Providing "Freeze Frames" of Cellular Processes by Thermal Stabilization of Samples: The Inactivation of Proteases, Preservation of Molecular Complexes, and Elimination of Apocryphal Spots On Two-Dimensional Gels

An understanding of cellular processes is facilitated only when methods such as two-dimensional gel electrophoresis (2DGE) are capable of isolating without bias the entire protein constituency of cells and tissues such that the analysis provides a comprehensive representation of the proteome. The regulated expression of proteins as it relates to normal or disease processes, the efficacy of therapeutic regime, or the identification of phenotypes requires that specific proteins are accurately quantified. Therefore, reliable methods of sample preparation become necessary to produce meaningful results. Since proteases regulate many cellular processes including the clearance of down-regulated proteins and the cleavage of precursors to produce functional proteins, it is essential to completely abolish all enzymatic activity to elicit a realistic “snapshot” of the proteome as it occurs in vivo, both qualitatively and quantitatively. Conventionally, proteolysis is attenuated by rapidly freezing the sample and by the inclusion of protease inhibitors and metal chelators. However, the formation of apocryphal protein spots in 2D gels indicates the inability to completely halt enzyme activity before protein fragmentation can occur. Further, sample processing artifacts and alterations of proteomes caused by in vitro degradation may obscure important information of a sample's biological state and prevent discovery of biomarkers. An alternative is to use rapid heat inactivation to eliminate enzymatic activity. Heat inactivation was evaluated and shown to avoid degradation and improve results by decreasing proteolytic fragments.