11st Organic Division SeminarYanxin Chen – Cai Group01/29/2018Trapped Ion Mobility: a New Dimension for Mass SpectrometryIntroductionIn chemical analysis, mass spectrometry (MS) and nuclear magnetic resonance (NMR) are thetwo most common analytical tools. They have their own specific advantages and disadvantages.Compared to MS, NMR provides more structural information. However, despite of the greatimprovement of sensitivity by recent technological advancement such as in high fieldsuperconducting magnets and miniaturized radiofrequency coils,1-3 NMR spectroscopy is stillinherently orders of magnitude less sensitive than MS.
Indeed, coupling MS with liquidchromatography (LC) or gas chromatography (GC) enables analysis of complex mixtures withfemtogram sensitivity.After over a hundred years of development, MS is now not only routinely used bysynthetic organic chemists,4,5but also widely used for the analysis of drug metabolites, and inproteomics, metabolomics and lipidomics research.6,7Since these applications involve highlycomplex mixtures, it is critical to separate the mixture to allow MS analysis of individualcompounds. Even using state-of-the-art GC and LC techniques to separate complex mixtures, coelutionof tens of compounds in a chromatography peak is often encountered. Recently, ionmobilityspectrometry (IMS) was developed, which served as a new dimension for rapidseparation of co-eluted ions based on their mobility in a carrier buffer gas.
8-12 The separation isbased on the cross section of the molecules flying through a drift cell containing buffer gas undera weak electric field (Figure 1). Among the various IMS methods,11,12 the recently developedtrapped ion mobility spectrometry (TIMS) method has the highest efficiency and resolution, andhas shown great potential to overcome many limitations of the current MS methods.Experimental ApproachFor analyzing complex mixture using conventional LC-MS, many precursors elute from thecolumn simultaneously, and are fragmented one at a time, whereas the others are discarded 2entirely. To solve this problem, Meier and coworkers employed TIMS on an orthogonalquadrupole-time-of-flight (QTOF) MS (Figure 1). In TIMS, all precursor ions are accumulated inparallel and released sequentially as a function of their ion mobility, and then adjusting thequadrupole to match the different m/z values.Figure 1. Schematic representation of the TIMS-QTOF instrument.13,14After liquid chromatography, the analytes are ionized by ESI and attracted together withthe 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 itexperiences from the gas flow matches the force of the electrical field. Because the ion storagecapacity of the TIMS device dictates the effective length of the trapping event, each TIMSanalysis takes place in tens of milliseconds, thus TIMS is most effectively coupled to the fastmass analyzers, such as a Q-TOF analyzer. The Q-TOF is a hybrid quadrupole TOF MS with acollision cell for collision induced dissociation (CID, Figure 1) of the ions. The quadrupole isoperated as an ion guide without CID and as mass selection device before CID. A reflectronTOF14 analyzer is placed orthogonally to the quadrupole and serves as a mass resolving devicefor the precursor and its fragment ions.In data dependent acquisition experiments, only around 20% of the co-eluting analytesare targeted by current mass spectrometers due to limitations in sequencing speed, sensitivity andresolution.
To overcome this problem, a technique called parallel accumulation serialfragmentation (PASEF) was developed.17TOF110 ?sMass Selection2.5–12 msIMS20–60 msLC1–10 sLC ESI TIMS Quadrupole CID TOFIonaccumulation3Figure 2.
Illustration of the PASEF method (B) in comparison with the standard TIMS method (A).17Figure 2A shows the selection of one precursor from a single TIMS scan, which allowsonly ions of a certain mass-to-charge ratio to reach the detector for a given m/z, while other ionshave 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 atdifferent m/z on the very same time scale. In this case, all targeted ions are fully used forfragmentation. Overall, the PASEF method is enabled by the efficient storage of ions of theintended precursor range, high ion mobility resolution, and the rapid switching of the quadrupolebetween 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 biologicalproperties, having antibacterial, antifungal and healing properties.15 Analysis of the propolissamples is difficult since it is a highly complex mixture, and there are overlapping compounds atm/z = 303.0499 Da, which are morin and quercetin (Figure 3A).16 They are commonly used asthe detection index of propolis samples.Figure 4. Chromatographic separation of propolis sample (A), MS spectrum with TIMS on for a sharpchromatographic peak (B), ion mobility heat map view of one time frame (0.1 second) of the chromatographic peakat the given MS scan ranges including m/z = 303.
0499 Da corresponding to morin and quercetin (C), and the 2Dplot of their mobility diagram (D).Figure 4A shows chromatographic separation of propolis sample. With TIMS on (Figure4B), 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 4indicating overlapping compounds at m/z = 303.0499 Da.
Figure 4D reveals an isomericcompound in the mixture (quercetin).As we can see from previous example, ion mobility?mass spectrometry adds anadditional dimension of separation to the standard MS scans. The TOF spectra from one scan canbe summed to determine the m/z and intensity of all the ions present. The advantage of PASEFwas manifested in the analysis of complex samples such as peptide mixtures from trypsincatalyzedhydrolysis 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 isolatedprecursors as in B and MS/MS spectrum. D.
Arrival time distribution of the summed fragment ions.17When analyzing a much more complex sample such as a combined tryptic digest ofalcohol dehydrogenase (ADH), BSA, phosphorylase b, and enolase, the advantage of PASEF 5method is clearly shown. Without ion mobility separation, this experiment would have yielded avery complex mass spectrum with multiple overlapping signals (right part of Figure 5A). Fourprecursors, 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 Figure5B). In the next step, the PASEF method was applied to performing MS/MS on the isolatedprecursors. 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, withfragment ion distributions very similar to their precursors (Figure 5D), which shows that thesignal of PASEF method is not missing compared to single precursor selection.ConclusionsCoupling IMS to MS gives a new dimension for separation of the analytes.
PASEF on atimsTOF 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 isthat the resulting spectra are fully precursor mass resolved, which makes PASEF compatiblewith ion based chemical multiplexing strategies.18 However, it should be noted that despitetimsTOF has the advantage mentioned above, there are still major shortages of currentinstrument, the most prominent one being the great difficulties to adjust the mass over itsspecified large range (50–40,000 amu) without reducing the resolution.References1.
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