MRTOF Applications

Evolution of MRTOF at MSC

In 2002, MSC came up with the proposal to arrange MRTOF between planar ion mirrors while determining the zigzag ion path by periodic lens [patents]. For a tighter folding of ion trajectory within compact mirrors we came up with a method of so-called double orthogonal ion injection.

This way we implemented high resolving MR-TOF at full mass range and coupled it to real-world continuous ion sources, like ESI an EI.

MSC put substantial efforts to implement MRTOF into products by LECO and by Waters. Leco’s ESI-MRTOF Pegasus was awarded Pittcon Gold in 2011.

The optical properties of ions mirrors were closely examined. We started with 3rd-order focusing ion mirrors. In time, we improved ion mirrors to the 5th and higher order focusing and developed a range of designs.

For some time MSC was exploring imaging properties of MRTOF for mass microscopes and mapping of matrix sources. Practice is limited by lack of matrix sources and low-cost detectors.

In experiments, we tested MRTOF  limits and reached R=1 million resolution. Initially (2006), in looped experiments, and later (in 2023) – at full mass range and low ppb mass accuracy.

MSC was repetitively attacking a weakest part of the MRTOF technology – low (<1%) duty cycle (DC) of orthogonal accelerators (OA). This work is described in MRTOF multiplexing story. An encoded frequent pulsing (eFP) brings the DC to 10% at DR=1E+5. Another method is to use trap converters, suitable for lower intensity beams, for example, MS2 stage in MSMS.

In 2017, MSC found yet more efficient way of improving the sensitivity and DR of MRTOF. With appearance of fast and high dynamic range detectors and fast ADCs, we returned back to a conventional OA and reached DC=10% for continuous ion beams and up to 40% in Pulsar mode. Perfecting all elements and using advanced peak centroiding allowed us to reach R=300K in a benchtop instrument with short (4-5 m) flight path. This made MR-TOF suitable for Top-Down analyses of mid-size proteins.

1 Million Resolving Power

In 2004 MSC made an 0.5m long MRTOF employing two planar mirrors 1 and 2, periodic lens 3, pulsed Cs ion gun 4, and an MCP detector 5. Pulsed operation of end lenses allowed to loop back and forth ion passage in the drift Z-direction, thus trapping ions for controlled number of cycles N. In 2005, the analyzer was coupled to ESI source and orthogonal accelerator.

In 2006, to test the MRTOF precision and voltage stability limits, we made experiments with looped trajectories. With wider ion packets past ESI source (10x2mm and 300eV energy spread), the looped MRTOF allowed to reach resolution R~100K at 2.5ms trapping time and N=5.

For smaller Cs pulsed packets (2mm x 20mrad x10eV) we reached a certain 300K resolution in averaged spectra. The peak was clearly moving at 50Hz of AC line. At individual shots or after drift removal at post-processing, the resolution exceeded 1 million. The result was not practical because of very limited mass range. Besides, we were not certain that ~30 ion packets were not shrinking due to the own space charge coalescence.

[A. Verenchikov, Concept of MRTOF, Sci Instrum (Ru), v16 (2006), #3, pp.3-20]

Resolving Power R=1M in Full Mass Range

The true 1M resolution was reached by MSC in 2023. A large scale MRTOF (0.7×1.4m) with a fifth order energy focusing and 40-elements periodic lens was constructed at <10um precision. The single ion path reached 80m and flight time of m/z=1000 ions – 2.5ms, still providing full mass range. The simulated aberration limit was predicted at >2M level for 5x1mm packets at 300eV energy spread. The analyzer was coupled to ESI source via a double orthogonal accelerator (OA). The OA was pulsed at average 30 kHz at time-encoded sequences, allowing to decode spectra within 1E+5 dynamic range and recovering the duty cycle to DC=10%. Multiple sources of vibrations were decoupled and power supplies were stabilized with sealed RC filters at 10Hz cut off frequency.

Fine peptide details could be recovered. Here we prove Ca adducts, earlier thought to be K.

In LCMS1 analyses of cell digests, two dimensional plots appear at much higher contrast as seen in zoom views (mid and right) when comparing plots at R=1M and R=100k.

In single LCMS1 analysis, R~1M allows to recover ~100k peptides in the 1E+5 dynamic range at RSD=80ppb mass accuracy for >3K proteins at FDR<1%

LC-MRTOF for Fast Quantitation of Cell Digests

Cell digests are separated by nano-LC (left) and full m/z range mass spectra are fast recorded on ESI-MRTOF. Thus detected 2D features in retention time (r.t.) and m/z space and interrogated against FASTA database and r.t. predicted by a machine learning algorithm. We used DirectMS1 search engine developed by Postgenome Technology Lab (Bulgaria).

We reliably detect and quantify LOQ ~1fmole (60pg) of BSA spike within 1ug of Baker’s Yeast.

The number of reliably identified proteins (FDR<1%, Human Cells) is moderate compared to the one in best MS/MS runs, however, the number of reliably filtered peptides is at least twice higher that improves the quantitation capability – the true goal of the most of bottom-up proteomics.

Tissue Imaging by Select Series MR-TOF (Waters Corporation)

High resolution MR-TOF platform was developed at MSC and adopted by Waters in their Select Series MR-TOF, applied to tissue imaging by DESI. 200k resolution helps resolving multiple isobaric features in MS-only spectra (without MS/MS) at fast DESI scanning.

Demonstration of the power of the high resolution MRT for imaging. A lipid signal corresponding to PC (33:1) is well-resolved from nearby lipid species but also from interfering background. On a lower resolution system the background would interfere with the PC (33:1) signal yielding a composite image.

GC-MRTOF with Z-OA

In 2016, MSC has departed from the double orthogonal injection (Y-OA) method in favor of conventional Z-OA, where the ion beam is oriented along the drift Z direction. This became possible with advances of fast and high dynamic range detectors and ADC, so as with improvements of continuous ion beams past RFG interface. The first compact Z-MRTOF instrument was constructed with an EI source, assisted with an RFG interface.

Dr. Yuri Khasin and Dr. Anatoly Verenchikov in front of Mini-1 GC-MRTOF, 2017. Right: Resolving power in the profile and centroiding modes in 300x500mm GC-MRTOF

The instrument has 10i/fg sensitivity which allows to record chromatographic traces of higher isotopes corresponding to low-fg injected quantities. Later on we developed a high dynamic range data acquisition system based on M-TOF detector and dual-gain amplifier. Spectra were recorded with a single channel SA230P digitizer in a time-shared method. With such DAS we demonstrated that GC-MRT has linear response at upper loads of 10ng, thus reaching dynamic range of 3E+6 (3fg to 10ng).

The combination of resolving power >25k, sensitivity of 10i/fg and DR=3E+6 appeared very valuable in trace analyses within complex matrices.

Petroleomics with GC and GCxGC MR-TOF

Crude oils are known to contain >100,000 compounds, formed by combinatory of N, O, and S-classes on top of rich variety of linear, branched and cyclic CH compounds.

Soft and Quantitative Ionization Methods at MSC

In 2000’s the only available high resolution MS was ESI with FTMS, known to discriminate between classes.  As a more viable alternative, MSC has developed soft ionization methods based on photo ionization (APPI) and conditioned glow discharge (CGD).

The MSC developed CGD appeared soft (with dominant M+ ions) and quantitative. CGD spectra for typical oil compounds (left) and quantitation by CGD (right).

Moreover, CGD appeared to be not discriminative between compound classes (oil sample).

Oil Analyses with GC-CGD-MRT

High resolution (R=100K) and accurate (<1ppm) mass allow to identify species elements and confirm them by fine isotopes within ~1E+5 dynamic range.

Identifications in C-DBE (eff. double bond) space revealed hundreds of species in the S-class

C-DBE plots for a few major oil classes in GC-CGD-MRTOF analyses. GC-MRTOF analyses of oils revealed a few ten of mixed classes and >10,000 species.

GCxGC-MRTOF with Soft Ionization

Leco Corporation provided GCxGC unit and methods. MSC coupled the GCxGC to CGD-MRT.

Soft ionization produce M+ ions, this way simplifying the compound identification with accurate mass, effectively providing a 4D-separation – in GC1, GC2, M and dM. While the total 4D space is extremely complex and corresponds to at least 300,000 species (including isomers), using accurate mass we could track individual classes and their C-DBE series.

Isomers could be separated in GCxGC. It was nice to learn that the number of separated isomers matched the number of knwon isomers in the NISt data base.

Thus, GCxGC with soft and quantitative ionization and high resolution MRT allows a multi-dimensional 4D separation estimated to have ~100B separation capacity. When applied to analysis of crude oils it revealed ~300k species, including their isomeric forms.