Breakthrough Technology for Bottom-Up Proteomics and Small Molecule Research

High mass resolution is a prerequisite for high mass accuracy. Both are key for the analysis of unknown samples. High resolution enables the separation and detection of isobaric signals that are not discernible under lower resolution conditions. With this technology, the mass-to-charge ratio (m/z) for individual ions is very accurately reported so that the elemental formula can be determined confidently. Consequently, there is a need for a technology that can offer resolutions of greater than 100,000 (defined by full-width half maximum [FWHM]).

The LTQ Orbitrap mass spectrometer (Thermo Electron Corp., Bremen, Germany) is a hybrid system combining the capabilities of the Finnigan LTQ linear ion trap mass spectrometer in tandem with the high resolution and high mass accuracy capabilities of the Orbitrap mass analyzer.1 The result is a technology that offers high resolving power; mass accuracy; and high dynamic range, both in MS and MSn experiments.

The LTQ Orbitrap supports a wide range of applications from routine compound identification to the most demanding analysis of very low-level components in complex mixtures. The instrument is well suited for applications in the areas of metabolomics; metabolite identification; proteomics; and drug discovery, in which high resolution and accurate mass measurements are the key to success.

Principle of operation

Figure 1 - Orbitrap.

The instrument combines a well-established LTQ mass spectrometer with the Orbitrap mass analyzer. The proof-of-principle of the LTQ Orbitrap was first described by Makarov.2 The LTQ Orbitrap consists of an inner (central) and an outer electrode, which are used to trap ions in an electrostatic potential (Figure 1). Interfacing of the Orbitrap to the LTQ mass spectrometer is made possible by a C-shaped ion storage trap (C-trap), which is used to store and collisionally cool ions prior to injection into the Orbitrap. Using this technique, it is possible to generate ion populations with intensity ranges of 104 in a single spectrum.

Figure 2 - LTQ Orbitrap operation.

In the hybrid instrument, ions generated in the atmospheric pressure ionization (API) ion source are trapped in the LTQ mass analyzer, where they are analyzed using the instrument’s MS and MSn scan modes. Following this, ions are axially ejected from the LTQ, stored in the C-trap, and then pulsed toward the central point of the C-trap arc that coincides with the Orbitrap entrance aperture. Ions are then captured in the Orbitrap by rapidly increasing the voltage on the inner electrode. The trapped ions assume circular trajectories around the inner electrode as well as axial oscillations along it (Figure 2).

After voltages are stabilized, the oscillating ions produce a signal on the outer electrodes, which is detected as an image current by a differential amplifier and converted into a frequency spectrum using a Fourier transform algorithm. Because the frequency of oscillations is directly related to the mass-to-charge ratio, the frequency spectrum is readily transformed into a mass spectrum using two-point calibration and processed using Xcalibursoftware (Thermo Electron Corp).

Dynamic range of accurate mass

The dynamic range over which accurate measurements of mass can be made is a central analytical figure-of-merit since it determines the true utility of the accurate mass capability of a mass spectrometer for real-life applications. With accurate mass analyzers coupled to LC devices, it is important to determine the range of intensities over which accurate masses can be determined at sufficient detection speed (e.g., when recorded at a rate of 1 spectrum/sec). For all analyzers, mass accuracy is limited statistically by too few ions or by space charging effects due to too many ions.

It was shown that the dynamic range of mass accuracy of the LTQ Orbitrap mass analyzer reaches 5000 (at least an order of magnitude higher than typical values for time-of-flight instruments). Due to the high resolving power of the instrument, the accurate mass of a signal can be determined as soon as the peak is reliably distinguished from noise (S/N peak-to-peak >2 . . . 3). From this point of view, the LTQ Orbitrap enables accurate mass measurements over a dynamic range that matches or exceeds the spread of signal intensities in the electrospray ion source.

Applications: Metabolomic profiling

Metabolomics involves generating a semiquantitative comparison between a collection of control and modified samples. One example of a metabolomics experiment involves comparing the small molecule metabolic profile of healthy individuals with the profile of individuals with a defined disease or toxicological insult. The response of the organism to a pathophysiological condition and the impact of a pathogen itself on an organism are reflected by a change in the metabolite profile contained in a biological specimen such as blood, urine, or tissue. The emphasis is on small molecules because many of the endogenous metabolites of interest have molecular weights of less than 250 u. In a metabolomics experiment, one goal is to locate endogenous components that are changing as a result of a stimulus (e.g., a drug dose). Once change is observed, the components need to be identified. These often require MS-MS for structure elucidation. High-efficiency low mass ion transmission is critically important for small molecule MS-MS experiments.


High-resolution LC-MS data were obtained with an LTQ Orbitrap with instrument parameters optimized for the transmission of low mass ions in the range 85–850 u. The standard caffeine, MRFA, and Ultramark (Bio-Rad Laboratories, Hercules, CA) tune mixture were used to calibrate the LTQ Orbitrap.


Figure 3 - MS-MS data collected for buspirone using the LTQ Orbitrap.

The LTQ Orbitrap showed good mass transmission independent of the acquired mass range. In general, it demonstrated high performance for metabolic profiling. Low mass transmission resulted in spectra very similar to those from the linear ion trap. Accurate MS and MS-MS data were collected for buspirone with only 10 pg loaded on a 4.6 × 150 mm column at 1 mL/min flow rates (Figure 3). Moreover, with the vast majority of measurements on large and small peaks falling within 3 ppm, the performance appeared well suited for small molecule structure elucidation work. Daily calibration of the LTQ Orbitrap is recommended in order to maintain these levels of mass accuracy.

Application: Protein identification

High mass accuracy helps to virtually eliminate the problem of false positive peptide identification in proteomics and to identify post-translational modifications much more easily than is currently possible.3


To characterize LC-MS-MS in the LTQ Orbitrap, 10 fmol of a digest of bovine serum albumin (BSA) were injected onto a 75-μm column, where a 30-min elution gradient was performed. One-second survey scans with a resolution of 60,000 (at m/z 400) and in parallel in the ion trap mass analyzer, six low-resolution MS-MS scans (0.25 sec each) were chosen, resulting in a cycle time of approximately 2.5 sec.


Figure 4 - Data evaluation with BioWorks using SEQUEST.

With the above approach, data evaluation with BioWorks™ (Thermo Electron Corp.) using SEQUEST® (University of Washington, Seattle, WA), a sequence coverage of >60% was obtained (Figure 4). BSA was identified as the first hit, and all peptides found showed excellent probability scoring as well as high XCorr values. High resolving power and good signal-to-noise were evident. The mass accuracy of the precursor ions helped to minimize database search time and increased confidence in search results.


The LTQ Orbitrap is a reliable, easy-to-use, robust hybrid mass spectrometer that combines patented Orbitrap technology with the Finnigan LTQ linear ion trap to offer high mass accuracy and high mass resolution together with a large dynamic range and very good detection power. It enables accurate mass measurements over a dynamic range that matches or exceeds the spread of signal intensities in the electrospray ion source. Experiments demonstrate that the performance of the LTQ Orbitrap, with regard to mass resolution, mass accuracy, and sensitivity, is well suited for small molecule structure elucidation and metabolomic profiling applications, as well as for bottom-up proteomics.


  1. Makarov, A.; Denisov, E.; Lange, O.; Kholomeev, A.; Horning, S. Proc. 53rd Conf. Am. Soc. Mass Spectrom., San Antonio, TX, June 5–9, 2005; poster no. 1885.
  2. Makarov, A. Electrostatic axially harmonic orbital trapping: a high- performance technique of mass analysis. Anal. Chem. 2000, 72, 1156–62.
  3. Jesper, V.; Olsen, J.; Lyris, M.F.; de Godoy, L.; Li, G.; Macek, B.; Mortensen, P.; Pesch, R.; Makarov, A.; Lange, O.; Horning, S.; Mann, M. Parts per million mass accuracy in an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell Proteomics Dec 2005, 4, 2010–21.

Dr. Makarov is Research Manager, Dr. Muenster is Director of Marketing, Thermo Electron Corp., Hanna-Kunath-Str. 11, 28199 Bremen, Germany; tel.: +49-421-5493300; fax: +49-421-5493426; e-mail: [email protected]