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

Abstract

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 α-synuclein (α-syn) has emerged as a promising biomarker candidate for PD, but a lack of analytical methods to measure complex diseaseassociated variants of α-syn has prevented its widespread use as a biomarker. Antibodybased 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 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 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.

CIUSuite 3: Next-Generation CCS Calibration and Automated Data Analysis Tools for Gas-Phase Protein Unfolding Data

“…However, with the inclusion of CIU 2 and performing multidimensional classification resulted in a higher probability of correct assignment of each class and increased the probability by up to 7% (Table S2). Overall, multidimensional classifications of serum albumins, in combination with prior reports demonstrating the utility of CIU 2 assays, corroborate and demonstrate the accuracy and applicability of this feature for CIU analysis.…”

“…CIU 2 experiments were performed using a Select Series Cyclic Ion Mobility-Mass Spectrometer from Waters (Milford, MA), as described previously. , Ions were generated by nESI at 1.0 kV using borosilicate capillary emitters pulled using a P97 micropipette puller (Sutter, Novato, CA) and coated in gold using a SC7620 mini sputter coater (Quorum Technologies, Laughton, UK). A single charge state was selected using the quadrupole and the collision energy of the trap collision cell was set to the voltage required to reach the desired unfolding intermediate (i.e., CIU 1 feature).…”

“…For instance, the development of cyclic IM-MS (cIM-MS) allows for the acquisition of a primary CIU data, followed by a timed ion selection event to capture unfolded intermediates of interest produced during the primary unfolding experiment. The selected ions can be subjected to a second CIU experiment (CIU 2 ), where ions are further activated in the prearray region of the cIM-MS platform. , Furthermore, a wide range of IM-MS instrument platforms capable of performing CIU have been made commercially available, many of which enable the rapid acquisition of CIU data, ,− necessitating a software solution capable of automating various aspects of CIU analyses in a manner that is cross-platform accessible. , Current CIU analysis software packages do not support higher-order CIU (e.g., CIU 2 ) and the seamless automation of CIU data analysis, which serves to limit the applications of CIU assays in the areas of biomarker analysis, pharmaceutical chemistry, and protein engineering.…”