Abstract
The crucial roles of proteoforms in biological processes and disease mechanisms have been increasingly recognized. However, the rate at which new proteoforms are being discovered using top-down proteomics has far outpaced the rate at which the functional significance of different proteoforms can be determined. Because of the close connection between protein folding and protein function, protein folding stability measurements on proteoforms have the potential to identify functionally significant proteoforms of a given protein. While a number of mass spectrometry-based proteomics methods for making protein folding stability measurements on the proteomic scale have been reported over the past decade, none have been interfaced with top-down proteomics. Described here is a top-down (TD) stability of proteins from the rates of oxidation (SPROX) approach for making proteoform specific folding stability measurements. This approach is validated using a mixture of three model proteins with well-characterized protein folding behavior by conventional SPROX as well as other more conventional biophysical techniques. The method is also used to evaluate the relative folding stabilities of the <30 kDa protein fraction isolated from an MCF-7 cell lysate. The relative folding stabilities of 150 proteoforms from 83 proteins were successfully characterized in the cell lysate analysis using the TD-SPROX approach.