Time-of-flight (TOF) systems are one of the most widely used mass analyzers in native mass spectrometry (nMS) for the analysis of non-covalent multiply charged bio-macromolecular assemblies (MMAs). Typically, microchannel plates (MCPs) are employed for high mass native ion detection in TOF MS. MCPs are well known for their reduced detection efficiency when impinged by large slow moving ions. Here, a position- and time-sensitive Timepix (TPX) detector has been added to the back of a dual MCP stack to study the key factors that affect MCP performance for MMA ions generated by nMS. The footprint size of the secondary electron cloud generated by the MCP on the TPX for each individual ion event is analyzed as a measure of MCP performance at each mass-to-charge (m/z) value and resulted in a Poisson distribution. This allowed us to investigate the dependency of ion mass, ion charge, ion velocity, acceleration voltage, and MCP bias voltage on MCP response in the high mass low velocity regime.
Abstract
Native mass spectrometry (native MS) has emerged as a powerful technique to study the structure and stoichiometry of large protein complexes. Traditionally, native MS has been performed on modified time-of-flight (TOF) systems combined with detectors that do not provide information on the arrival coordinates of each ion at the detector. In this study, we describe the implementation of a Timepix (TPX) pixelated detector on a modified orthogonal TOF (O-TOF) mass spectrometer for the analysis and imaging of native protein complexes. In this unique experimental setup, we have used the impact positions of the ions at the detector to visualize the effects of various ion optical parameters on the flight path of ions. We also demonstrate the ability to unambiguously detect and image individual ion events, providing the first report of single-ion imaging of protein complexes in native MS. Furthermore, the simultaneous space- and time-sensitive nature of the TPX detector was critical