This chapter will provide an overview of electron capture dissociation (ECD) and electron transfer dissociation (ETD) in the context of the sequence analysis of polypeptides ionized under denaturing conditions. ECD and ETD require multiply charged cations (typically protonated: [M+nH]ⁿ⁺ where n is the number of added protons) to generate sequence informative product ions, and modern proteomic applications are often based on the coupling of reversed-phase liquid chromatography (RPLC) and mass spectrometry (MS). Therefore, throughout this chapter we will implicitly refer to experiments where electrospray ionization (ESI) is used to generate polypeptide cations in the presence of organic solvents (e.g., methanol, acetonitrile) and mild acids (e.g., formic acid, acetic acid). Here we do not intend to discuss in detail the principles and theory of ECD and ETD, and instead we redirect the readers to the previous chapter of this volume for a thorough description of their proposed mechanisms. However, it is important to first lay the foundation of the topics covered here by describing the general characteristics of ECD and ETD experiments and the products they generate, in addition to the instrumentation requirements for performing these gas-phase reactions. Next, we will analyze the role played by specific features of the analytes, namely charge density and sequence composition, and discuss their effects on the ECD/ETD process. We will then begin to decipher the importance of residual higher-order structure in directing polypeptide fragmentation. Finally, we will examine the most relevant applications of these ion dissociation techniques, distinguishing them as a function of the mass of the analyte under consideration. In doing so, we will discuss the scenarios where ECD and ETD can act as powerful tools for bottom-up, middle-down and top-down proteomics, and also for the investigation of post-translational modifications.