Skip to main content
image





Clemmer Group

Multidimensional IMS Instrumentation



  1. Introduction
  2. Basic Experimental Setup
  3. Example Datasets
  4. Selected References


Introduction



The Group has previously developed a simple split-field drift tubeimage
A simple split-field drift tube design. See Pub. 99 for more information.
for ion mobility/mass spectrometry. This early instrumental design allowed for mixtures of precursor ions to be separated in a low-field region before being exposed to a short variable-field region prior to mass analysis. When this variable-field region was operated at an applied field equivalent to that of the drift region, the precursor ions would be allowed into the mass analyzer. When the applied fields in this variable-field region were increased, the precursor ions could be fragmented prior to mass analysis, and then by modulating these low- and high-field conditions, mass spectra for both precursor and fragment ions could be obtained for complex mixtures of ions without initial mass selection.

Further development of this idea led to the constuction of a new IMS instrument that allows a mixture of precursor ions to be dispersed based upon differences in mobility. Once dispersed, it is then possible to select an ion of a specific mobility and subject it to further collisional activation resulting in fragmentation of the precursor ion, changes in the conformation of the ions, or in certain instances, both. These new fragment ions (or conformers) are then allowed to separate again in a second (or third) low-field region prior to mass analysis. This IMS–IMS approach is analogous in many ways to MS–MS strategies; however, the separations of the initial precursor and first dissociation products are based on cross section-to-charge (Ω/z) rather than mass-to-charge (m/z) ratios.



Experimental



image
Figure 1: Schematic of the IMS–IMS–IMS–TOFMS instrument. Ions can be gated (G1–G3) and activated (IA1–IA3) in three regions of the instrument. The design of the instrument allows for multiple operation methods, including one-dimensional IMS–MS, two-dimensional IMS–IMS–MS, and three-dimensional IMS–IMS–IMS–MS. Example instrumental setups for each of these methods are illustrated here, although it is noted that other configurations are possible.

Note: Both of our current linear IMS instruments are capable of multidimensional IMS experiments, however, for the purposes of this discussion, the experimental summary focuses on the use of our longer 3m instrument as opposed to the original 2m apparatus. More information about the 2m instrument can be found here.

Briefly, a continuous beam of electrosprayed ions is introduced directly into an ion funnel region (F1) and accumulated. Periodically the concentrated ion packet is gated into the first drift tube (D1). The drift tube contains 3 Torr He buffer gas; ions migrate across the tube under the influence of a weak electric field, and different species separate due to differences in their mobilities through the gas. As ions exit the first drift region, they enter another ion funnel that is used to radially focus the diffuse ion clouds and transmit species into the front of a second drift region (D2). The entrance of D2 contains an ion gate and ion activation region such that it is possible to select and energize specific components of the ion mixture. There is a third drift region (D3) that operates in an analogous fashion. Ions exit the drift tube into a vacuum chamber and are focused into a time-of-flight mass spectrometer for m/z analysis.

The entire drift tube assembly is ~300 cm long. Ions are selectively gated by raising or lowering fields in the gate regions (G1–G3), as shown in Figure 1 at specific delay times that are controlled by a pulse delay generator. Collisional activation in any activation region (IA1, IA2, or IA3) is achieved by increasing the voltage between the last two lenses of each funnel.

Another way to visualize multi-dimensional IMS instrumentation is shown below using a theoretical protein ion. If the animation progresses too fast, and alternate collection of images can be found here.

image
 
Figure 2: An illustrative animation of multi-dimensional IMS techniques. As shown here, a narrow selection of a broad distribution can be selected at G2, activated at IA2, and then allowed to separate based on mobility through D2 and D3. Ions selected and activated after D1 can also be selected and activated after D2 as well, allowing for multiple experiments to be conducted, and ultimately elicidating a large amount of structural information.


Example Datasets



It is instructive to highlight several example datasets from our previous multidimensional IMS experiments. These types of experiments are extremely varied, allowing for a wealth of potential information to be obtained.

image
Figure 3: Two-dimensional td(m/z) plot of mobility-dispersed fragment ions (left) obtained by selection of the [M+13H]13+ ion of electrosprayed ubiquitin at G2 and activation of IA2. Fragment ions formed upon activation of the [M+13H]13+ as well as charge states formed via charge transfer are shown. The (y58)9+ fragment ion is mobility selected at G3 (middle). Fragment ions produced upon activation of the (y58)9+ in IA3 are separated based on their mobilities through D3 (right). Diagonal lines correspond to mass spectra of fragment ion families shown.


image
Figure 4: Cross section distributions for the 11+ charge state of ubiquitin obtained upon selection and activation of specific mobility regions at G2 and IA2, respectively. Four stable conformations (labeled A�D) are observed within the initial distribution of ions produced by ESI (top). Mobility-selected ion distributions (with no activation) indicate the presence of stable and unstable ion populations (left). Activation of these ions with 80 V (middle) and 100 V (right) produces a new distribution that is unique to the selected ions. Distributions are labeled according to the cross section that was selected (indicated by the arrow in the distribution), and dashed lines delineate the regions corresponding to the compact, partially-folded, and elongated conformations. Distributions are obtained by extracting integrated (m/z) slices from two-dimensional tD(m/z) data sets across all drift times (tD).

More information and discussion of these figures can be found in the associated references listed below, all of which can also be found on the Publications page as well.



Selected References


  1. Liu, X.; Plasencia, M.; Ragg, S.; Valentine, S. J.; Clemmer, D. E. Development of High-Throughput Dispersive LC–Ion Mobility–TOFMS Techniques for Analyzing the Human Plasma Proteome, Brief Funct. Genomic Proteomic 2004, 3, 177–186.
  2. Koeniger, S. L.; Merenbloom, S. I.; Valentine, S. J.; Jarrold, M. F.; Udseth, H.; Smith, R.; Clemmer, D. E. An IMS–IMS Analogue of MS–MS, Anal. Chem. 2006, 78, 4161–4174.
  3. Merenbloom, S. I.; Koeniger, S. L.; Valentine, S. J.; Plasencia, M. D.; Clemmer, D. E. IMS–IMS and IMS–IMS–IMS/MS for Separating Peptide and Protein Fragment Ions, Anal. Chem. 2006, 78, 2802–2809.
  4. Koeniger, S. L.; Clemmer, D. E. Resolution and Structural Transitions of Elongated States of Ubiquitin. J. Am. Soc. Mass Spectrom. 2007, 18, 322–331.