Enhancing Mass Spectrometry Capabilities

From Spectroglyph, LLC

Jump to: navigation, search

IMS Orbitrap

Ion Mobility Spectrometry (IMS) has been widely used for structural characterization of biochemical components in proteomic profiling. Orbitrap™ mass spectrometry offers unsurpassed analytical performance for both denatured and intact proteins, and has been successfully applied to detection and identification of large protein complexes, based on both accurate mass measurements and sequence identification of the constituent backbone fragments. Due to a fundamental mismatch between IMS and Orbitrap signal acquisition rates, no IMS structural characterization has been enabled yet in intact protein analysis with an Orbitrap. Herewith, we bridge the gap between IMS and Orbitrap capabilities with the novel analytical system capable of both structural and sequence characterization of intact proteins at high resolution and mass accuracy.

Figure 1 shows a novel hybrid IMS-Orbitrap system for obtaining both structural and sequence information of biochemical compounds.

Figure 1: A novel hybrid IMS-Orbitrap system.


Our novel IMS instrument has been coupled to a Thermo Fisher Scientific Q Exactive Orbitrap™ mass spectrometer using ESI in direct infusion mode. The IMS incorporates an interface for orthogonal ion injection, followed by an ion funnel trap, a 100 cm-long drift tube and a stacked-ring ion guide with an IMS Exit Gate. Per each IMS experiment, 2N-1 ions packets were injected into the drift tube using a waveform based on a pseudo-random sequence of N-bits. The same waveform was applied to the Exit Gate and shifted sequentially in time. At each delay step, Orbitrap spectra were acquired, exhibiting signals of concurrently detected N-ion packets. The acquired multiplexed spectra were inverse transformed to reconstruct the original data vector.


Our front-end interface encompassing orthogonal ion injection provides highly efficient capture, desolvation and transmission of the ESI-generated ions to the ion funnel trap, accompanied by concurrent removal of large undesolvated droplets and neutrals. While in the trap, the ions exhibited a trapping efficiency of 50% for a variety of peptide and protein species. Complete purging of an ion cloud from the trap is accomplished in 200 us, which ensures lower peak dispersion in the IMS domain. Multiple ion packet injections on the time scale of a single IMS separation, or Multiplexed Mode, result in high sensitivity detection and structural characterization of complex biological samples.

IMS instrument was coupled to a Q ExactiveTM OrbitrapTM Mass Spectrometer (Thermo Fisher Scientific). IMS-OrbitrapTM Q Exactive system has been validated in experiments with a variety of samples, including a calibration mixture, a bovine serum albumin tryptic digest, and different solutions of denatured and native proteins. The acquired multiplexed IMS-Orbitrap signals were successfully reconstructed using an inverse matrix transform. Reconstructed data vectors revealed an IMS resolving power of 50 to 80, a mass resolving power of 60,000 (and above) and a mass accuracy of 1 ppm.

Figures 2-4 show 500 nM Bovine Serum Albumin (BSA) encoded and reconstructed multiplexed IMS-Orbitrap signals. Signal reconstruction was performed using an inverse matrix transform.

Figure 2. BSA Digest: Encoded multiplexed IMS-Orbitrap signals
Figure 3. IMS-Orbitrap BSA Digest signals
reconstructed using an inverse matrix transform
Figure 4. One of the BSA peptides at m/z 710.42

In addition to signal acquisitions in the multiplexed dispersion mode, targeted IMS-MS/MS mode is available. Once the drift times of ions of interest are determined, IMS-Orbitrap system is then programmatically controlled to transmit mobility-selected, m/z-selected precursor ions to the Higher Collision Energy (HCD) cell for the following fragmentation and spectra acquisition at high resolution and mass accuracy, thereby providing both structural and sequence information of the precursor species.

Contact Us

Requests for Quotation should be sent to info@spectroglyph.com

Mail Spectroglyph