Proteases for Proteomics

The term proteome refers to all proteins expressed in a specific cell, tissue or organism.  Proteomes form extremely complex and highly integrated networks of interactions.  These networks are dynamic and vary with cell type, metabolic state and disease state.  The complex and dynamic nature of proteomes makes them rich with useful information. 

Current methodologies remain inadequate for achieving the full potential of proteomic analysis.  The basic enzymological tool for characterizing a proteome is the protease. Proteases are already an essential part of proteomic analysis, but more sophisticated tools are needed to identify low abundance proteins in highly complex samples. 

Our goal is to develop a suite of site-specific proteases that cut with high specificity but with different frequency.  The basic idea is to cut a sub-population of proteins that contain a specific sequence motif and then to resolve the population of cleaved proteins from the uncleaved.  This would produce a sequence-filtered slice of a proteome. The identity of this subset of proteins would be known from searching protein databases for the cognate motif. 

Two basic characteristics will determine the effectiveness of a protease for this type of proteomic analysis:  1) Frequency – how often the cognate motif occurs in a proteome and 2) Specificity – the activity of the protease against the cognate motif relative to others.  Frequency determines resolution. 

When every protein is cut, there is no resolution in the sequence dimension. The lower the frequency of cutting the higher the resolving power of the protease. At the extreme, a protease may be engineered to cut only a single protein (e.g., a biomarker) in a given proteome allowing its detection without fractionation. 

The specificity of the protease determines the background it produces.  The higher the specificity, the greater the ability of the protease to detect low abundance proteins in a complex mixture.  Our goal is to produce a tool box of high-specificity proteases that cut amino acid sequence motifs with different frequencies of occurrence.