The Approach and Design of a Surrogate Protease Substrate for Disease Biomarker Discovery
Cell Preservation Technology
Biomarker discovery investigations utilizing sera samples typically show differences in peptides originating from high abundant proteins. These peptides, produced from the activity of one or more protease(s), by themselves may not reflect a disease-associated biomarker(s), but in fact, show the downstream effect of disease specific protease(s). The ability to select “true” biomarkers of disease is mired by the inter- and intrapatient variability typically observed in a proteomics approach to serum biomarker discovery. As an example, serum from an individual was collected with three types of serum separator tubes (SST) and stored at 4°C for prolonged periods of time. The postcollection integrity of the serum was examined using microcapillary nanoflow reversed-phase liquid chromatography (nano-RPLC) tandem mass spectrometry (MS/MS) of the trypsin-digested, low molecular weight protein fraction. An increase in unique peptides per protein was observed for the highest abundant proteins with prolonged storage times. Of note was the presence of truncated variations of high abundant parent peptides, which presumably arise from the action of endogenous exo- and endo-peptidases in human serum. Based on these observations, a surrogate peptide was synthesized with a central affinity label as a means to screen enzymatic activity in serum. The surrogate peptide was used to screen cell-free media of two human breast cancer cell lines, SKBr3 and MDA-MB-231, for differential enzymatic activity as an initial disease model. In light of these results, the use of a “spiked” protease substrate may provide an additional means to differentiate between sera from diseased versus healthy individuals.
Biomarker, disease, discovery, surrogate, protease, substrate
Stewart, Nicolas A.; Prieto, Darue A.; Cosentino, Louis M.; Issaq, Haleem J.; and Veenstra, Timothy D., "The Approach and Design of a Surrogate Protease Substrate for Disease Biomarker Discovery" (2007). Pharmaceutical Sciences Faculty Publications. 340.