1 chemical analysis, mass spectrometry (MS) and nuclear

1st Organic Division Seminar
Yanxin Chen – Cai Group
Trapped Ion Mobility: a New Dimension for Mass Spectrometry
In chemical analysis, mass spectrometry (MS) and nuclear magnetic resonance (NMR) are the
two most common analytical tools. They have their own specific advantages and disadvantages.
Compared to MS, NMR provides more structural information. However, despite of the great
improvement of sensitivity by recent technological advancement such as in high field
superconducting magnets and miniaturized radiofrequency coils,
1-3 NMR spectroscopy is still
inherently orders of magnitude less sensitive than MS. Indeed, coupling MS with liquid
chromatography (LC) or gas chromatography (GC) enables analysis of complex mixtures with
femtogram sensitivity.
After over a hundred years of development, MS is now not only routinely used by
synthetic organic chemists,4,5
but also widely used for the analysis of drug metabolites, and in
proteomics, metabolomics and lipidomics research.6,7
Since these applications involve highly
complex mixtures, it is critical to separate the mixture to allow MS analysis of individual
compounds. Even using state-of-the-art GC and LC techniques to separate complex mixtures, coelution
of tens of compounds in a chromatography peak is often encountered. Recently, ionmobility
spectrometry (IMS) was developed, which served as a new dimension for rapid
separation of co-eluted ions based on their mobility in a carrier buffer gas.
8-12 The separation is
based on the cross section of the molecules flying through a drift cell containing buffer gas under
a weak electric field (Figure 1). Among the various IMS methods,11,12 the recently developed
trapped ion mobility spectrometry (TIMS) method has the highest efficiency and resolution, and
has shown great potential to overcome many limitations of the current MS methods.
Experimental Approach
For analyzing complex mixture using conventional LC-MS, many precursors elute from the
column simultaneously, and are fragmented one at a time, whereas the others are discarded
entirely. To solve this problem, Meier and coworkers employed TIMS on an orthogonal
quadrupole-time-of-flight (QTOF) MS (Figure 1). In TIMS, all precursor ions are accumulated in
parallel and released sequentially as a function of their ion mobility, and then adjusting the
quadrupole to match the different m/z values.
Figure 1. Schematic representation of the TIMS-QTOF instrument.13,14
After liquid chromatography, the analytes are ionized by ESI and attracted together with
the gas into the TIMS device by vacuum, in which an axial electronic field gradient (EFG)
profile is tailored to trap and then release the ions with different mobility (Figure 1). Specifically,
an electrical field controls each ion from moving beyond the position where the push that it
experiences from the gas flow matches the force of the electrical field. Because the ion storage
capacity of the TIMS device dictates the effective length of the trapping event, each TIMS
analysis takes place in tens of milliseconds, thus TIMS is most effectively coupled to the fast
mass analyzers, such as a Q-TOF analyzer. The Q-TOF is a hybrid quadrupole TOF MS with a
collision cell for collision induced dissociation (CID, Figure 1) of the ions. The quadrupole is
operated as an ion guide without CID and as mass selection device before CID. A reflectron
TOF14 analyzer is placed orthogonally to the quadrupole and serves as a mass resolving device
for the precursor and its fragment ions.
In data dependent acquisition experiments, only around 20% of the co-eluting analytes
are targeted by current mass spectrometers due to limitations in sequencing speed, sensitivity and
resolution. To overcome this problem, a technique called parallel accumulation serial
fragmentation (PASEF) was developed.17
110 ?s
Mass Selection
2.5–12 ms
20–60 ms
1–10 s
Figure 2. Illustration of the PASEF method (B) in comparison with the standard TIMS method (A).
Figure 2A shows the selection of one precursor from a single TIMS scan, which allows
only ions of a certain mass-to-charge ratio to reach the detector for a given m/z, while other ions
have unstable trajectories and collide with the rods. Conversely, the PASEF method (Figure 2B)
involves rapid switching of the quadrupole mass position to select multiple precursors at
different m/z on the very same time scale. In this case, all targeted ions are fully used for
fragmentation. Overall, the PASEF method is enabled by the efficient storage of ions of the
intended precursor range, high ion mobility resolution, and the rapid switching of the quadrupole
between precursors on the time scale of a single TIMS scan.
Figure 3. Molecular structure of morin and quercetin.
Propolis is a gum gathered by bees from various plants. It is known for its biological
properties, having antibacterial, antifungal and healing properties.15 Analysis of the propolis
samples is difficult since it is a highly complex mixture, and there are overlapping compounds at
m/z = 303.0499 Da, which are morin and quercetin (Figure 3A).
16 They are commonly used as
the detection index of propolis samples.
Figure 4. Chromatographic separation of propolis sample (A), MS spectrum with TIMS on for a sharp
chromatographic peak (B), ion mobility heat map view of one time frame (0.1 second) of the chromatographic peak
at the given MS scan ranges including m/z = 303.0499 Da corresponding to morin and quercetin (C), and the 2D
plot of their mobility diagram (D).
Figure 4A shows chromatographic separation of propolis sample. With TIMS on (Figure
4B), the MS spectrum increases confidence in compound identification and the heat map view
(Figure 4C) of the analysis displays a broad peak in the mobility dimension, potentially
indicating overlapping compounds at m/z = 303.0499 Da. Figure 4D reveals an isomeric
compound in the mixture (quercetin).
As we can see from previous example, ion mobility?mass spectrometry adds an
additional dimension of separation to the standard MS scans. The TOF spectra from one scan can
be summed to determine the m/z and intensity of all the ions present. The advantage of PASEF
was manifested in the analysis of complex samples such as peptide mixtures from trypsincatalyzed
hydrolysis of protein BSA.
Figure 5. A. Full scan IMS heat map and MS spectrum of combined tryptic digest of ADH, BSA, phosphorylase b,
and enolase. B. Sequential isolation of four ions at different m/z after parallel accumulation. C. Four isolated
precursors as in B and MS/MS spectrum. D. Arrival time distribution of the summed fragment ions.17
When analyzing a much more complex sample such as a combined tryptic digest of
alcohol dehydrogenase (ADH), BSA, phosphorylase b, and enolase, the advantage of PASEF
method is clearly shown. Without ion mobility separation, this experiment would have yielded a
very complex mass spectrum with multiple overlapping signals (right part of Figure 5A). Four
precursors, corresponding mass positions at m/z 810.3 (1), 714.3 (2), 559.3 (3) and 560.6 (4)
were selected, and the appropriate switching times were uploaded to the instrument controller,
the quadrupole correctly isolated these precursors on the TIMS time scale (right part of Figure
5B). In the next step, the PASEF method was applied to performing MS/MS on the isolated
precursors. This led to a characteristic ladder of fragment ions at each precursor (Figure 5C).
Projection of all fragment ions onto the ion mobility axis shows coherence in arrival times, with
fragment ion distributions very similar to their precursors (Figure 5D), which shows that the
signal of PASEF method is not missing compared to single precursor selection.
Coupling IMS to MS gives a new dimension for separation of the analytes. PASEF on a
timsTOF instrument makes it is possible to investigate samples with limited amounts (e.g.
clinical samples) at an unprecedented depth of insight. Another important advantage of PASEF is
that the resulting spectra are fully precursor mass resolved, which makes PASEF compatible
with ion based chemical multiplexing strategies.
18 However, it should be noted that despite
timsTOF has the advantage mentioned above, there are still major shortages of current
instrument, the most prominent one being the great difficulties to adjust the mass over its
specified large range (50–40,000 amu) without reducing the resolution.
1. Felli, I. C.; Brutscher, B., Recent Advances in Solution NMR: Fast Methods and Heteronuclear Direct Detection.
ChemPhysChem 2009, 10 (9–10), 1356–1368.
2. Kentgens, A. P. M.; Bart, J.; Bentum, P. J. M. v.; Brinkmann, A.; Eck, E. R. H. v.; Gardeniers, J. G. E.; Janssen, J.
W. G.; Knijn, P.; Vasa, S.; Verkuijlen, M. H. W., High-resolution liquid- and solid-state nuclear magnetic resonance
of nanoliter sample volumes using microcoil detectors. The Journal of Chemical Physics 2008, 128 (5), 052202.
3. Juul, T.; Palm, F.; Nielsen, P. M.; Bertelsen, L. B.; Laustsen, C., Ex vivo hyperpolarized MR spectroscopy on
isolated renal tubular cells: A novel technique for cell energy phenotyping. Magnetic Resonance in Medicine 2017,
78 (2), 457–461.
4. Altelaar, A. F. M.; Munoz, J.; Heck, A. J. R., Next-generation proteomics: towards an integrative view of
proteome dynamics. Nature Reviews Genetics 2012, 14, 35.
5. Parkins, W. E., The Uranium Bomb, the Calutron, and the Space-Charge Problem. Physics Today 2005, 58 (5),
6. Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey,
S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J.,
Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents.
Molecular & Cellular Proteomics 2004, 3 (12), 1154–1169.
7. Creaser, C. S.; Griffiths, J. R.; Bramwell, C. J.; Noreen, S.; Hill, C. A.; Thomas, C. L. P., Ion mobility
spectrometry: a review. Part 1. Structural analysis by mobility measurement. Analyst 2004, 129 (11), 984–994.
8. Kanu, A. B.; Dwivedi, P.; Tam, M.; Matz, L.; Hill, H. H., Jr., Ion mobility-mass spectrometry. Journal of mass
spectrometry : JMS 2008, 43 (1), 1–22.
9. Price, P., Standard Definitions of Terms Relating to Mass Spectrometry: A report from the Committee on
Measurements and Standards of the American Society for Mass Spectrometry. Journal of the American Society for
Mass Spectrometry 1991, 2 (4), 336–348.
10. Guevremont, R., High-field asymmetric waveform ion mobility spectrometry: a new tool for mass spectrometry.
Journal of Chromatography A 2004, 1058 (1), 3–19.
11. Kentgens, A. P.; Bart, J.; van Bentum, P. J.; Brinkmann, A.; van Eck, E. R.; Gardeniers, J. G.; Janssen, J. W.;
Knijn, P.; Vasa, S.; Verkuijlen, M. H., High-resolution liquid- and solid-state nuclear magnetic resonance of
nanoliter sample volumes using microcoil detectors. J Chem Phys 2008, 128 (5), 052202.
12. Pu, Y.; Ridgeway, M. E.; Glaskin, R. S.; Park, M. A.; Costello, C. E.; Lin, C., Separation and Identification of
Isomeric Glycans by Selected Accumulation-Trapped Ion Mobility Spectrometry-Electron Activated Dissociation
Tandem Mass Spectrometry. Analytical Chemistry 2016, 88 (7), 3440–3443.
13. Toyoda, M., Development of Multi-Turn Time-of-Flight Mass Spectrometers and Their Applications. European
Journal of Mass Spectrometry 2010, 16 (3), 397–406.
14. Uzel, A.; Sorkun, K.; Oncag, O.; Cogulu, D.; Gencay, O.; Salih, B., Chemical compositions and antimicrobial
activities of four different Anatolian propolis samples. Microbiological research 2005, 160 (2), 189–95.
15. Hsiu, S. L.; Tsao, C. W.; Tsai, Y. C.; Ho, H. J.; Chao, P. D., Determinations of morin, quercetin and their
conjugate metabolites in serum. Biological & pharmaceutical bulletin 2001, 24 (8), 967–9.
16. Meier, F.; Beck, S.; Grassl, N.; Lubeck, M.; Park, M. A.; Raether, O.; Mann, M., Parallel Accumulation–Serial
Fragmentation (PASEF): Multiplying Sequencing Speed and Sensitivity by Synchronized Scans in a Trapped Ion
Mobility Device. Journal of Proteome Research 2015, 14 (12), 5378–5387.
17. Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.;
Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J.,
Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.
Molecular & cellular proteomics : MCP 2004, 3 (12), 1154–69.
18. Mao, L.; Chen, Y.; Xin, Y.; Chen, Y.; Zheng, L.; Kaiser, N. K.; Marshall, A. G.; Xu, W., Collision Cross
Section Measurements for Biomolecules within a High-Resolution Fourier Transform Ion Cyclotron Resonance Cell.
Analytical Chemistry 2015, 87 (8), 4072–4075.