The development of synthetic biomarkers may broaden the applicability of biomarkers to include noninvasive urinary monitoring.
Kwong et al. describe the action of synthetic urinary biomarkers created by combining nanoparticles with mass-encoded peptides. Since many disease states are characterized by dysregulated protease activity, the synthetic probe acts by stimulating this response. The nanoparticle is introduced and allowed to accumulate in diseased tissue where it is cleaved by the overactive proteases. This cleavage releases the mass-encoded peptides into the host’s urine, where it can be detected using mass spectrometry. While the researchers in this study used this method to monitor liver fibrosis and detect early stage cancer, this noninvasive urinary monitoring could prove applicable to many diseases that feature protease dysregulation, including cancer, atherosclerosis, inflammation, and Alzheimer’s disease.
To create and evaluate the synthetic urinary biomarker, the researchers first determined the specific peptide substrates related to the proteases actively dysregulated in liver fibrosis and cancer. They then conjugated fluorescein-labeled derivative of approximately 50 peptide substrates to nanoworm nanoparticles. After incubating these with recombinant proteases commonly upregulated in disease and blood-borne proteases to discern cross-reactivity, Kwong et al. analyzed each substrate. This allowed them to choose 10 peptide substrates for use as peptide-nanoworm biomarkers. Using the fluorophore-labeled peptide glutamate-fibrinopeptide B as a prototype, the researchers noted that, when introduced intravenously to a mouse host, the peptide alone demonstrated rapid clearance into the urine of both fibrotic and healthy mice. By contrast, nanoworms without peptides congregated in the liver and did not clear into the urine. The prototypic peptide-nanoworm biomarkers, on the other hand, localized in the livers of the mice and triggered protease cleavage, allowing the peptide fragments to clear into the urine of the fibrotic mice with upregulated protease activity.
Kwong et al. then applied an encoding method they call isobar coded reporters, or iCORE, to produce a family of mass encoders from Glu-fib with a shared parent mass to enable the researchers to easily collect and identify peptides using mass spectrometry. Each of the 10 protease substrates were also fitted with mass codes located on the y6 ion that were produced by enriching the hexamer with heavy amino acids to produce 10 individual mass codes. This resulted in 10 peptides with identical parent masses and individuated mass codes on the y6 ion.
To evaluate the iCORE reporters, the researchers used liquid chromatography mass spectrometry (Thermo Scientific) to demonstrate that the peptide library first appeared as a single peak but then resolved into 10 distinct peaks. When the 10 protease substrates were tagged with iCORE reporters and coupled with nanoworms to produce synthetic biomarkers, the peptides cleaved by the upregulated proteases were introduced to UV light to unbind the reporters for quantification with mass spectrometry. The resulting spectrum included individual y6 peaks, indicating that iCORE-encoded nanoworms are useful for analysis.
The researchers next turned to a mouse model of liver fibrosis to determine if the 10 synthetic urinary biomarkers could act as an alternative to the invasive needle biopsies and histological analyses that now form the basis of treatment. Overall, the seventh biomarker (G7) demonstrated excellent trackability with the kinetics of the disease model, although biomarkers G4, G5, and G6 also demonstrated high sensitivity to the disease. Kwong et al. also evaluated combination biomarkers, revealing that G5/G7 is the best dual combination and G5/G6/G7 is the best triple probe combination.
In the case of a mouse model for colorectal cancer, the researchers determined that the best-performing biomarkers were G1, G2, and G3, with the dual G1/G2 and triple G1/G2/G3 combinations showing even better performance. This highlighted the clinical value of using a family of diverse probes to increase sensitivity and performance. In both aspects of the study, Kwong et al. found that synthetic urinary biomarkers are effective, when multiplexed, for the noninvasive diagnosis and monitoring of liver fibrosis and may even detect colorectal cancer earlier than traditional biomarkers.
Kwong, G., et al. (2012) ‘Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease’, Nature Biotechnology, published online December 16, 2012. doi: 10.1038/nbt.2464