The discovery of protein biomarkers is severely hindered by the high abundance proteins which are uninteresting and can mask the presence of the putative marker. Due to the increased sensitivity of low molecular weight component analysis, the presence of a protein can oftentimes be determined by measurement of its derivative products, even when no protein marker is evident. SeraFILE™ provides such a rational tiered method for isolating the causative 'needle in the haystack' protein(s). SeraFILE™ can deliver multi-faceted proteomic signatures combining elements of protein abundance as well as functional activities.
These elements can be used to "score" the various pools for enrichment of desirable activity. Each sub-proteome is scored - by bioassay, HPLC, NMR, or mass spectrometry, relative to its total protein content. The high-score fractions are selectively applied to additional tiers of enrichment; each tier producing successively greater levels of purification. Because functional activity is maintained in all pools, Functional Proteome Prospecting™ can be applied to any measurable metabolite or byproduct. Also, through pool recombination, Functional Proteome Prospecting™ techniques can be applied to discover important enzyme regulating factors..
Functional Proteome Prospecting for Specific Activity
In this example, an enzyme profile of Alkaline Phosphatase in Sheep Serum compares the activity to the total protein content for each SeraFILE™ derived sub-proteomes. The numbers on the x axis designate different SeraFILE™ surfaces. By comparing specific activities, an enrichment factor can be calculated showing which sub-proteome had the highest enrichment score. In this case, it was surface number 8, with the second elution.
Low Abundance Protein Enrichment
The SeraFILE™ surface library can be used in serial strategies for enrichment of low abundance proteins. Like peeling an onion, one surface can void one or more high abundance proteins (see serum example), and concentrate the low abundance proteins. If necessary, more than one surface can be used in series, to provide maximal enrichment and resolution of proteins from gel electrophoresis visualization.
SeraFILE™
