S.R. Piersma1, K.J. Gotink2, M. Labots2, R. de Haas2, H. Broxterman2, H. Verheul2, C.R. Jiménez1
1OncoProteomics Laboratory and 2Angiogenesis Laboratory, Dept. Medical Oncology, VU University Medical Center
Grant: VUmc CCA
Abstract
Background Protein phosphorylation is one of the key post translational modifications in intracellular signal transduction. It has many regulatory functions like homeostasis of body's water content, protein-protein interaction and degradation of proteins. Kinases (518 distinct enzymes in human) catalyze the addition of a phosphate molecule from ATP to a serine, threonine and tyrosine -OH group of a protein. This covalent modification is recognized by downstream targets of the phosphorylated protein and can induce propagation of the signal. Kinases themselves are often phosphorylated and this phosphorylation can be correlated with kinase activity.
Phosphorylation analysis by proteomics is centered around isolation, detection and analysis of phosphopeptides. First, (Phospho)proteins in a cell lysate are enzymatically digested in to (phospho)peptides. The critical step is selective capture of phosphopeptides from an excess of non-phosphorylated peptides. Several techniques are available for global phosphopeptide capture: immobilized metal affinity capture (IMAC), Titanium dioxide (TiOx) or strong cation exchange (SCX) chromatography. More recently also peptide immunoaffinity capture is being used, initially for selective phosphotyrosine (pY) capture, and now also for specific Ser/Thr motifs (pS/T). After phosphopeptide enrichment, peptides are separated by C18 nanoHPLC and detected by MS/MS and database searching.
Aim 1. Implement methods for global tyrosine phosphopeptide capture and 2 application of phosphotyrosine profiling to the analysis of the effects of targeted kinase inhibitors in cancer cell lines
Results and outlook. We have implemented an antibody-based method for precipitation of phosphotyrosine peptides that can profile hundreds of phosphotyrosine peptides in a cancer cell lysate. In conjunction with an optimized database search pipeline, over a thousand phosphopeptides were identified in a single analysis. Currently we are applying phosphor-tyrosine proteomics to the analysis of sensitive and resistant colorectal cancer cell lines +/- sunitinib. We expect that these experiments will provide novel insights into resistence mechanisms and identify novel biomarkers (key targets and phosphorylation events) for patient stratification.