MSe Collision Energy Optimization for the Analysis of Membrane Proteins Using HDX-cIMS.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has evolved as an essential technique in structural proteomics. The use of ion mobility separation (IMS) coupled to HDX-MS has increased the applicability of the technique to more complex systems and has been shown to improve data quality and robustness. The first step when running any HDX-MS workflow is to confirm the sequence and retention time of the peptides resulting from the proteolytic digestion of the nondeuterated protein. Here, we optimized the collision energy ramp of HDMSE experiments for membrane proteins using a Waters SELECT SERIES cIMS-QTOF system following an HDX workflow using Phosphorylase B, XylE transporter, and Smoothened receptor (SMO) as model systems. Although collision energy (CE) ramp 10-50 eV gave the highest amount of positive identified peptides when using Phosphorylase B, XylE, and SMO, results suggest optimal CE ramps are protein specific, and different ramps can produce a unique set of peptides. We recommend cIMS users use different CE ramps in their HDMSE experiments and pool the results to ensure maximum peptide identifications. The results show how selecting an appropriate CE ramp can change the sequence coverage of proteins ranging from 4 to 94%.

Modulating Antibody Structure and Function through Directed Mutations and Chemical Rescue.

Monoclonal antibody therapeutics have revolutionized the treatment of diseases such as cancer and autoimmune disorders, and also serve as research reagents for diverse and unparalleled applications. To extend their utility in both contexts, we have begun development of tunable antibodies, whose activity can be controlled by addition of a small molecule. Conceptually, we envision that incorporating cavity-forming mutations into an antibody can disrupt its structure, thereby reducing its affinity for antigen; addition of a small molecule may then restore the active structure, and thus rescue antigen binding. As a first proof of concept toward implementing this strategy, we have incorporated individual tryptophan to glycine mutations into FITC-E2, an anti-fluorescein single-chain variable fragment (scFv). We find that these can disrupt the protein structure and diminish antigen binding, and further that both structure and function can be rescued by addition of indole to complement the deleted side chain. While the magnitude of the affinity difference triggered by indole is modest in this first model system, it nonetheless provides a framework for future mutation/ligand pairs that may induce more dramatic responses. Disrupting and subsequently rescuing antibody activity, as exemplified by this first example, may represent a new approach to "design in" fine-tuned control of antibody activity for a variety of future applications.