Proteomic Strategies To Identify Protein-Ligand And Protein-Cell Interactions

Proteins mediate virtually all biological process. They function through interactions with other proteins as well as other molecules. In this dissertation, studies have been made on setting up new proteomic strategies for identification of real-time protein-ligand interactions and for global analyses of protein-cell interactions.The4Facts strategy-Fast Fix, Fish, and Filter, can be used to comprehensively, sensitively and precisely identify real-time protein-protein interactions without prior intervention. In this strategy, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncross-linked complexes and simultaneously providing molecular weight information for identification. Only proteins that simultaneously satisfy the following three criteria can be considered true ligands:1. the target protein must be identified in the same slice as its ligands;2. the ligands must be identified in slices for the experimental group but not in the corresponding, neighboring or higher control slices;3. the ligands appear in the range of molecular weights equal to or greater than the sum of the theoretical molecular weights of the target and the ligand. Using albumin and transferrin as examples, the4Facts strategy was applied to identify protein-ligand interactions in human blood. Human blood was directly added to10%formaldehyde for5s of cross-linking. The use of this strategy identified35ligands for albumin and52ligands for transferrin. Comparison with three major previous studies of the albuminome revealed that68.57%of the35albumin ligands identified in our study were also identified in these other studies. Since fast fixing of5s was achieved, this strategy can be potentially used in identification of dynamic protein-protein interactions, and become a new standard in future.The4S strategy-Surface Shaving and Species Specificity, can be used to achieve global analyses of protein-cell interactions between different species. Bovine serum-human Ca Ski cell interaction was used as an example to illustrate this strategy. Cells cultured in bovine serum were washed three times with PBS and then incubated with trypsin. The supernatant after cell surface shaving were send to MS analysis. Bovine serum and Ca Ski cell lysate were analyzed as control. Many proteins were reported in protein groups and cannot be quantified because of shared peptides. We assigned SpC (spectral counts) of shared peptides to different proteins according to the SpC ratio of species specific peptides in those proteins. Bovine proteins (proteins with bovine specific peptides) and human proteins (proteins with human specific peptides) were quantified by spectral counting using NSAF values. Using this strategy,30bovine proteins were identified as proteins that interact with Ca Ski cells as they were enriched comparing with bovine serum. They might play important roles in cell culture. Most of them have binding function with GO annotation. In addition,216human proteins were enriched comparing with cell lysate. They might be cell membrane proteins or secreted proteins. This strategy achieved global analyses of protein-cell interactions between different species, and can be potentially used in identification of pathogen-host microenvironment interactions, etc.