Quantitative Phosphoproteomics of Colon Cancer Initiation and Progression

Phosphoproteomics is a valuable tool for mapping molecular signaling events and dissecting the interactions among biological pathways. Despite many advances in the sample preparation methods and instrument technologies used for liquid chromatography – mass spectrometry analyses of protein phosphorylation, there is still a lack of consensus regarding best practices in this field. In particular, the chemistry used to enrich phosphopeptides from complex cell lysates is especially critical to a comprehensive, sensitive, and accurate phosphoproteomics experiment. This dissertation compared two popular phosphoproteomics enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO2)-based metal oxide affinity chromatography (MOAC). A multi-step enrichment protocol incorporating benchtop high-pH reverse phase fractionation, developed in the Hummon lab, minimizes the amount of peptide starting material required for maximal recovery from both IMAC and TiO2. The two chemistries were found to enrich phosphopeptides equally well, but favor unique subgroups of peptides. TiO2 enriches peptides that are slightly shorter, more acidic, and more hydrophobic than IMAC does. The improved enrichment protocol was applied to two cancer-relevant signaling networks. The first examined constitutive phosphorylation differences between a primary tumor-derived cell line and its patient-matched lymph node metastasis cell line. This study of the phosphoproteomes of SW480 and SW620 revealed that the lines differ significantly in their regulation of cellular adhesion, mitosis, and mRNA translation. In the second study, a single-step TiO2 enrichment allowed characterization of signaling networks affected by low dose and high dose ionizing radiation in non-malignant breast cell line MCF10A. This revealed that ATM & DNAPK kinases are induced by 10 Gy of γ-rays but not 0.05 Gy. In contrast, the MAPK signaling response to IR was dose-independent. Taken together, this work demonstrates the utility of the IMAC – TiO2 – high pH reverse phase workflow to maximize phosphoproteome coverage and to generate novel biological insights.