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 Xcalibur™ software
(Thermo Electron Corp).
Dynamic range of accurate
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
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.
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.
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
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.
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
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
- 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.
- Makarov, A. Electrostatic axially harmonic orbital trapping: a
high- performance technique of mass analysis. Anal. Chem.
2000, 72, 1156–62.
- 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]