Methodology of ion-mobility mass spectrometry

Ion-mobility mass spectrometry (IMS) was first introduced in the 1970s as a portable economical alternative to MS for detection of airborne volatiles,

e.g., environmental and industrial process contaminants, explosives, chemical warfare agents, and drugs ^[271]. Over the past decade, two major technical developments have raised and broadened the analytical importance of IMS: (i) the effective coupling of IMS to MS ^[272], especially the advent of IMS-TOF instrumentation that enables a parallel dispersion of ion mixtures in mobility and mass/charge (m/z) dimensions ^{[273, 274]}, and (ii) interfacing of ESI and MALDI soft ionization sources to IMS ^[275] and IMS-MS ^{[276, 277]} which has enabled applications to biopolymer chemistry and biomedicine.

Ion-mobility spectrometry and mass spectrometry share a similar theoretical background and the historical development of both was presented recently by Uetrecht and co-workers ^[278]. Mass spectrometric methods are limited to the separation of ions measured by mass-to-charge (m/z) ratios and ion intensities, while ion-mobility separates ions based on their mobility and measures the time that it takes an ion to migrate through a buffer gas in the presence of a low electric field (drift time). The velocity of the ion, ν (drift velocity) is directly proportional to E with the proportionality constant K called ion-mobility, and is determined by measuring the time required, tD to traverse a drift cell of known dimensions, d. IMS-MS enables separation of species according to their collisional cross section (CCS) (Ω) which represents the orientationally averaged area of the ion capable to interact with a buffer gas. For large ions (e.g. proteins), Ω can be approximated computationally ^[279]. Ions with a larger apparent diameter undergo a greater number of collisions with the buffer gas, consequently their passage through the drift cell is retarded in comparison to smaller ions which experience less friction ^[278]. IMS-MS implements a new mode of separation that allows the differentiation of protein conformational states; therefore is ideally suited to the study of protein folding, unfolding and aggregation [243, 280, 281].

Recently, “traveling wave” ion-mobility mass spectrometry (TWIMS) ^[282,

283] has been developed as a new tool to probe complex biomolecular structures, due to the potential of IMS-MS for separation of mixtures of protein complexes by conformation state, spatial shape and topology [237, 238, 284-286]. Unlike the traditional “drift time” IMS, in which a low electrical field is applied

continuously to the cell, a high electrical field is applied to one segment of the cell, thus ions are moved through the mobility cell in pulses as waves of the electrical field pass through them [239, 287, 288]. IMS spectra are obtained in milli seconds and TOF mass spectra are obtained in micro seconds, so that thousands of mass spectra can be obtained for each ion-mobility spectrum producing a two-dimensional array in which both mobility and mass of ions are recorded ^[239].

IMS-MS was applied to the direct analysis of mixtures of αSyn aggregation products in vitro during prolonged incubation times. IMS-MS determinations were performed at Waters Corporation (Manchester, UK) with a Synapt High Definition Mass Spectrometry (HDMS) QTOF-MS equipped with a nano-electrospray ionization source (Figure 32).

ESI source

Detector Trap Ion mobility

separation Transfer

T-Wave

Ion Guide Quadrupole

TRIWAVE

TOF

Figure 32: Adapted scheme of ion-mobility MS Waters Synapt high-definition mass spectrometer (HDMS) system ^[282]: Ion-mobility MS system consists of an electrospray ionization (ESI) source, quadrupole mass spectrometer, ion gate, drift region (Triwave), time-of-flight mass spectrometer for a two-dimensional analysis

Ions were allowed to first pass through a quadrupole that is set to either transmit a substantial mass range, or to select a particular m/z before entering the Triwave ion-mobility cell. Triwave consists of a series of planar electrodes arranged orthogonally to the ion transmission axis. Opposite phases of radio-frequency (rf) voltage are applied to adjacent electrodes to provide radial confinement of the ions, resulting in high ion transmission. A traveling voltage wave can be incorporated into the rf ion guide to minimize the ion transit time.

The Triwave system is composed of three T-Wave devices (Figure 33a). The Trap T-Wave accumulates ions which are stored and gated (500 µsec) into the second device. The final electrode on the trap ion guide is dc only and its voltage is modulated to periodically gate ions into the IMS. Typically no traveling wave is used in the trap ion guide. In the ion-mobility T-Wave cell the ions are separated according to their cross section-dependent mobilities. The transfer T-Wave cell is used to transfer the mobility separated ions to the MS analysis, accomplished by an orthogonal acceleration time-of-flight (oa-TOF) analyzer. In order to ensure that the mobility separation is maintained on transit to the oa-TOF, the transfer ion guide has a continual running traveling voltage wave. As both the trap and transfer ion guides operate at approximately standard collision cell pressures, it is possible to fragment ions in either one of these regions.

They share a common gas supply (typically Argon, 10^-2 mbar). Using the DriftScope feature, it is possible to extract m/z and drift time information for any ion of interest and thus obtain a much better drift time separation. The extracted drift time distributions for a two dimensional plot of drift time versus (vs.) mass-to-charge (m/z) snapshot clearly revealed the separation of components.

Generally, the speed with which an ion traverses the drift region depends on its CCS (gas phase size); ions with larger CCSs proceed more slowly than ions with smaller ones.

2.3.3.2 Identification of full-length and proteolytic fragments of