| The rapid development of proteomic techniques in the post-genomics era has allowed much attention to be paid to the understanding of molecular mechanisms of functional regulatory proteins. This amazingly fast transformation from "know-how" to "know-why" has allowed the determination of if functional regulation is based on up- or down-regulation, or based on any particular chemical modification on the amino acid residues of protein targets.;Here, different biologically important regulatory proteomes or protein targets were investigated using comprehensive combinations of proteomics techniques. A large emphasis was placed on expression techniques suitable for protein and protein binding partner affinity purification for targeted analysis of phospho-proteins. The main protein of interesting these particular studies was an important transcriptional factor in cardiac development, serum response factor-1. Several proteomics methods were examined and, along with the difficulties encountered, the results are presented. Small molecule-protein interaction was examined with the cell cycle regulatory protein Cdc25B incubated with an inhibitor. Post-translational modification-related functional proteomics were also studied, first in a rodent early hemorrhage model wherein S-nitrosylation of plasma proteins were determined, then the phosphorylation of serum response factor-1. Finally, general proteome mapping was represented by a detailed and very successful analysis of the proteins found enriched in brain tumor cell pseudopodia.;This dissertation illustrates the vast range of potential of proteomics technologies in studying function-related biological systems. Meanwhile, some pioneering work using targeted functional proteomics strategies was achieved, and the results will help in part to advance the field of "targeted functional proteomics", the combination of cell and molecular biological techniques with chemical, affinity and mass spectrometric approaches to study regulation of biological systems. |
