Extended Data Fig. 2: Denaturing and native top-down mass spectrometry measurements offer distinct data for proteoform identification.

a, Schematic overview of a typical denaturing top-down mass spectrometry measurement. Protein ions are generated via electrospray ionization from acidified mixtures of aqueous and organic solvents, often following online separation (for example, liquid chromatography). The resulting ions are typically monomeric, highly-charged, and often small and homogenous, facilitating analysis by high- resolution mass spectrometry. Fragmenting protein ions from denaturing solutions generally yields product ion spectra with high signal-to-noise ratios. The masses of the precursor and fragments can be used to identify the precursor ion by searching a library of theoretical proteoforms, such as through a traditional open search with a ±1–500 Da precursor tolerance. b, Schematic overview of a native top-down mass spectrometry (nTDMS) measurement. Intact protein complexes are ionized from electrolyte solutions at physiological pH, typically using a static nanoelectrospray ion source. The resulting ions can comprise of multiple subunits (it is often not possible to dissociate individual subunits in the gas phase), each potentially bearing diverse modifications, affording a heterogenous set of unresolvable molecular species. Product ion spectra are typically of lower quality when compared to denaturing analyses, with reduced sequence coverage. Additionally, in targeted nTDMS studies, proteoforms with unexpected or uncommon post- translational modifications (PTMs) are frequently of particular interest—these PTMs are not represented in standard proteoform libraries. PrSM, proteoform–spectrum match.