PhD defence: Mass Spectrometry-Based Phosphoproteomics: From Method Development to Application

PLEASE NOTE: If a candidate gives a layman's talk, the livestream will start fifteen minutes earlier.

Phosphorylation is one of the most ubiquitous and dynamic post-translational modifications in biology, functioning as a molecular switch that regulates protein activity, localization, interactions, and turnover across virtually all cellular pathways. Through reversible phosphate-group addition and removal, cells rapidly adapt to internal and external signals. Mass spectrometry (MS) has become the method of choice for studying phosphorylation because it enables site-specific identification and high-throughput quantification of phosphorylation events across complex proteomes.

This thesis aimed to develop and apply advanced MS-based approaches to better understand phosphorylation signaling in diverse biological contexts, including cancer, cellular secretion, and infection. The work is organized around three central themes: optimization of sample preparation workflows, development of novel mass spectrometry strategies, and integration of phosphoproteomics with biological assays to interpret signaling mechanisms and phenotype changes.

Chapter 1 provides an overview of protein phosphorylation biology and the technological foundations of MS-based phosphoproteomics. Chapter 2 describes the optimization of the suspension trapping workflow for phosphoproteomics, demonstrating that replacing phosphoric acid with trifluoroacetic acid significantly improves phosphopeptide enrichment while maintaining global proteome performance, particularly benefiting low-input extracellular vesicle samples. Chapter 3 introduces Targeted Signal Amplification of Phosphopeptides (TSAP), a strategy combining tandem mass tag labeling with real-time MS3 acquisition to improve quantification sensitivity for low-abundance phosphopeptides while retaining discovery-driven profiling. Chapter 4 investigates distinct proteomic and phosphoproteomic responses of PIK3CA H1047R and E545K mutants, revealing mutation-specific adaptations and showing that insulin can rescue PI3K inhibitor effects through pathway reactivation and adaptive signaling. Chapter 5 summarizes key findings and outlines future opportunities, highlighting how continued advances in phosphoproteomics will support the development of personalized medicine.