LC-MS/MS-based methods for characterizing proteins

A method for characterizing the stability of a target protein in a simulated digestion assay, comprising the steps of:

• (a) subjecting a target protein to pepsin digestion;
• (b) obtaining a plurality of samples of peptides resulting from pepsin digestion at a plurality of time points and collecting LC-MS/MS data directly for each of the plurality of pepsin digestion samples at each of the plurality of time points without first enriching for membrane or microsomal fractions or separating the target protein by electrophoresis;
• (c) subjecting the plurality of samples of pepsin digestion samples to complete trypsin digestion and collecting LC-MS/MS data directly for each of the plurality of pepsin-trypsin digestion samples after trypsin digestion is completed for each of the plurality of time points without first enriching for membrane or microsomal fractions or separating the target protein by electrophoresis;
• and
• (d) determining from the LC-MS/MS data the kinetics of target protein digestion and the susceptibility to proteolysis of specific regions of the target protein by comparing the data from each of the plurality of time points of the pepsin digestion with the data from the corresponding dual pepsin-trypsin digestion, wherein the decline of tryptic peptides obtained from the dual pepsin-trypsin digestion is used as a proxy for intact target protein, and the appearance and disappearance of peptic peptides is used to indicate the in vitro digestibility of the target protein.