ProFACT :: Rational Proteome Prospecting™

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, Rational Proteome Prospecting™ can be applied to any measurable metabolite or byproduct. Also, through pool recombination, Rational Proteome Prospecting™ techniques can be applied to discover important enzyme regulating factors..

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Ubiquitin/Proteasome Pathway (UPP) Characterization.
Background.The 20S proteasome forms the catalytic core of the UPP complex, responsible for proteolytic activity. The 19S regulatory complex associates with either or both ends of the 20S complex. The 19S regulator is involved in the recognition, binding and de-ubiquitinylation of ubiquitinylated proteins tagged for destruction, and stimulates its proteolytic activity.

Results. Western Blot analysis of SeraFILE™ -derived yeast sub-proteomes. Ub(n) regions visualize proteins conjugated to Ubiquitin; Numbers 1-11 correspond to the SeraFILE sub-proteomes; RPT1 regions are indicative of 19S; Bottom Line numbers are measurements of specific proteolytic activity of the 20S particle. Control Proteolytic Activity (without SeraFILE™ treatment) was 82 Units.

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.

Our Basic Enrichment Screening Service is applicable when low abundance protein enrichment is desirable either globally or specifically; globally for proteomic comparison profiling, or specifically for eventual purification of a pre-identified factor. After separations with a panel of 6 SeraFILE™ surfaces, Flow-through, Eluate, and Bead-bound sub-proteome pools are delivered back to the client for subsequent analysis by Western blot, enzymatic assay, and/or 1&2 Dimensional Electrophoresis.