Figure 1. Schematic workflow of the hybrid MS approaches for the structural analysis of protein heterogeneity.

High-resolution native MS is used to qualitatively and quantitatively profile all co-appearing proteoforms and middle-down proteomic experiments are performed to characterize each modification site. The data from the site-specific PTM characterization obtained by middle-down proteomics is used to construct a native MS spectrum. A standard Pearson correlation test is conducted between the constructed and experimental native MS spectra, or between different native MS spectra to quantitatively assess the structural similarities.