Gram-negative bacteria have evolved an array of secretion systems for protein transport across their membranes. The ability to secrete a diverse set of proteins to their target of choice provides bacteria with a menacing tool for pathogenesis. This work aims to enhance our understanding of bacterial pathogenesis by characterizing key aspects of bacterial secretion systems using mass spectrometry. Chapter 1 provides a general introduction of protein secretion pathways in Gran-negative bacteria, with a special emphasis on type III and type VI secretion systems. Chapter 2 highlights chemical cross-linking and mass spectrometry (CXMS) as a technique for studying protein-protein interactions in large molecular complexes. Chapter 3 describes a novel pipeline for acquisition and analysis of CXMS data. In Chapter 4, key interactions between the basal components of type III secretion systems (T3SS) are identified, and used to create a model of the core structure of T3SS. In Chapter 5,...