November 1, 2016 (Vol. 36, No. 19)

A New High-Performance, Yet Flexible, Method for Life Science Applications

Ion-mobility spectrometry (IMS) separates and characterizes ions in the gas phase based on their mobility within a carrier gas. IMS has recently being coupled with mass spectrometry (MS) and high-performance liquid chromatography (HPLC) to provide even greater separation and more detailed analysis.

Trapped ion mobility spectrometry-time-of-flight MS (timsTOF™), commercialized by Bruker, features adjustable ion mobility separation through imeX™ (Ion Mobility Expansion), a proprietary feature of tims-MS systems.

TIMS differs from conventional IMS in that:

• rather than transporting ions through a stationary gas, TIMS holds ions stationary against a moving gas;

• this allow for a physically small analyzer (10 cm vs 100 cm); operating at much lower voltages (300 V vs 3,000 V);

• nonetheless basic performance specifications are significantly better than competing technologies (e.g. mobility resolving power as much as nine times greater than t-wave);

• with “parallel accumulation,” duty cycles of 100% can be achieved; and

• perhaps most importantly, TIMS is far more flexible than competing technologies—allowing for the development of new workflows (e.g. PASEF).

TIMS adds an additional separation dimension to LC-MS. In TIMS, a gas propels ions forward through the TIMS tunnel with a force proportional to the ions’ collisional cross-section.

Simultaneously, an electric field pushes in the opposite direction, holding back ions according to their charge. The combination of electric field and gas causes species to separate and collect according to the combined characteristics of charge and cross-section. Ions are then eluted according to their mobilities—low mobility first—by ramping down the electrical field.

timsTOF components include (Figure 1):

  • a TOF analyzer with a resolving power of 50,000;
  • a TIMS tunnel, where a high-resolution ion mobility separation occurs, including parallel accumulation for up to 100% ion acquisition duty cycle and very high collection efficiency;
  • a quadrupole analyzer for selecting precursor ions; and
  • a collision cell, optimized for ion fragmentation and efficient ion transfer.

Data files acquired on timsTOF have an open format, *.tdf, that supports transparent and tailored analyses based on the open source SQLite relational database platform. SQLite thus enables users to develop their own analytical algorithms. Thus data from timsTOF are easily incorporated into novel data processing and customized visualization reports.

Ion mobility provides separation capability that MS alone or LC-MS cannot. Ions that are indistinguishable by MS alone, for example isomers and conformers, may be resolved through ion mobility and characterized by IM-MS. It also provides insight into molecular flexibility and folding mechanisms. It thus extends the capabilities of MS to the three-dimensional structure of ions, while simultaneously increasing peak capacity and therefore confidence.

Additionally, sensitivity in conventional IM-MS has been hampered by a limited duty cycle, a consequence of which is significant loss of ions. timsTOF provides a 100% duty cycle through its unique parallel accumulation capability. Users may enable/disable TIMS through software control without losing QTOF performance.

Figure 1. Configuration of the timsTOF ion path including the TIMS tunnel, where ions are mobility analyzed, an improved analytical quadrupole for selecting precursor ions, a collision cell optimized to work with TIMS, and a high-resolution TOF analyzer for rapid analysis of precursors and fragment ions.


During its development, evaluation, and beta-testing stage timsTOF has matured into a valued extension of MS for the analysis of polymers, proteins, peptides, and genes.

Applications of timsTOF to molecules of interest in the life sciences—particularly proteins, peptides, and genes—has been amply demonstrated. Scientists at Bruker have employed TIMS to uncover the conformational behavior of several model peptides, including bradykinin. At resolving power in excess of 250, investigators uncovered new features that remained hidden when using conventional low-resolution IMS analyzers.

TIMS provided resolving power up to three or eight times greater than competing drift tube or traveling wave IMS, respectively. Additionally the technique resolved “congested” (poorly resolved) features through the molecules’ charge-neutral collisional cross sections (Figure 2). This information, if generalized to other therapeutic peptides in solution phase, could lead to new strategies for exploiting the potential of both new and established therapeutic proteins.

A similar approach was taken with ubiquitin, a 76-residue, 8565-amu regulatory protein that exists in all eukaryotic cells. Ubiquitin was known to display previously unresolved conformations during “soft” electrospray ionization. Relevant species of ubiquitin M+6H through M+13H, identical to the unresolved molecular forms reported in the literature, include compact, partially folded, and elongated states. TIMS resolved all these conformers.

Yet the authors cautioned that “the microheterogeneity within a particular conformational family and the relative state-to-state abundance can be altered by solvent memory, energetic, and kinetic effects.” They note as well that continued improvements in TIMS methodology could improve the ability to characterize solution conformations as well.

In a research collaboration between Germany’s Max Planck Institute of Biochemistry, Martinsried, and Bruker a technique around timsTOF, parallel accumulation-serial fragmentation (PASEF) is under development. Using PASEF, Matthias Mann and coworkers seek to overcome a limitation of LC-MS-based shotgun proteomics, namely that many precursor species elute simultaneously; some ions fragment one at a time while others are discarded. This is essentially the duty-cycle issue mentioned previously.

Through parallel accumulation, ions in TIMS of the same charge and collisional diameter collect until they are released. According to the researchers’ estimates, PASEF has the potential to generate hundreds of MS/MS spectra per second without loss of sensitivity, thus bringing about a tenfold gain in sequencing speed.

“An important advantage of PASEF is that the resulting spectra—in addition to the ion mobility dimension—are fully precursor mass resolved, unlike recently proposed data independent strategies. This also makes PASEF compatible with reporter ion-based chemical multiplexing strategies, such as iTRAQ or TMT. The about ten-fold gain that should be achievable by PASEF in shotgun proteomics experiments can be employed as increased sequencing speed without decrease in sensitivity,” reports Mann.

The Max Planck group has also noted the flexibility of accumulating and analyzing ions in parallel. In a paper co-authored with Bruker scientists, they note that the parallel accumulation design retains flexibility regarding the exchange of mobility range, resolution, and duty cycle, while maintaining “superior ion utilization efficiency.”

According to the authors in full duty-cycle mode timsTOF traps ions of m/z 622 to 2722 with an average efficiency of around 70%, which is constant across accumulation times of up to 85 msec. Compared with transmission mode (MS-only), the authors report improvements in peak intensities greater than a factor of ten.

In its current implementation timsTOF is considered an “expert” MS platform suitable for core laboratories and research groups with considerable MS expertise. As it evolves toward greater versatility and user-friendliness, the technique will continue to be adopted for solving scientific problems that have been hitherto inaccessible. 

Figure 2. The analysis of complex samples by LC-MS alone often fails to reveal all details. For example, the structural isomers Quercetin and Morin are here separated only in combination with TIMS.

Lanucara F et al. Nature Chemistry 6, 281–294 (2014).
Michelmann K et al. J. Am. Soc. Mass Spectrom. (2015) 26:14Y24.
Silveira J. et al, J Am Soc Mass Spectrom. 2016 Apr;27(4):585-95.
Silveira J. et al. Anal. Chem., 2014, 86 (12), pp 5624–5627/
Mann M et al. J Proteome Res. 2015 Dec 4;14(12):5378-87.
Silveira J. et al. Intl J. Mass Spectr. Available online 14 March 2016. Accessed at:

Melvin A. Park, Ph.D. ([email protected]), is director of research at Bruker
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