Size Exclusion Chromatography Strategies and MASH Explorer for Large Proteoform Characterization.

Top-down mass spectrometry (MS)-based analysis of larger proteoforms (>50 kDa) is typically challenging due to an exponential decay in the signal-to-noise ratio with increasing protein molecular weight (MW) and coelution with low-MW proteoforms. Size exclusion chromatography (SEC) fractionates proteins based on their size, separating larger proteoforms from those of smaller size in the proteome. In this protocol, we initially describe the use of SEC to fractionate high-MW proteoforms from low-MW proteoforms. Subsequently, the SEC fractions containing the proteoforms of interest are subjected to reverse-phase liquid chromatography (RPLC) coupled online with high-resolution MS. Finally, proteoforms are characterized using MASH Explorer, a user-friendly software environment for in-depth proteoform characterization.