Grant: Discovery Grants Program - Individual

The Human Genome Project ushered transformative approaches to study human health. A startling reveal of this project was the low number of genes (~20,500) in our genome - being a fraction of what was anticipated. This confirmed that our biological diversity is captured through variations in gene products, i.e. proteins. A proteome is a dynamic system; discrete proteins change concentration or functional activity to correlate with a particular physiological state. Such changes may mark the progression of a disease (biomarkers); mapping the proteome may tailor new therapeutic approaches (personalized medicine). *** *We speculate that over a million unique human proteins may exist. This diversity is created in part through chemical modifications (e.g. methylation, phosphorylation) which manifests unique protein forms. Proteoform' characterization (as we now call these chemically distinct proteins) is a driving feature behind the emerging field of top-down proteomics. This approach employs a combination of separation, high resolution tandem mass spectrometry (MS/MS) and computational tools to profile intact proteins. Proteoform characterization is still fraught with challenges: proteins are prone to degradation, aggregation or unintended chemical modification; separation strategies for intact proteins lack the needed resolution and can exaggerate sample loss; MS detection is subject to interference. Improved analytical technologies are urgently needed to establish proteoform analysis as a viable strategy to diagnose, monitor, and potentially cure diseases. *** *Our group has pioneered multiple approaches for proteome analysis using MS. Of note are platforms for intact protein separation, methods to purify proteins, and improved insights on how to maintain proteome stability. Such developments have advance MS-based proteomics, particularly in top-down format. We still have a long way to go. A pivotal aspect of the current proposal is the use of sodium dodecyl sulfate (SDS), a surfactant beneficial to solubilize, stabilize and separate the proteome. Unfortunately, SDS is also detrimental to MS and for this reason many researchers avoid this detergent. We propose an automated platform based on electrophoresis to isolate proteins from SDS ahead of MS. We also evaluate acetone precipitation as a promising approach for proteome purification and preservation, aiming to improve our fundamental understanding of the process (kinetic studies, mechanism of aggregation). Methods to fractionate proteoforms are proposed, combining electrophoresis with solid phase protein capture. These studies aim to provide powerful tools to profile biological systems. Ultimately, these innovations establish proteomics alongside genetic testing, thus improving the health outcomes of Canadians. **