True understanding of biological function at the molecular level requires thorough examination of protein variation resulting from differences in genetics, protein structure or post-translational modifications (PTMs). Top-down MS (TDMS) is an invaluable tool for the in-depth characterization of these protein variants, termed proteoforms. In TDMS, ion activation is used to dissociate the covalent bonds making up the protein backbone. These covalent bonds break in a predictable fashion, enabling the amino acid sequence of the protein, including modifications to its residues, to be determined from the molecular masses of the observed fragments. Under the right conditions, TDMS can provide almost complete sequence coverage, and therefore comprehensively characterize PTMs, single nucleotide polymorphisms (SNPs), and splice or truncation variants. Whilst cleavage of every inter-residue bond in a protein to enable complete coverage is desirable in characterizing these modifications, it is rarely achieved in practice, with the exception of very small model proteins. This is attributed to the relatively low fragmentation efficiency of protein species as they get larger and adopt relatively low charge states. As such, considerable effort in recent years has been placed in novel instrumentation and approaches to overcome these limitations in fragmentation efficiency. Ignited by these developments, sufficient sequence coverage has been achieved to prove the power of TDMS to identify proteins and characterize proteoforms in a variety of biological areas, including in complex systems such as human biofluids and tissue samples. Historically, to maximize sequence coverage and increase proteoform identification, TDMS has been performed under denaturing conditions, making the protein backbone more accessible for fragmentation. Recently, however, native top-down mass spectrometry (nTDMS) has come to the fore, in which proteins are introduced into the mass spectrometer using native sample preparation and soft ionization conditions to retain their solution-state characteristics. The major benefit of nTDMS is that protein-protein and protein-ligand interactions remain intact, and therefore proteoforms are characterized within this context. While protein fragmentation patterns observed in TDMS have provided exquisite proteoform detail to date, this advent of nTDMS has added an extra dimension to the analysis by enabling attempts to derive structural information from fragmentation patterns. Since their development in the late 1990s, electron-based fragmentation techniques have proved particularly fruitful in this endeavor, with the product ions produced accepted to be sensitive to protein structure. Similar experiments have even been used to successfully map key biological features on the protein backbone such as protein complex interfaces, regions of helicity, and ligand-binding sites. Ultraviolet Photodissociation (UVPD) has similarly shown exciting promise for linking protein fragmentation to 3-dimensional structure. Notable structural insights ranging from characterization of protein-protein interaction and ligand binding regions through to documenting unfolding pathways have been revealed using the UVPD approach. Furthermore, recent attempts have proven successful in linking protein structural moieties to fragmentation patterns obtained from infrared photon-based modalities, alongside collision-based methods previously thought to be unable to report on conformational elements. A key limitation in the use of nTDMS to inform on protein conformation, however, is that the majority of instruments currently used in such studies lack ion mobility (IM) separation and the ability to separate and isolate conformers prior to activation. While this is not an issue for primary structural characterization, it becomes problematic when co-existing conformers exist, as the fragmentation patterns will be a composite of the two conformers rather than a reflection of a single structure. This inability to link top-down information to individual conformers hampers biological interpretation of the data, especially when structural differences are key to protein function or disease pathogenesis. Proteins with multiple conformations which have different activity are treated as a single species, rather than being studied individually for their unique characteristics. Similarly, in the case of protein misfolding, distinctive features of incorrectly folded proteins would be indistinguishable from the correctly folded analog. Depending on whether a protein or protein -ligand complex is analyzed, different workflows can be followed. For a protein with more than one conformation, tandem ion mobility can be u sed to isolate each conformer for ECD fragmentation, thus obtaining conformer-specific top-down data (blue line). For protein-ligand complexes, two workflows exist. ECD fragmentation of the complex, which can reveal structural changes due to ligand binding (orange line), or dissociation of the complex followed by isolation of each component using ion mobility, followed by ECD or CID fragmentation (red line). This workflow allows identification of the bound ligand.