This thesis describes the development of a new, powerful concept and approach to study signalling pathways based on top-down quantitative phosphoproteomics. This approach, called the Phospho-SILAC strategy, combines a first step of in-house developed phosphoprotein enrichment using Immobilized Affinity Metal Chromatography (IMAC), and Stable Isotope Labelling in Cell Culture (SILAC) with labelled arginine containing six 13C atoms. NRK49F cells grown in medium containing 13C6 Arg (heavy arginine, hR) were stimulated for short times with EGF and the cell lysate obtained was mixed in a ratio 1:1 with the lysate from unlabelled cells, used as control. Phosphoproteins were then enriched from the protein mixture using IMAC and displayed on 2D gels, allowing multiple isoform separation. Individual post-translational changes in different proteins and in different isoforms of the same protein upon EGF stimulation are described. Major focus was given to Hsp8 isoforms, as they were found to be differentially involved in EGF signalling when tested with antibodies against MAPK/CDK and PCK substrates. A potential MAPK phosphorylation site on Hsp8 N-terminal sequence was identified. The Phospho-SILAC strategy was also applied to Rho and Rab GTPases and their corresponding GDI (guanidine nucleotide dissociation inhibitor) molecules. Phosphorylation might regulate the binding between GTPase-GDI. The results obtained also suggested that RhoGDI-1 activity could be regulated by arginine methylation. A potent new concept and methodology is outlined for wider investigations of molecular dynamics and for guiding the selection of candidate proteins and their modification for further studies.