Proteolytic Fingerprinting of Complex Biological Samples Using Combinatorial Libraries of Fluorogenic Probes

Abstract

Proteases have garnered interest as candidate biomarkers and therapeutic targets for many human diseases. A key challenge is the identification and characterization of disease-relevant proteases in the complex milieu of biological fluids such as serum, plasma and bronchoalveolar lavage, in which a multitude of hydrolases act in concert. We have successfully utilized a concise combinatorial library of internally quenched fluorogenic peptide probes (IQFPs) to map the global proteolytic substrate specificities of complex biological samples. The substrate profiling approach provides a global and a quantitative comparison of protease specificities between different biological samples. Such a comparative analysis can lead to the identification of disease-specific ‘fingerprints’ of proteolytic activities with potential utility in diagnosis and therapy.

Sample collection in clinical proteomics—Proteolytic activity profile of serum and plasma

“…Several caveats should be noted when interpreting our data based on the library characteristics and are discussed in our previous publications . While the results presented here are observed from a single donor, the results match the previous observations .…”

Intracellular Action of a Secreted Peptide Required for Fungal Virulence

“…Protease assays were performed with the specified substrates as previously described (Jambunathan et al, 2012). …”

Proteolytic Enzymes as Biomarkers of Focal Segmental Glomerulosclerosis

“…We profiled the plasma proteolytic activity under classical serine protease conditions for two important reasons: first, serine proteases are the most abundant class of proteases in plasma, and second, evidence points to serine class protease involvement in FSGS [Carraro et al., ]. Moreover, our preliminary optimization of buffer space using nonspecific probes indicated that maximal proteolytic activity for plasma was observed under serine protease buffer conditions [Jambunathan et al., ].…”

“…Thus, each well of the library contains an equimolar mixture of up to eight individual IQFPs. A more detailed description of this library is provided elsewhere [Watson et al, 2011, Jambunathan et al, 2012a.…”

“…Thus, each well of the library contains an equimolar mixture of up to eight individual peptides. This library has been validated previously through substrate specificity profiling of recombinant proteases from each of the major protease classes [Thomas et al., ; Jambunathan et al., ,b].…”