Cyclic Ion Mobility-Mass Spectrometry and Tandem Collision Induced Unfolding for Quantification of Elusive Protein Biomarkers

Sensitive analytical techniques that are capable of detecting and quantifying disease-associated biomolecules are indispensable in our efforts to understand disease mechanisms and guide therapeutic intervention through the early detection, accurate diagnosis, and effective monitoring of disease. Parkinson’s Disease (PD), for example, is one of the most prominent neurodegenerative disorders in the world, but the diagnosis of PD has primarily been based on the observation of clinical symptoms. The protein α-synuclein (α-syn) has emerged as a promising biomarker candidate for PD, but a lack of analytical methods to measure complex disease-associated variants of α-syn has prevented its widespread use as a biomarker. Antibody-based methods such as immunoassays and mass spectrometry-based approaches have been used to measure a limited number of α-syn forms; however, these methods fail to differentiate variants of α-syn that only display subtle differences in sequence and structure. In this work, we have developed a cyclic ion mobility-MS method that combines multiple stages of activation and IM selection to quantify α-syn variants using both mass- and structure-based measurements. This method can allow for the quantification of several α-syn variants present at physiological levels in biological fluid. Taken together, this approach can be used to galvanize future efforts aimed at understanding the underlying mechanisms of PD and serves as a starting point for the development of future protein structure-based diagnostics and therapeutic interventions.