Searching the Secretome
Daniel Chelsky, Ph.D., CSO at Caprion Proteomics, presented his company’s proteomics methodology to biomarker discovery and validation. “We combine a search for biomarkers in biological fluids with discovery in tissues, driven by highly multiplexed assays.”
Dr. Chelsky’s overall developmental strategy is based on MRM, a mass spectrometry assay that can specifically target large numbers of proteins. Promising biomarkers can be assembled by trolling the literature, proteomics assays, and transcript profiling. Through a validation funnel, the candidates are screened and winnowed down to the most promising possibilities.
“From this data, expression in the source tissue predicts expression in blood,” Dr. Chelsky said. “We can also determine the specificity of these proteins by building a ‘Body Atlas’ catalog of shared vs. specific secreted proteins from a wide range of tissues. We believe that this approach, combined with highly multiplexed multiple reaction monitoring, provides a powerful tool for biomarker verification.”
The company is focused on biomarker discovery using label-free, gel-free, quantitative mass spectrometry—a nonhypothesis-driven strategy, or “fishing expedition” in which thousands of proteins are profiled, narrowing down to dozens or hundreds of differentially expressed proteins. Subsequently, these are verified through a targeted approach involving multiplexed, quantitative multiple-reaction monitoring by mass spectrometry. This strategy allows large numbers of samples to be analyzed with a final aim of validation for regulatory compliance.
Caprion has pursued a number of malignant conditions such as lymphoma within the CNS. Diagnosis of this condition is fraught with difficulty, requiring expensive imaging or invasive biopsies that are dangerous and not always possible. As an alternative, cerebrospinal fluid from patients with lymphoma and matched controls was investigated, resulting in some 76 candidate markers. Among these, antithrombin III levels were found to be an accurate predictor of disease progression, providing better sensitivity and specificity than the conventional cytology, according to the company.
Given that secreted proteins are perhaps a thousand times more concentrated in the Golgi apparatus than in the blood, targeting this subcellular organelle and the attendant secretory vesicles to reveal the secretome is another promising strategy to detect markers present in very low concentrations. Caprion staff investigated the presence of biomarker proteins in prostate tumors and their normal counterpart.
Among 60 differentially expressed proteins, they were able to identify seven that were previously identified by other investigators in plasma. Two of these, prostate specific antigen and macrophage migration inhibitory factor, were measured by ELISA in the plasma and were strongly correlated with measurements in the patients’ tumors.
Another series of investigations identified secreted protein indicative of progressive type 2 diabetes. By focusing on those secreted proteins found only in the beta cells, Caprion researchers identified almost 300 proteins specific to the beta cells that are currently being evaluated as candidate biomarkers.