Proteolysis and Mass Spectrometry for Structural Characterization of Adnectin Inclusion Bodies

Abstract

Inclusion bodies (IBs) are cellular aggregates that commonly form upon overexpression of protein in heterologous hosts. The formation of IBs is of broad interest for protein production for research, medical or biotechnological applications, and soluble or insoluble forms such as self-immobilized catalysts, drug delivery systems, and functional protein release systems. These aggregates are known to be heterogeneous, containing different conformations of the constituent protein. Adnectins are engineered binding proteins with high global thermostability and expression levels but varying propensity to form IBs, providing an attractive system for studying IB formation. A single-point mutation and two sets of multiple-point mutations with increasing charged residues were designed to alter the Adnectin IB structure. Fourier Transform Infrared spectroscopy (FTIR) showed an increased amyloid-like signal for the four-point mutations. To further analyze IB structure, we combined protease digestion with mass spectrometry (MS). Proteinase K, as a non-specific protease, can serve as an analytical tool by conducting parallel experiments digesting unlabeled and 15N-labelled IBs, followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. This procedure facilitated the identification of digested fragments. Comparing the fragments from the Adnectin variant IBs reveals both similarities between the IBs as well as different protease-accessible sites indicating structural differences. This new procedure for protease digestion analysis provides a valuable complementary tool to help overcome challenges in characterizing heterogeneous aggregation processes and advance the understanding and, ultimately, control of IB structure.