Top-down protein mass spectrometry can provide unique insights into protein sequence and structure, including precise proteoform identification and study of protein–ligand and protein–protein interactions. In contrast with the commonly applied bottom-up approach, top-down approaches do not include digestion of the protein of interest into small peptides, but instead rely on the ionization and subsequent fragmentation of intact proteins. As such, it is fundamentally the only way to fully characterize the composition of a proteoform. Here, we provide an overview of how a top-down protein mass spectrometry experiment is performed and point out recent applications from the literature to the reader. While some parts of the top-down workflow are broadly applicable, different research questions are best addressed with specific experimental designs. The most important divide is between studies that prioritize sequence information (i.e., proteoform identification) versus structural information (e.g., conformational studies, or mapping protein–protein or protein–ligand interactions). Another important consideration is whether to work under native or denaturing solution conditions, and the overall complexity of the sample also needs to be taken into account, as it determines whether (chromatographic) separation is required prior to MS analysis. In this review, we aim to provide enough information to support both newcomers and more experienced readers in the decision process of how to answer a potential research question most efficiently and to provide an overview of the methods that exist to answer these questions.

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