aberrantly expressed membrane proteins of melanoma cells sets the stage for the future investigation of these proteins in the progression of human melanoma. The method held a great potential in the comprehensive identification of tumor progression makers, as well as in the discovery of protein-based new therapeutic targets.
In Chapter five, a comparative study of protein expression in human leukemia cells upon 6-thioguanine treatment was performed by using a MS-based proteomic method together with SILAC. The biological implications of the changed expression of some proteins were discussed. This study may offer new insights into the molecular mechanisms of action of this drug.
