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 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 alpha-synuclein (alpha-syn) has emerged as a promising biomarker candidate for PD, but a lack of analytical methods to measure complex disease-associated variants of alpha-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 alpha-syn forms; however, these methods fail to differentiate variants of alpha-syn that display subtle differences in only the sequence and structure. In this work, we developed a cyclic ion mobility-mass spectrometry method that combines multiple stages of activation and timed ion selection to quantify alpha-syn variants using both mass- and structure-based measurements. This method can allow for the quantification of several alpha-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.