Characterization of microchannel plate detector response for the detection of native multiply charged high mass single ions in orthogonal‐time‐of‐flight mass spectrometry using a Timepix detector

Abstract

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. The study of measurement ranges; ion mass = 195 to 802,000 Da, ion velocity = 8.4 to 67.4 km/s, and ion charge = 1+ to 72+, extended the previously examined mass range and characterized MCP performance for multiply charged species. We derived a MCP performance equation based on two independent ion properties, ion mass and charge, from these results, which enables rapid MCP tuning for single MMA ion detection.

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

“…The significantly higher S/N ratio of TPX3 mass spectrum in Figure 2c,d compared to MCP and phosphorus screen spectrum is mainly due to the larger oversampling factor, which depends on various parameters such as MCP bias voltage, phosphor screen voltage, and camera aperture f-stop value. 20,21 Next, we evaluated the performance of TPX3CAM at higher m/z values utilizing intact IgM (∼970 kDa). IgM exists as a pentamer, in which each of the five monomers is composed of two LCs (∼25 kDa) and two HCs (∼70 kDa).…”

“…Smaller pixel clusters (a parameter that is related to the MCP detection efficiency) were observed for neutrals compared to the precursor ions. This is caused by a reduced ion-to-electron conversion efficiency when neutrals impact the MCP . The average pixel cluster size for metastable product neutrals and precursor ions are 3.5 and 5.5 pixels, respectively (Figure S7).…”

“…Typical TOT values for these experiments were ∼25–1050 (in ns) ( Figure S3 ), which means each ion event is oversampled by a factor of 25–1050 (in ns) in Figure 2 d. The significantly higher S/N ratio of TPX3 mass spectrum in Figure 2 c,d compared to MCP and phosphorus screen spectrum is mainly due to the larger oversampling factor, which depends on various parameters such as MCP bias voltage, phosphor screen voltage, and camera aperture f -stop value. 20 , 21 …”

“… 1 − 3 Although TPX detectors originate from high-energy physics, the integration of TPX with microchannel plate (MCP) amplifiers has enabled the detection of low-energy particles 4 − 6 that extended their scope to biomolecular mass spectrometry (MS). 7 − 21 MCP-TPX detection systems were previously coupled to time-of-flight (TOF) mass spectrometers equipped with nano/micro-electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), or secondary ion MS (SIMS) sources, where the arrival time information at the TPX detector was used for the generation of the mass spectrum. The additional impact position information provided by the TPX compared to conventional MS detectors has previously been utilized to obtain insight into the ion transport properties and ion optical processes within the MS, 14 , 16 , 18 , 20 as well as to improve the spatial resolution and throughput of MS imaging via stigmatic ion imaging.…”

“…Previous investigations conducted on the Ultraflex III MS coupled with MCP-TPX assembly and the modified LCT (nanoESI-orthogonal acceleration reflectron TOF) MS coupled with HV-floating TPX quad system demonstrated the ability of TPX to detect intact protein ions with m / z values up to 400,000 Da 11 and multiply charged non-covalent protein complexes of molecular weight up to 800,000 Da ( m / z < 13,000 Da). 20 , 21 Here, we extend the m / z detection range of TPX family up to 970,000 Da by the measurement of single-charged intact immunoglobulin M (IgM) ions using the MCP-phosphor screen-TPX3CAM assembly, which has previously been used with great success in velocity map imaging to investigate small-molecule reaction dynamics. 25 − 32 In addition, we demonstrate the utilization of multiple information provided by TPX3 such as TOA, pixel coordinates, and TOT for better visualization of the mass spectrum.…”