The combined use of electrospray ionization run in so-called "native mode" with 
top-down mass spectrometry (nTDMS) is enhancing both structural biology and 
discovery proteomics by providing three levels of information in a single 
experiment: the intact mass of a protein or complex, the masses of its subunits 
and non-covalent cofactors, and fragment ion masses from direct dissociation of 
subunits that capture the primary sequence and combinations of diverse 
post-translational modifications (PTMs). While intact mass data are readily 
deconvoluted using well-known software options, the analysis of fragmentation 
data that result from a tandem MS experiment - essential for proteoform 
characterization - is not yet standardized. In this tutorial, we offer a 
decision-tree for the analysis of nTDMS experiments on protein complexes and 
diverse bioassemblies. We include an overview of strategies to navigate this 
type of analysis, provide example data sets, and highlight software for the 
hypothesis-driven interrogation of fragment ions for localization of PTMs, 
metals, and cofactors on native proteoforms. Throughout we have emphasized the 
key features (deconvolution, search mode, validation, other) that the reader can 
consider when deciding upon their specific experimental and data processing 
design using both open-access and commercial software.