Methods for Characterization of Protein Aggregates

: Physicochemical characterization of protein aggregates is important on one hand, due to its large impact in understanding many diseases for which formation of protein aggregates is one of the pathological hallmarks. On the other hand, recently it has been observed that bacterial inclusion bodies (IBs) are also highly pure proteinaceous aggregates of a few hundred nanometers produced by recombinant bacteria supporting the biological activities of the embedded polypeptides. From this fact arises a wide spectrum of uses of IBs as functional and biocompatible materials upon convenient engineering but very few is known about their physicochemical properties. In this chapter we present methods for the characterization of protein aggregates as particulate materials relevant to their physicochemical and nanoscale properties. Specifically, we describe the use of infrared spectroscopy (IR) for the determination of the secondary structure, dynamic light scattering (DLS) for sizing, nanosight for sizing and counting, and Z-potential measurements for the determination of colloidal stability. To study their morphology we present the use of atomic force microscopy (AFM). Cryo-transmission electron microscopy will be used for the determination of the internal structuration. Moreover, wettability and nanomechanical characterization can be performed using contact angle (CA) and force spectroscopic AFM measurements of the proteinaceous nanoparticles, respectively. The physical principles of the methods are briefly described and examples of data for real samples and how that data is interpreted are given to help clarify capabilities of each technique.