Time-Resolved Imaging of High Mass Proteins and Metastable Fragments Using Matrix-Assisted Laser Desorption/Ionization, Axial Time-of-Flight Mass Spectrometry, and TPX3CAM

Abstract

The Timepix (TPX) is a position-and time-sensitive pixelated charge detector that can be coupled with time-of-flight mass spectrometry (TOF MS) in combination with microchannel plates (MCPs) for the spatially and temporally resolved detection of biomolecules. Earlier generation TPX detectors used in previous studies were limited by a moderate time resolution (at best 10 ns) and single-stop detection for each pixel that hampered the detection of ions with high mass-to-charge (m/z) values at high pixel occupancies. In this study, we have coupled an MCP-phosphor screen-TPX3CAM detection assembly that contains a silicon-coated TPX3 chip to a matrix-assisted laser desorption/ ionization (MALDI)-axial TOF MS. A time resolution of 1.5625 ns, per-pixel multihit functionality, simultaneous measurement of TOF and time-over-threshold (TOT) values, and kHz readout rates of the TPX3 extended the m/z detection range of the TPX detector family. The detection of singly charged intact Immunoglobulin M ions of m/z value approaching 1 × 10 6 Da has been demonstrated. We also discuss the utilization of additional information on impact coordinates and TOT provided by the TPX3 compared to conventional MS detectors for the enhancement of the quality of the mass spectrum in terms of signal-to-noise (S/N) ratio. We show how the reduced dead time and event-based readout in TPX3 compared to the TPX improves the sensitivity of high m/z detection in both low and high mass measurements (m/z range: 757−970,000 Da). We further exploit the imaging capabilities of the TPX3 detector for the spatial and temporal separation of neutral fragments generated by metastable decay at different locations along the field-free flight region by simultaneous application of deflection and retarding fields.

An Orbitrap/Time-of-Flight Mass Spectrometer for Photofragment Ion Imaging and High-Resolution Mass Analysis of Native Macromolecular Assemblies

“…Subsequently, the influence of the custom-built TOF analyzer and the associated ion optics on the ion trajectories was investigated by the visualization of the spatial distribution of the ion cloud with axial and orthogonal imaging detectors. The MCP-TPX quad − ,,− and MCP-P47-TPX3CAM ,,− assemblies coupled with the Orbitrap/TOF instrument are deployed for this purpose (Configuration 1b, Figures S1–S2). Singly charged CsI ions of mass 5589 Da were selected using the quadrupole mass filter.…”

“…The imaging assembly mounted on the orthogonal TOF MS consists of an MCP-P47-TPX3CAM detector system (Configurations 1b and 2) that was previously employed in Ultraflex III MS. 53 Briefly, each ion impact on the MCP-P47-TPX3CAM detection assembly leads to a cascade of secondary electrons within the MCP that is subsequently converted to photons by the scintillator (P47). The photons create local electron−hole pairs in the Si-coated TPX3 56 within the TPX3CAM that results in a detectable current on individual pixels of the TPX3 chip.…”

“…Here, we discuss the design and development of an innovative MS-based instrument that targets comprehensive molecular and structural analysis of MMAs at the same time using only picomoles of sample from solution . A combination of a Thermo Scientific Q Exactive ultrahigh mass range (UHMR) hybrid quadrupole-Orbitrap mass spectrometer − and a custom-designed orthogonal time-of-flight (TOF) mass analyzer with an integrated 193 nm excimer laser ,− and two position-and-time sensitive Timepix (TPX) detectors − was used for the initial characterization of the system presented here. The Orbitrap/TOF system allows the m / z -resolved imaging of UV-generated products from the precursor MMA ions, which can be utilized to understand the energetics of the MMA dissociation process as the TOF analyzer can retain the relative positions of the product ions following the fragmentation process until they reach the TPX imaging detector.…”

Technological developments in mass spectrometry towards molecular structural elucidation of macromolecular assemblies

“…The imaging assembly mounted on the orthogonal TOF MS consists of an MCP-P47-TPX3CAM detector system that was previously employed in Ultraflex III MS. 322 Briefly, each ion impact on the MCP-P47-TPX3CAM detection assembly leads to a cascade of secondary electrons within the MCP that is subsequently converted to photons by the scintillator (P47). The photons create local electron-hole pairs in the Si-coated TPX3 137 within the TPX3CAM that results in a detectable current on individual pixels of the TPX3 chip.…”

“…Here, we discuss the design and development of an innovative MS-based instrument that allows comprehensive molecular and structural analysis of MMAs at the same time using only picomoles of sample from solution. 320 A combination of a Thermo Scientific TM Q Exactive TM ultra-high mass range (UHMR) hybrid quadrupole-Orbitrap mass spectrometer 74,108,169,186,224 and a custom-designed orthogonal time-of-flight (TOF) mass analyzer, with an integrated 193 nm excimer laser [167][168][169]321 and two position-and-time sensitive Timepix (TPX) detectors 71,143,146,152,290,322 were used for the initial characterization of the system presented here. The Orbitrap/TOF system allows the m/z-resolved imaging of UV generated products from the precursor MMA ions, which can be utilized to understand the energetics of the MMA dissociation process as the TOF analyzer can retain the relative positions of the product ions following the fragmentation process until they reach the imaging detector.…”