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Fourier Transform Mass Spectrometry

Michaela Scigelova‡¶, Martin Hornshaw§, Anastassios Giannakopulos‡,
and Alexander Makarov‡
This article provides an introduction to Fourier transform- sients produced by the image current. Consequently, they are
based mass spectrometry. The key performance charac- often referred to as Fourier transform-based mass spectro-
teristics of Fourier transform-based mass spectrometry, meters (FTMS).
mass accuracy and resolution, are presented in the view The combination of FTMS with ion preselection and frag-
of how they impact the interpretation of measurements in mentation devices and their coupling to reversed-phase liquid
proteomic applications. The theory and principles of op-
chromatography (LC) represents a ubiquitous approach to
eration of two types of mass analyzer, Fourier transform
both small molecule and proteomic analyses. Multidimen-
ion cyclotron resonance and Orbitrap, are described. Ma-
jor benefits as well as limitations of Fourier transform- sional LC separations have an important role to play in pro-
based mass spectrometry technology are discussed in teomics applications for reducing sample complexity, and
the context of practical sample analysis, and illustrated complement well the high dynamic range of detection in an
with examples included as figures in this text and in the acquisition offered by FTMS instruments.
accompanying slide set. Comparisons highlighting the The current article does not intend to be an exhaustive
performance differences between the two mass analyzers review paper on FTMS techniques; it is intended as teaching
are made where deemed useful in assisting the user with material for scientists without a physics background to assist
choosing the most appropriate technology for an applica-
them with understanding the mass spectrometry tools they
tion. Recent developments of these high-performing
are called to use in their everyday laboratory work. Thus, only
mass spectrometers are mentioned to provide a future
outlook. Molecular & Cellular Proteomics 10: 10.1074/ the fundamental aspects of FTMS which are useful to a non-
mcp.M111.009431, 1–19, 2011. physicist are introduced. The discussion then focuses on
examples of where this technology can be applied and what
are the benefits as well as limitations of this technology for
Mass spectrometers have been used for a long time in a practical proteomic applications.
variety of biological applications. Recent years, however, Need for High Resolution and Mass Accuracy—The mass
have witnessed a significant increase in the employment of accuracy is the ratio of the m/z measurement error to the true
mass spectrometers such as of time-of-flight, Fourier trans- m/z, usually quoted in parts per million (ppm). The mass
form ion cyclotron resonance (FTICR)1, and Orbitrap, which resolving power (resolution) is the measure of the ability to
provide accurate mass of analytes over wide mass range. The distinguish two peaks of slightly different m/z, herein under-
FTICR and Orbitrap analyzers outperform any other com- stood as full width at half maximum (FWHM). Linearity of de-
monly used mass spectrometer with respect to the maximum tection and very high fidelity in the determination of frequency
mass resolution and accuracy routinely achievable even for are inherent to FTMS and allow very high resolving power, mass
small numbers of ions. Both these mass spectrometers share accuracy, and dynamic range to be achieved. But why would
certain features, such as an image current detection system one need high mass accuracy and high resolution?
and the application of Fourier transform mathematical oper- The benefit of measuring a compound’s mass with ade-
ations for generating mass spectra from time domain tran- quately high accuracy can directly determine its elemental
composition. Accurate mass thus acts as a powerful “filter”
From the ‡Thermo Fisher Scientific, Bremen, Germany; §Thermo useful for confirming the identity of a compound or even
Fisher Scientific, Hemel Hempstead, UK identification of an unknown. This is illustrated in Fig. 1 show-
Received March 8, 2011 ing the fragmentation (tandem mass spectrometry (MS/MS))
Published, MCP Papers in Press, May 9, 2011, DOI 10.1074/
spectrum of flavonoid quercetin (m/z 303). Mass deviation of
mcp.M111.009431
1
The abbreviations used are: FTMS, Fourier transform-based mass less than 3 ppm for any detected fragment together with the
spectrometry; FTICR, Fourier transform ion cyclotron resonance; richness of the fragmentation spectra itself enable confirming
FWHM, full width at half maximum; MS/MS, tandem mass spectrome- the elemental composition of the starting compound as well
try; mmu, millimass unit; RF, radiofrequency; SWIFT, stored waveform as providing useful hints to its structure.
inverse Fourier transform; MECA, multiple excitation collisional activa-
tion; SORI, sustained off-resonance irradiation; IR, infra red; IRMPD,
In many cases, however, additional information other than
infra red multiphoton dissociation; ECD, electron capture dissociation; just the accurate mass measurement will be needed to obtain
MSn, multiple levels of fragmentation; LTQ, linear trap quadrupole. correct elemental composition. This includes, among others,
Molecular & Cellular Proteomics 10.7 10.1074/mcp.M111.009431–1

FIG. 1. Fragmentation (MS/MS)

spectrum of flavonoid quercetin (m/z
303). Mass deviation of less than 3 ppm
for any detected fragment together with
the richness of the fragmentation spec-
tra itself enable confirming the elemental
composition of the starting compound
as well as providing useful hints to its
structure. Assignment of the peaks per-
formed using software package Mass
FrontierTM.
FIG. 2. Effect of mass accuracy on

peptide identification, an example of
tryptic peptides from a human protein
database. A, Graph shows how many
peptides would match a search criterion
based on the accurate mass of the pep-
tide alone, considering mass deviation of
50, 20, 10, 5, 1, 0.1, and 0.01 ppm, re-
spectively. B, Detail of graph A. Slicing
the analyzed mass range into bins with a
width corresponding to the respective
mass deviation and counting number of
peptide hits in each such bin reveals that
even with a really low mass deviation
one would still encounter many bins with
more than 1 peptide in it. C, Histograms
for mass bin width of 5, 2, 1 and 0.1
ppm, respectively, where number of
peptide matches in a bin is on the x axis
and the frequency of an occurrence on
the y axis. Courtesy of David Fenyo,
Rockefeller University.
restrictions for the number of elements considered, Lewis and viation) the number of possible peptide candidates drops
Senior chemical rules, or isotopic patterns (1–3). Other as- significantly. The accurate mass acts as a “filter” that is re-
pects of the usefulness of mass accuracy for small molecule flected in later statistical evaluations of the results through a
analysis include the application of mass defect (4). significant reduction in the number of potential false positive
In the context of proteomics the precursor masses are used identifications, thus resulting in a higher confidence for pep-
as constraints for database searches. Fig. 2 illustrates this on tide identification.
the example of human protein database (provided by D. Fe- The other observation that can be made from Fig. 2 is that
nyo). It shows how many peptides would match a database even with a very low mass deviation of 0.1 or 0.01 ppm (at
search based on the accurate mass of the peptide alone, present not attainable on any routinely used mass spectro-
considering mass deviations of 50, 20, 10, 5, 1, 0.1, and 0.01 meter) one will still encounter many cases in the human da-
ppm, respectively. From the inspection of Figs. 2A and 2B it is tabase in which more than one peptide will fit into the bin of a
clear that with an improved mass accuracy (lower mass de- selected peptide mass plus or minus its respective mass
10.1074/mcp.M111.009431–2 Molecular & Cellular Proteomics 10.7

TABLE I
Effect of mass resolution on the confidence interval of mass accuracy
(mass tolerance). Example of pesticide Pirimicarb (C11H18N4O2;
关M⫹H兴⫹ ⫽ 239.15025) illustrating how the mass tolerance can limit
the number of elemental composition suggestions, and hence greatly
assist a compound identification. Resolution 15,000 and 80,000
(FWHM) and CHNO elements considered in this example
Number of elemental
Resolution Mass tolerance (mmu) composition
suggestions
15,000 ⫹/⫺ 9 14
80,000 ⫹/⫺1.7 1
deviation. This is summarized in the histograms in Fig. 2C

wherein the x axis shows the number of peptide matches in a
bin whereas the y axis denotes the frequency of occurrence. FIG. 3. High resolution results in better mass accuracy. Pesti-
Thus, for a tolerance of ⫾5 ppm one can observe that a cide Pirimicarb was measured in a mixture of other 115 pesticides
majority of peptide mass bins will have more than one and food toxins in a horse feed matrix. Top panel shows a mass
peptide in it, whereas for a tolerance of ⫾ 0.1 ppm a large spectrum taken at the time of the elution of Pirimicarb acquired at
proportion of peptide mass bins contain only one peptide. resolving power 15,000 FWHM. The unresolved interferences caused
the measured mass of Pirimicarb to be skewed toward a slightly
(Note: this calculation does not consider peptide modifica- higher value resulting in a mass deviation of 6.5 ppm. The same
tions that would add a large number of candidates.) A sample was then acquired at resolution 80,000 FWHM (bottom panel).
significant proportion of peptide mass bins, however, still This resulted in improved accuracy of both mass measurement (0.3
have more than one peptide. Thus, other information is ppm mass deviation) and quantitation. Courtesy of Markus Kellmann,
needed to support peptide identification than just its accu- Thermo Fisher Scientific.
rately measured mass.
The mass accuracy is therefore a very important parameter power 15,000 (FWHM). In this particular case, the unresolved
in proteomics experiments; incorrect determination of mass interferences are causing the measured mass of Pirimicarb to
can lead either to identification statistics that are worse than be skewed toward a slightly higher value resulting in a mass
they need to be if the mass accuracy window for a database deviation of 6.5 ppm. Acquiring data at higher resolution, a
search is set too wide, or to missed identification (false neg- much improved accuracy of mass measurement (0.3 ppm
atives) if the window is set too narrow (5). mass deviation) is achieved.
It is perhaps less obvious what the role of resolution is, and The above mentioned effect of resolution limiting the
how resolution and accurate mass “play together.” High res- achievable mass accuracy has very important consequences
olution is needed because one can not perform mass mea- for screening and quantitation experiments. If, for instance,
surement or quantitation of a peak accurately if the constitu- the extraction window is set too wide, compensating for pos-
ent components remain insufficiently resolved. In this context, sible matrix interferences and/or inadequate resolving power,
the resolution settings used in the analysis define a confi- then 1) mass accuracy will be compromised, and 2) hidden
dence interval (mass tolerance) within which we can trust our interferences will contribute to the integrated peak area de-
mass measurements. Table I illustrates this point considering tected for the compound of interest. In effect, there is a
an example of a pesticide Pirimicarb (m/z 239). Analysis per- serious risk of having a case of overquantitation or even of a
formed at resolution 15,000 (FWHM) defines a confidence false positive. If, on the other hand, the user sets an extraction
interval for mass measurement as ⫾9 mmu, while using window that is too narrow, the compound showing a higher
80,000 resolution narrows this interval to ⫾1.7 mmu. mass deviation than expected because of the presence of an
The reasoning behind this is that in a complex mixture many unresolved interference could go completely undetected.
matrix components and other analytes could co-elute with the There is a risk of a false negative.
compound of interest, and some of them could have a very In summary, for a given sample analyzed, the resolving power
similar mass. The overlap of such co-eluting peaks of a very is a key parameter affecting the correct assignment of masses
similar mass will skew the peak shape of our compound of for analytes. The accurate mass measurement can only be
interest, and the peak centroid will no longer correspond to its relied on when measured at sufficiently high resolution. In the
accurate mass. This is shown with the example of Pirimicarb analysis of complex mixtures the resolution impacts the reliabil-
mixed with 115 other pesticides and toxins into a horse feed ity of a compound’s detection and quantitation. In biomarker
matrix (Fig. 3). The top panel shows a mass spectrum taken at discovery studies, better mass accuracy translates into im-
the time of the elution of Pirimicarb acquired at resolving proved alignment and quantitation across spectra (6).


Fourier Transform—The roots of the Fourier transform (FT)
are found in a Fourier series in which complicated periodic
functions are written as the sum of simple waves mathemat-
ically represented by sines and cosines. Because of the prop-
erties of sine and cosine it is possible to recover the contri-
bution of each wave in the sum by an integral.
In FTMS, masses are represented by frequencies, and be-
cause frequencies can be measured very accurately, FTMS
can offer potentially very high mass measurement accuracy.
To illustrate the signal complexity that one encounters in
FTMS, consider an example of four frequencies representing
four different masses: frequency “v” of an intensity 1, fre-
quency “2v” of an intensity 0.5, frequency “5v” of an intensity
1.5, and frequency “8v” of an intensity 0.2 (Fig. 4A). Their
respective waveforms are displayed all together in Fig. 4B. An
FTMS detector will see all these four waveforms combined
into a signal looking similar to that in Fig. 4C.
Real sample analysis is, however, a far more complicated
affair; it is not unusual in a proteomic experiment to have
hundreds or even thousands of molecular species present in
a single mass spectrum. In addition, each molecular species
has also its isotopomers. The complexity of the resulting
signal being detected thus is considerable.
Time Domain Signal—In FTMS the ions are observed (im-
age current is induced on detection electrodes, digitized, and
recorded into a file) over a period of time potentially extending
up to several seconds. This time domain signal containing all
the characteristic frequencies of the measured ions at inten-
sities corresponding to the amount of the molecular species in
the sample is treated with the mathematical tool of the Fourier
transform.
The Fourier transform operation converts the time domain
signal into a complex (in the mathematical sense, i.e. contain- FIG. 4. Illustration of signal complexity encountered in FTMS. A,
Four frequencies representing four different masses are considered
ing a real and an imaginary part) spectrum. When the phase is here: frequency “v” of an intensity 1, frequency “2v” of an intensity
zero the real part of the frequency domain spectrum shows 0.5, frequency “5v” of an intensity 1.5, and frequency “8v” of an
what we call an absorption mode line, and in the case of the intensity 0.2. B, Displayed waveforms for the considered intensities
exponentially decaying signal it is known as absorption mode and frequencies. C, All four waveforms combined into a signal emu-
lating the time domain signal detected by an FTMS.
Lorentzian. The imaginary part of the spectrum gives a line
shape known as the dispersion mode Lorentzian, which is
broader than the absorption mode and also has positive and Attempts have been made to control or determine the phase
negative parts. This occurs only when the phase shift of the of the ion motion, and the topic is further developed in the
time domain signal is zero. Fig. 5 illustrates the real and Section Improving the Performance Characteristics of FTMS.
imaginary parts of the solution with their position and height Further Signal Processing—In most proteomics applica-
being phase dependent. tions, where dynamic range within the spectrum is of impor-
To eliminate this phase dependence, a square root of the tance, further signal processing needs to be carried out,
sum of the square of the real and imaginary parts is used, namely apodization and zero-filling.
known as the “magnitude spectrum” Apodization, literally meaning “removing the foot,” is a
transformation of transient to smooth the discontinuities at its
magnitude ⫽ 冑Re2 ⫹ Im2
beginning and end. It is used to remove artifact peaks adja-
(Eq. 1)
cent to real peaks at the base of the spectrum (Fig. 6).
The drawback is that the magnitude spectrum has only about To transform the time domain signal (detected by FTMS
half the resolution obtainable from that particular data (Fig. 5). and digitized) accurately into its constituent frequencies, long

FIG. 7. Ion motion within an ICR cell. A, Force affecting a charged

particle moving in the homogeneous magnetic field causes the par-
FIG. 5. Phase dependence of the real and imaginary part of ticle to assume circular trajectory. B, As the magnetic field can
Fourier transform solution. A, At initial time phase equal to zero, the confine the ions only in the radial direction, no confinement exists in
real and imaginary part of the complex (real and imaginary) frequency the axial direction along the magnetic field.
spectrum represents the pure absorption and dispersion mode spec-
trum, respectively. B, For any other initial time domain phase, the literature are often given for a nonapodized spectrum whereas
complex (real and imaginary) components represent a linear combi- quotations of the Orbitrap performance always refer to an
nation of absorption and dispersion modes, and the resulting peak
apodized one.
shapes are asymmetric. Employing a ”magnitude spectrum” removes
the initial time domain phase dependence with a penalty of obtaining Zero-filling is another treatment of the spectrum, which
a spectrum with only half the resolution achievable for that particular corresponds to adding zero-filled interval to the transient. It
data. enables easier interpolation between points in the frequency
domain, and therefore results in an improved peak shape.
Fourier Transform Ion Cyclotron Resonance

Principle of Operation—A charged particle in a magnetic
field with a velocity vector perpendicular to the magnetic field
experiences a force perpendicular to the plane defined by the
velocity and the magnetic field. This force, which is known as
the “Lorentz force,” induces a change in the direction of the
velocity vector but not in its magnitude (Fig. 7A). This force is
always perpendicular to the direction of the velocity vector.
FIG. 6. Example of apodized and nonapodized simulated spec- Following the vector of velocity we see that it describes a
tral peak. Whereas apodization improves dynamic range and appear- circle and as a result the charged particle (an ion) is trapped
ance of FTMS spectra, it impacts negatively on its resolution. Cour- by the magnetic field on a circular trajectory. The frequency of
tesy of R. Malek, Thermo Fisher Scientific.
this rotation is characteristic for each mass and magnetic
field, and in a uniform magnetic field it is not dependent on
periods of observation (long transients) are required. In prac-
initial coordinates and velocities of ions.
tice, however, transients can not be very long (limited to a
An instrument in which ions are excited to a larger trajectory
maximum of several seconds) because of time restrictions
in the presence of the magnetic field is called an “ion cyclo-
(e.g. eluting chromatographic peaks) or, more importantly,
tron resonance” (ICR) mass spectrometer. The theory of cy-
because of the loss of signal caused by collisions of the ions
clotron resonance was developed by Lawrence in the 1930s
under observation with residual gas. Such finite observation
and awarded the Nobel prize in 1939. Other designs followed
intervals cause the appearance of artifact peaks (spectral
producing instruments that were used principally to study
leakage) after the Fourier transform. Weighing functions, re-
ion-molecule reactions (7). The Fourier transform, introduced
ferred to as windows, are thus applied to the data to reduce
by Comisarow and Marshall in 1974, offers the crucial advan-
these artifact peaks. The most commonly used window in
tage of detecting all frequencies simultaneously rather than
FTMS is the “raised cosine” from Hann apodization, which
detecting one frequency at a time (8).
has the advantage of low aliasing.
Ion Motion Inside the ICR Cell—The angular cyclotron fre-
By removing the small artifact peaks from the spectrum the
quency on an FTICR that consists of two infinite parallel
apodized spectra allow for a higher dynamic range. This
electrodes and contains only a magnetic field is described by
comes for a price; the resolution of an apodized spectrum is
about half the resolution of a nonapodized one. Please note qB
␻c ⫽ (in radian/sec) (Eq. 2)
that the performance characteristics of FTICR quoted in the m

and one revolution is equal to 2␲ radian
␻ ⫽ 2␲␯ (Eq. 3)
wherein “␯“ is the frequency of revolutions measured in Hz,

“␻c” is the angular frequency in radian/second, “q” is the ion
charge, “m” the mass, and “B” the strength of the magnetic
field. This formula shows that there is an inversely proportional
dependence between m/q and the observed orbital frequency.
The magnetic field alone can confine the ions only in the
radial direction and no confinement exists in the axial direc-
tion along the magnetic field (Fig. 7B). Static electric fields
with potential wells on the order of a few volts are used to
confine the ions in the axial direction, and the ions start
executing a harmonic trapping oscillation between the two
electrodes at a trapping frequency
␻z ⫽ 冑 2qVtrap␣
ma2
(Eq. 4)
wherein “Vtrap” is the trapping potential in the axial direction,

“␣” is a value characteristic of the trap geometry (2.77 for a
cubic (9), 2.84 for a cylindrical (10, 11), and 3.87 for an open
trap (12)) and “a” is the trapping electrode separation. The
interference of this field with the cyclotron motion of the ions
produces deviations from the unperturbed cyclotron motion
and as a result lowers the frequency to
␻⫹ ⫽
␻c
2
⫹ 冑冉冊
␻c
2
2
⫺
␻z2
2
(Eq. 5)
FIG. 8. Techniques applied in FTICR to excite a whole range of
m/z simultaneously.
As the deviation from the unperturbed cyclotron frequency detectable image current on the detection plates. Ions need
depends strongly on the trapping potential, trapping voltages to be coherently excited to a larger radius for a signal to be
as low as possible are used. detected. The ions will remain at this radius after the exci-
Finally, there is also a low frequency precession motion tation signal has been stopped until their energy is dissi-
known as “magnetron” described by pated by collisions with residual gas. This together with
dephasing of ion coherence, in which ions of the same
␻⫺ ⫽
␻c
2
⫺ 冑冉冊
␻c
2
2
⫺
␻z2
2
(Eq. 6)
cyclotron frequency become distributed out of phase at the
same cyclotron radius, limits the extent of detection time.
The larger the size of the molecule and the higher the
The magnetron and trapping frequencies are usually much residual gas pressure inside the ICR cell, the shorter is the
lower than the cyclotron frequency. They are generally not time available for detection.
detected unless there is a small misalignment of the instru- All masses should receive a similar excitation to avoid mass
ment axis with the magnetic field, or the ion motion amplitude dependent detection conditions. A radiofrequency (RF) poten-
is so large that it becomes comparable with the dimension of tial is normally applied to a pair of excitation plates on oppo-
the ICR cell. Then they demonstrate themselves as small site sides of the ICR cell. In FTICR, instead of exciting and
sidebands of the peaks in the spectrum. detecting one mass (frequency) at a time, a whole range of
In conclusion, ions should enter the ICR cell at low kinetic m/z is excited simultaneously by usually applying one of the
energies so that they can be confined by relatively low trap- following techniques (Fig. 8).
ping potentials, and they should be located preferably in the Frequency sweep (chirp) excitation rapidly scans the fre-
center of the cell to take full advantage of the excitation quency range corresponding to the masses that need to be
conditions and the cell dimensions. excited (13, 14). A wide m/z range can be excited and a
Excitation of Ions in the ICR Cell—Because of the kinetic reasonably flat response can be achieved. However, shoul-
energy of the thermal motion, ions will have some very small ders remain at each end of the excitation range.
cyclotron motion even at room temperature and without any Because Fourier transforms work also in reverse, another
excitation. Such conditions, however, do not generate a way to produce an excitation waveform is to consider the

masses or ranges of masses that need to be excited, and

carry out an inverse Fourier transform from the frequency
domain to the time domain. The corresponding transforma-
tion can be recorded and played back for excitation. This is
known as stored waveform inverse Fourier transform (SWIFT)
(15). This method offers a good excitation response. In addi-
tion, unwanted masses can be completely ejected from the
cell during this step. SWIFT waveforms can also be used for
ion isolation before MS/MS analysis in the ICR cell, as dem-
onstrated for a single isotopomer of ubiquitin (8.6 kDa) and
carbonic anhydrase (29 kDa) (16). Another example was an
analysis of intact bovine histone H4 in which a mass selec-
tivity of 0.1 m/z unit for isolation of the 18⫹ charge state was
achieved (17).
Mass Calibration—As mentioned earlier, the trapping po-
tentials change the ICR’s intrinsically simple mass to fre-
quency relationship (equation (2)) to a more complicated form
(18)
m A B
⫽ ⫹ 2 (Eq. 7)
z ␯⫹ ␯⫹
wherein “A” and “B” are constants that can be obtained by

fitting a set of experimental frequencies to m/z values. Addi-
tion of more parameters into the equation does not improve
significantly the outcome (19).
The limiting condition for which the above equation is valid
is that the space charge should be negligible, i.e. a very small FIG. 9. Grid cell used to minimize the excitation field perturba-
number of ions should be present. Nevertheless, the field tions. Graphical rendering of the ICR cell of the LTQ FT Ultra instru-
perturbations affect both the calibrant and the ions, and this ment depicting the grids placed inside the cylindrical electrodes over
the entire length of the ICR cell. The excitation waveforms are sup-
equation can be extended also for large numbers of ions as
plied to these grids so that the excitation field extends well past the
long as their number in the FTICR cell is always kept the trapping region. The trapping rings are segmented because the po-
same. For this reason, FTICR mass spectrometers that have tentials applied to the segment behind the grids have to be 4.6-fold
automatic control of the number of ions being injected into the higher than those applied to the grid-free segments in order to es-
ICR cell have a clear advantage (20). This is especially impor- tablish the same trapping potential.
tant in proteomic experiments, in which the FTICR instrument
is often coupled to an LC, and the number of ions can fluc- segmented into four sections because the potentials applied
tuate dramatically from spectrum to spectrum. to the segment behind the grids has to be 4.6-fold higher than
Issues Due to Electrical Field—Both the electrostatic fields those applied to the grid-free segments to establish the same
(i.e. the trapping voltage) as well as RF fields used for exci- trapping potential (Fig. 9).
tation can create problems for mass analysis, and uniformity The introduction of the “grid cell” is an interesting proposition
of both these types of field is called for. Various solutions have also from the perspective of mass accuracy achievable on the
been proposed to deal with the axial trapping field (21, 22). In instrument. Going back to the mass calibration formula (equa-
the late 1980s, Wang and Marshall used a grounded mesh tion (7) in Section Mass Calibration) and taking into account that
just in front of the axial trapping electrodes (23). In this case, the parameter B therein relates to the electric field errors, it can
the trapping field penetrating through the grid can have an be seen that the electric field causes an error ⌬B of the calibra-
effect only when the ions get very close to the trapping tion electric field-dependent parameter B resulting in a mass
electrodes. assignment error ⌬m. To improve mass accuracy, one either
The simple approach of using a grid on an open-ended ICR has to reduce ⌬B or increase the cyclotron frequency by means
resolves the problem with homogeneity of the excitation field of a higher magnetic field. The grid cell reduces ⌬B and, con-
as well. The grids are placed inside the cylindrical electrodes sequently, ⌬m approximately by a factor of 4. If one then real-
over the entire length of the ICR cell. The excitation wave- izes that the same effect on ⌬m could be achieved by exchang-
forms are supplied to these grids so that the excitation field ing the 7 T magnet for a 14 T one (increasing the cyclotron
extends well past the trapping region. The trapping rings are frequency by a factor of 2), the design employing the grid cell is

FIG. 10. Effect of magnetic field

strength on resolution. Resolution
(FWHM) achievable for a range of
masses for a 1 s transient employing
magnetic field strengths of 1 to 40 T is
presented. The resolution shown for
FTICR is nonapodized. Please note that
both axes are logarithmic.
an elegant solution. There are, nevertheless, also other methods fields. High kinetic energy would be useful for collision-in-
that can be implemented to achieve homogeneity of the exci- duced dissociation (CID). For example, in a 3 T magnet using
tation field (24 –26). argon as a collision gas, an ion of 1000 Da at 1 cm excitation
Effect of Magnetic Field on Resolution (High Field Mag- radius has center of mass kinetic energy of 1.67 eV, whereas
nets)—The resolution at low pressure is given by (27, 28) in a 9.4 T magnet it would have 16.4 eV. This would translate
to a more effective fragmentation. But as mentioned already,
m 1.274 ⫻ 107 zB0Taqn
⫽ (Eq. 8) to carry out CID in the ICR cell, gas has to be admitted and the
⌬m m
pressure has to be raised. To resume analysis afterward,
wherein m/z is given in ␮ per elementary charge. Fig. 10 enough time needs to be allowed for the gas to be dissipated
shows the resolution achievable within a 1 s transient for and the ICR cell to return to the low pressure required for
magnetic field strengths of 1 to 40 T and for different masses. excitation and detection. It is much more practical to carry out
The values in this graph are for a nonapodized spectrum, CID outside of the ultra-high vacuum region in an external
which means that resolution of an apodized spectrum would analyzer or collision cell.
drop to about a half (in most cases it is not possible to infer d) Ion trapping duration—In the presence of collisions with
exact values for an apodized spectrum as each manufacturer residual gas in the ICR cell the ions lose kinetic energy. This
uses a different apodization algorithm). A detailed discussion means that the cyclotron radius decreases rapidly with time,
of the characteristics and advantages of high field magnets and simultaneously, the magnetron radius slowly increases.
can be found in Marshall et al. (29). Here, we will summarize The motion of ions becomes unstable when magnetron radius
some of the main points: happens to be significant in comparison to the cell radius. The
a) Mass resolving power and mass accuracy—The formula longer it takes for this to happen, the longer the time domain
(8) above shows clearly that resolving power (m/⌬m) will in- transient that can be achieved, and the higher the subsequent
crease linearly with increasing magnetic field (Fig. 10). Higher resolution. Employing higher magnetic field strength is advan-
mass accuracy can be achieved as a consequence of in- tageous as the time required for the ion magnetron radius to
creased resolving power. expand to the radius of the ICR cell increases in a quadratic
b) Data acquisition speed—The same formula also shows dependence with B.
that the time needed to acquire a time domain signal of a Another mechanism for ion loss in ICR is dephasing. Ions
given mass resolving power varies as 1/B. This can represent within the cloud of the same m/z value lose their coherence
a considerable gain in time. On some instrument designs this and over time become distributed at different cyclotron phase
benefit might not make a practical difference, however, as angles at the same radius. Ion cloud density, columbic
some fragmentation techniques performed in the ICR cell interactions with other ion packets, and magnetic field
require introduction of gas (for fragmentation or axialization). strength are all parameters that can affect dephasing.
This translates into an added time overhead of extra pumping e) Charge-related phenomena—In an ICR cell the existence
in order for the instrument to drop back to good vacuum of concentrated charge can be responsible for shifting and
levels before ion excitation and detection. broadening mass spectral peaks, sometimes to the extent of
c) Kinetic energy—Higher maximum ion kinetic energy for a merging peaks of similar mass into one. Such an artifact is
given excitation radius can be achieved with higher magnetic known as peak coalescence. The tendency of two ion packets

FIG. 11. Effect of transient duration

on resolution. Resolution (FWHM)
achievable for a range of masses with
magnets of 7 and 21 T and for durations
of transients of 1 and 5 s is provided for
comparison. The resolution shown for
FTICR is nonapodized. Please note that
both axes are logarithmic.
to coalesce drops with increasing magnetic field strength. In gies, carry out fragmentation experiments, and re-analyze the
practice, this (and not just a mere increase in resolution and products. Alternatively, the ions can be allowed to interact at
mass accuracy, which could be achieved by other means as low kinetic energies, preferably thermal, with electrons or
well, for instance by an improved ICR cell design as mentioned photons. This makes the ICR cell a reaction vessel, which, with
in Section Problems with Potential Field) tends to be the main some added time overhead, can be a tandem mass spectro-
reason one would consider using a bigger magnet having in meter in its own right. Here we mention the three main fragmen-
mind all the negative aspects this brings with it (e.g. consider- tation techniques used in the ICR cell, and discuss the advan-
able increase in cost and handling difficulties). tages and disadvantages of implementing them.
A different manifestation of charge-related effect can be Collisional-induced Dissociation—A technique traditionally
observed at low numbers of ions (⬍1000). Ions “evaporate” used in FTICR is the collision induced dissociation. Ions have
from the ion packet and a “comet tail” is formed (30). After to enter the ICR cell at low kinetic energies to be trapped
some time, depending on a particular ICR cell geometry and effectively by the trapping potentials being as low as possible.
magnetic field strength, ions of the ion packet spread into a These conditions, however, do not promote any fragmenta-
ring with no ability to induce image current on the detection tion. Ions need to be excited to larger radii to gain higher
plates. A limit on resolution is thus reached because of a kinetic energy. This excitation can be done resonantly for
shortened transient duration. The “comet tail” extension each mass employing one of the following techniques: multi-
rate will diminish at higher magnetic fields due to the de- ple excitation collisional activation (MECA) (31) where ions are
crease in the magnetron frequency which is responsible for resonantly excited and then allowed to relax by collisions;
this effect. very low-energy CID (32), which uses 180o phase shifts in the
Effect of Transient Time Duration on Resolution—Another excitation wave form; or the simplest to implement and at the
way to increase the resolution without using a bigger magnet is same time the most robust technique known as sustained
to allow for a longer transient acquisition. Fig. 11 compares the off-resonance irradiation (SORI) (33). In SORI, ions of a spe-
resolution achievable with magnets of 7 and 21 T for durations cific mass are excited by an RF field that is slightly off-
of transients of 1 and 5 s. Of course, there are limits because: resonance with the cyclotron frequency. As a result, ions are
a) long acquisition times might not be always practical, alternatively accelerated and decelerated with a period that is
particularly in the case of coupling to LC. reciprocal to the frequency mismatch.
b) there is only a limited amount of time the transient can be All these methods activate the ions not in a single event but
recorded before it dies out because of collisions with residual by using multiple collisions. Because collision gas is used in
gas. Although all mass spectrometers require vacuum for the the form of a pulsed valve opening on a millisecond time
analysis and detection of ions, the performance of FTMS is scale, a delay of several seconds is normally used to allow the
more sensitive to pressure than other mass analyzers. Vac- gas pressure to drop before resuming FTICR mass analysis.
uum of 10⫺9 to 10⫺10 Torr (1 Torr ⫽ 133.3 Pa) is required to Using activation by gas collisions the ions absorb energy in
achieve high resolution, vacuum requirements being more multiple steps, and because there is enough time for redistri-
stringent for higher-field magnets. bution of this energy in the molecule (a process defined as
Fragmentation in the ICR Cell—The ICR cell is able to “ergodic”) the weakest bonds tend to break first. As many
confine different masses, excite them to higher kinetic ener- post-translational modifications are labile, including phosphor-

FIG. 12. ECD fragmentation of an intact protein. Charge state 12⫹ of ubiquitin was fragmented using ECD. From the 72 theoretically
cleavable peptide bonds within ubiquitin sequence 71 have been cleaved obtaining 147 fragment ions in total. Courtesy of M. Zeller, Thermo
Fisher Scientific.
ylation, such moieties tend to break first. The fragmentation of cathode placed behind the ICR cell. Electron capture pro-
a peptide would not proceed any further because the rest of duces a radical cation [M⫹nH](n⫺1)⫹. which can dissociate via
the peptide can not be anymore excited by the original ap- a rapid, facile fragmentation of the N-C bond of the peptide
plied excitation frequency (the mass of the peptide has chain, producing mainly c and z type fragment ions (38).
changed by losing the modification moiety and its cyclotron ECD has some remarkable advantages as the fragmenta-
frequency is now different). tion is not directed by peptide bond protonation and shows a
Infra-red Multiphoton Dissociation—Another method to de- nonergodic nature (“nonergodic” means that the process
posit energy into the molecules is by photon absorption. Ions adds internal energy to the precursor faster than is the rate of
have been activated in the ICR cell by using both infrared (34, energy randomization). As a consequence, the fragmentation
35) and ultraviolet (36) photons. Although infrared multiphoton does not have to happen on the weakest bond within the
dissociation (IRMPD) was used initially for small molecules, it compound as would be the case with CID or IRMPD tech-
soon found an application in sequencing biomolecules (37). niques. Thus, fragile post-translational modifications within
The fragmentation spectra produced are similar to collision the peptide and protein sequence are preserved.
induced dissociation but with the added benefit of not requir- ECD allows site specific analysis of phosphorylation (39,
ing pulses of gas that spoil the vacuum conditions. Another 40), O- and N-linked glycosylation (41), or sulfation (42). ECD
benefit is that the ions start the mass analysis from the center holds much promise as a supplementary dissociation tech-
of the ICR cell (thus simplifying excitation and maximizing nique to CID for unambiguous protein identification, de novo
signal) because they have to be there for a successful inter- sequencing (43) and detailed protein characterization (44).
action with the laser beam. Nevertheless, a few hundred The method has been shown to distinguish leucine and iso-
millisecond up to a few second interaction time is required. leucine residues (45) and even differentiate between peptides
Electron Capture Dissociation—Electron capture dissocia- containing a D- or L-amino acid within its sequence (46). ECD
tion (ECD) has recently evolved as an alternate activation had also been applied to intact protein analysis (47) delivering
method, especially for peptide and protein sequencing. With a good level of fragmentation. Fig. 12 depicts sequence cov-
ECD, multiply charged (usually but not exclusively) cations are erage of ubiquitin (charge state 12⫹) obtained with ECD. From
irradiated with low energy electrons produced by an emitter the 72 theoretically cleavable peptide bonds (ubiquitin has 75

peptide bonds in total, but the presence of three proline

residues means that even after these three peptide bonds had
fragmented the opened ring structure of proline holds the
respective fragments together) 71 were cleaved obtaining a
total of 147 identifiable fragment ions.
Summarizing the above, CID and IRMPD induce dissocia-
tion by vibrational excitation of the precursor resulting usually
in the cleavage of the weakest bond. Within peptides, the
backbone amide bond has the lowest energy barrier to dis-
sociation resulting in predominantly b- and y-type fragment
ions being formed. Unfortunately, substituents such as many
co- and post-translational modifications often have lower en-
ergy barriers than those of backbone cleavage. This can result
in more complex tandem mass spectra and, potentially, the
loss of information on the attachment site of these substitu-
ents. In contrast, co- and post-translational modifications are
preserved when using ECD fragmentation technique.
Orbitrap Mass Spectrometer—The Orbitrap mass spec-
FIG. 13. Orbitrap mass analyzer. Ions are captured in a quadro-
trometer was first described in 2000 and has now reached the logarithmic electrostatic field (see the equation insert). An outer elec-
status of a mainstream mass spectrometry technique. The trode enclosing a central spindle electrode consists of two halves
combination of the Orbitrap mass spectrometer with an ex- separated by a dielectric material. The image current of ions moving
ternal accumulation device such as a linear ion trap enables as concentric rings along the central electrode (oscillations in axial
direction denoted as z in the drawing) is picked up by the outer
multiple levels of fragmentation (MSn) for the elucidation of
electrode sections.
analyte structure and allows coupling with continuous ioniza-
tion sources such as atmospheric pressure chemical ioniza- centrifugal force compensates the force created by Er Sec-
tion sources, electrospray (ESI) or nanoelectrospray. The an- ond, the axial field strength Ez is at zero in the equator plane
alytical performance (mass accuracy and resolution) of the of the Orbitrap analyzer but increases uniformly in opposing
Orbitrap combined with the ease of use and small footprint directions along the z axis as the two coaxial electrodes
(benchtop versions are available) can support a wide range of become progressively closer. This means that the axial elec-
applications from routine compound identification and se- tric field directs the ions toward the equator of the trap with
quencing, to the analysis of trace-level components in com- the force proportional to the distance from the equator. It
plex mixtures, be it in proteomics, drug metabolism, doping accelerates ions toward the equator and then the ions con-
control or detection of food and feed contaminants (48, 49, tinue to migrate through the equator (point of zero force) along
50, 51). the z axis, but decelerate as they continue toward the oppo-
Motion of Trapped Ions—The Orbitrap mass spectrometer site end of the Orbitrap expending the axial velocity previously
consists of a spindle-like central electrode elongated along an gained in traversing the electric field gradient from the starting
axis, kept at high voltage when the ions are being trapped, point to the equator. Having “spent” their axial velocity, the
surrounded by an outer barrel-like electrode kept at ground ions are accelerated back toward the equator of the trap by
potential. The outer electrode is split in two halves to form the symmetric electric field along the z axis. In this way, the
pick-up electrodes for image current detection (Fig. 13). The ions oscillate naturally along the z axis in a manner analogous
electrodes are shaped in such a way that the quadro-loga- to pulling back a pendulum bob and then releasing it to
rithmic potential distribution is formed with very high accu- oscillate. It is this property of the electric field that causes the
racy. When ions start their motion at the correct energy and mass-dependent harmonic oscillation of the ions along the z
radius, stable trajectories are formed that combine rotation axis. This oscillation is then combined with a more compli-
around the central electrode with oscillations along the axis cated rotational motion.
and have a shape of a complicated spiral. It is important to Rotational and radial frequencies are strongly dependent on
note that axial motion is completely independent of rotational initial energy, position or angle of ions, while the frequency of
motion. axial oscillations is completely independent of initial velocities
The electrodes of the Orbitrap mass spectrometer create an and coordinates of the ions. Therefore, only the frequency of
electric field that is inhomogeneous in two directions, radial axial oscillations can be used for determination of mass-to-
and axial. First, the radial field Er attracts ions toward the charge ratios m/z:
central electrode, this field being stronger near the central
electrode. To provide a circular trajectory, the tangential ve-
locity of ions needs to be adjusted to such a value that the
␻⫽ 冑 e
共m/z兲
䡠k (Eq. 9)

FIG. 14. Resolution achievable with

FTICR and Orbitrap analyzers. A tran-
sient duration of 1 s is considered for
FTICR with field strength 9.4 and 21 T,
and for two different designs of the
Orbitrap analyzer. The resolution shown
for FTICR is nonapodized whereas the
resolution shown for Orbitrap is apo-
dized. In both types of FTMS the reso-
lution drops with increasing the mass of
an analyte. Within experimental settings
used in chromatographic applications in
proteomics (1 s transient) the Orbitrap
system can outperform the FTICR with
even very strong magnets for analytes
with large m/z.
wherein constant “k” is proportional to the potential difference oscillations, i.e. ions should oscillate coherently. Radial and
between the central and the outer electrodes. rotational frequencies do not appear in the frequency spec-
Image Current Detection—Axial oscillation frequencies can trum precisely because of the fast loss of coherence caused
be directly detected by measuring the image current from the by dependence of these frequencies on the initial parameters
outer Orbitrap electrodes after it has been amplified. A broad- of the ions.
band detection is followed by a Fourier transform to convert Getting the Ions Inside—A very short packet of ions ejected
the recorded time-domain signal into a mass-to-charge spec- from an RF-only gas filled multipole (known as the C-trap in
trum (51, 52). the current instrument implementations because it is shaped
The image current is amplified and processed in a similar like a letter “C”) enters the Orbitrap analyzer off its plane of
way as for FTICR, resulting in comparable sensitivity and symmetry through a tiny hole in one of the outer electrodes.
signal-to-noise ratio. There is, however, an important distinc- As ions enter they experience an attractive field of the central
tion between the two FTMS systems; the square-root de- electrode, which increases very fast with time. This field
pendence originating from the electrostatic nature of the field bends their trajectories into circular arcs. The radii of the arcs
in the Orbitrap analyzer causes a much slower drop in resolv- become progressively smaller with every microsecond so that
ing power observed for ions of increased m/z. As a result, the ions cannot return to the entrance radius to hit the outer
Orbitrap analyzer may theoretically outperform FTICR in this electrode and thus be lost for detection.
respect for ions above a particular m/z (typically, above m/z The axial component of the electric field is proportional to the
1000 –2000) for the same length of time of acquisition. Fig. 14 distance from the plane of symmetry and so reaches substantial
contrasts the rate of the theoretical resolution drop in the two values at the point of entry. It starts to pull all ions toward the
types of FTMS as the mass of the ions under investigation plane of symmetry as soon as they enter the field. This initiates
increases. For that comparison a 1 s acquisition time was axial oscillations without any additional excitation.
used. Please note that the resolution quoted for the FTICR in Because of the pulsed extraction from the RF multipole,
this figure is nonapodized while the resolution of the Orbitrap which sends the ions into the Orbitrap, ions of each m/z (ion
is apodized (which means that the apodized resolution for the packet) enter the field practically simultaneously. The axial
FTICR would drop to about half of that displayed in this size of the ion packet remains almost unchanged and good
graph). coherence is provided automatically thus fulfilling this impor-
Sensitivity of image current detection is determined by the tant prerequisite for successful image current detection.
internal thermal noise of electronic components of the image After all ion packets across the mass range of interest have
current preamplifier. For the best present-day solutions it entered the field, ramping of the electric field stops, and the
corresponds experimentally to a limit of detection equal to potential of the central electrode is stabilized so that no fre-
5–10 elementary charges for a 1 s acquisition. quency drift can take place during the subsequent image
It is important to note that for ensembles of ions a nonzero current detection.
image current can be detected only if ions are moving in- Because of the strong dependence of rotational frequen-
phase with each other. This means that ions of each m/z cies on ion energies, angles, and initial positions, each ion
inside the Orbitrap analyzer should be concentrated in pack- packet quickly spreads over the angular coordinate and forms
ets that have an axial length shorter than the amplitude of a thin rotating ring. The whole ring then oscillates along the

central electrode harmonically with frequency dependent only Because of a weak dependence of sensitivity on detection
on the m/z of the ions. time, Orbitrap analyzers have an important advantage for
The very short time ions stay concentrated before forming chromatography: the dynamic range goes down much slower
the rotating ring means that space charge effects do not have with an increase of repetition rate in comparison to other
time to develop. Moreover, the existence of the central accurate-mass analyzers (e.g. TOF) (54). In this context it is
electrode in the middle of the ring effectively shields one part worth mentioning that the Orbitrap analyzer shows an insig-
of the ring from the other. A high space charge capacity can nificant trade-off in sensitivity versus resolving power.
be attained (millions of ions) before unwanted effects on the Linking the FTMS to Other Mass Spectrometers - Hybrid
mass spectrum (i.e. coalescence effects, loss of resolution, Systems—Although FTICR is an MS/MS instrument in its own
and mass accuracy) are observed. In FTICR for comparison, right and can achieve high resolution precursor ion selec-
all ions of the same mass remain in one line, and higher field tion—albeit with a time penalty for the isolation-fragmenta-
magnets need to be employed to mitigate the space charge tion-axialization-remeasurement process, and ion intensity
effects. penalty for high resolution precursor ion selection—there is a
Factors Limiting the Mass Resolving Power—For a com- significant advantage when a precursor ion selection device
mercial Orbitrap analyzer, nominal resolving power (apodized) such as a transmitting quadrupole and collision cell or a linear
of 100,000 FWHM at m/z 400 requires 1.07 s detection time. trap quadrupole with MSn capability is used. In the case of the
Under ideal conditions, the ions could remain in the analyzer linear trap quadrupole (LTQ) which carries its own detector,
indefinitely, and there would be no limit to the resolution MSn experiments at lower resolution can be carried out while
achievable. Unfortunately, collisions with residual gas, as well the high resolution measurement in the FTICR takes place.
as minuscule imperfections of the electrode manufacturing The situation is different for the Orbitrap analyzer. When an
limit the time a signal can be detected, and thus the maximum ion decays inside the Orbitrap analyzer its fragments will
resolving power of the analyzer. retain the same velocity as the parent ion had. As their energy
Collisions with residual gas cause a loss of coherence by remains proportional to their individual m/z, their trajectories
scattering the ions. First, the loss of ion momentum in colli- will become highly elliptical. Most of the fragments (except for
sions causes the coherent ion packet to “diffuse,” thus in- those that have m/z similar to that of the parent ion) will hit one
creasing aberrations and accelerating further “diffusion” due of the electrodes and thus be lost for detection. Although
to other factors like field imperfections. Second, collisions can there are technological answers to achieving MS/MS capabil-
lead to prompt or metastable ion fragmentation, which could ity within the Orbitrap field (51), the first commercial system
also lead to a direct loss of ions if the latter hit an electrode. introduced in 2005 was made by coupling the Orbitrap ana-
Both processes are random in time and, therefore, produce lyzer to a linear ion trap (LTQ) (Fig. 15). The resulting hybrid
noncoherent clouds of ions that cannot be detected by image MS system has an MSn capability and can use multiple frag-
current detection, even if ions are still stable within the trap. mentation methods (CID, higher energy collisional dissocia-
The time between collisions is inversely proportional to the tion (HCD), ETD). Another reason the linear ion trap was
residual pressure inside the trap and to the cross-section of chosen was because of its high sensitivity, accurate control of
an ion. For a pressure of 10⫺10 mbar (1 bar ⫽ 100 kPa), the the ion population, short cycle time, and high charge capacity
time interval between ion and gas collisions ranges from (55). A detailed description of the hybrid linear ion trap-Orbitrap
several seconds for small molecules to ⬍1 s for small- and instrument (LTQ Orbitrap) performance can be found in Ma-
medium-size proteins. One needs to remember that although karov et al. (56).
the time between collisions decreases for larger molecules Depending on the analysis requirements, the two mass
(i.e. higher collisional cross section) the percentage energy analyzers (the FTMS part and the linear ion trap part of a
loss per collision is smaller and multiple collisions need to hybrid instrument) can be used independently or in concert.
take place for the kinetic energy to be dissipated. By improv- The MSn spectra recorded by either of the analyzers are very
ing the Orbitrap vacuum below this level, isotopic resolution of similar, the only major difference being the resolution and
protein ions up to several tens kDa has been demonstrated mass accuracy of the observed peaks. A true parallel opera-
(53). tion is achieved by using the initial part of the transient still
In practice, resolving power achievable over a limited time being measured in the FTICR/Orbitrap analyzer to define the
frame characteristic for LC separations (typically 0.5 or 1 s) parent ion masses for the linear ion trap to fragment while the
rather than the best achievable resolution over an unlimited detection of the image current in the FTICR/Orbitrap analyzer
time frame becomes an important parameter. Resolving continues till the specified final resolution is reached. This
power is then limited only by the number of ion oscillations parallel mode of operation offers the ability for acquiring mul-
over this time frame, which is proportional to an axial fre- tiple MS/MS unit resolution spectra in the linear trap for every
quency of oscillations. As the axial frequency is inversely high resolution spectrum acquired in the FTICR/Orbitrap,
proportional to square root of m/z, resolving power over a which makes these systems so powerful for analyses of com-
limited time-frame also diminishes as (m/z)⫺1/2. plex mixtures.

FIG. 15. Schematic representation

of a hybrid ion trap-Orbitrap mass
spectrometer. The main parts of a com-
mercially available hybrid FTMS instru-
ment, the LTQ Orbitrap, are highlighted
on the diagram.
TABLE II
Performance Characteristics of FTMS
Resolution achievable by FTICR and Orbitrap mass spectrometers for
Mass Accuracy—One of the most coveted attributes of a various m/z values considering a 1 second acquisition time. 9.4 T field
strength and two different Orbitrap analyzer designs (56, 62) are
mass analyzer is undoubtedly its mass accuracy. FTMS can
considered in these calculations. Please note that the values for FTICR
reliably deliver mass accuracy below 1 ppm (54, 57– 60). This are non-apodized while the values for Orbitrap are apodized.
accomplishment can be aided by exploiting the use of certain
Standard Compact
ubiquitously present background ions. Precision in peptide m/z 9.4 T FTICR
Orbitrap (56) Orbitrap (62)
mass measurement typically within 100 parts per billion (ppb)
(nonapodized) (apodized) (apodized)
have been achieved for many peptides in the LC/MS run
100 1,500,000 200,000 400,000
without requiring an internal standard. Often, such a result 1000 120,000 63,000 120,000
limits the peptide to a single elemental composition and, 5000 24,000 28,000 56,000
therefore, represents the highest useful accuracy.
Acquiring tandem mass spectra with a high mass accuracy
is an interesting alternative to classical data acquisition species with m/z 1000 matches that of the nonapodized 9.4 T
schemes where fragment ions are detected at much lower FTICR, and for a species with m/z 5000 it even outperforms it.
mass accuracy and resolution in the linear ion trap or triple Another challenge related to mass spectrometric detection
quadrupole. A higher mass accuracy of the detected frag- of large molecules is that each molecular species appears in
ments adds to the specificity of identifications. This makes up multiple charge states. Fig. 16 shows a spectrum of a mono-
for a lower number of MS/MS spectra acquired at high reso- clonal antibody (approximate mass 147 kDa) acquired during
lution compared with the acquisition on a faster but mere unit LC/MS analysis with Orbitrap detection (63). Each peak in the
resolution instrument such as an ion trap. A much greater spectrum corresponds to a specific charge state with the one
degree of confidence is, however, a decisive advantage in the in the middle being charge state ⫹55, with increasing charge
case of peptides with unexpected modifications (61). states to the left and decreasing to the right thereof. Each
Resolving Power—Very high resolution, indeed, can be charge state consists then of multiple peaks representing
achieved when long transients are recorded. On a 9.4 T FTICR adducts, many of them being various cations from salts and
mass spectrometer, for example, a 5 s transient could yield buffers, and each of these has in turn a large isotopic distri-
resolution of 6,000,000 at m/z 100, 600,000 at m/z 1000, or bution (a sort of a Russian doll effect, as illustrated with
120,000 at m/z 5000. One has to note that multiply charged myoglobin spectrum example in Fig. 17).
intact proteins would mainly appear in the 1000 –5000 m/z All this effectively means that our initial sample signal has
window; this makes the technique well suited for the study of split over a very large number of peaks, with subsequent
large protein molecules spurring recent interest in drug dis- negative impact on apparent sensitivity of detection. This
covery (biologicals). The problem is that achieving transients feature is intrinsic to all mass spectrometers using electro-
longer than 5 s for large proteins with corresponding large spray ionization producing multiple charged ions. It might be
collision cross sections is by no means routine even at the pertinent to note that using a matrix assisted laser desorption
very low pressures required for FTICR. ionization (MALDI) would not simplify the matter; an FTMS
It might be useful to compare resolution achieved by an detection of a species with m/z 150,000 (singly charged ions
FTICR and Orbitrap for various m/z values. Considering a 1 s are produced by MALDI) would require an extremely long
acquisition time which is fairly typical in proteomics experi- transient to reach any usable resolution. Employing a time-of-
ments as it matches the chromatographic scale of peptide/ flight (TOF) detector with MALDI source would fare no better
protein separations, Table II contrasts the performance of 9.4 because ions would have to be accelerated to many tens of
T FTICR with that of a standard (56) and a newer compact (62) keV for such a massive analyte to produce a measurable
Orbitrap design. The performance of the Orbitrap analyzer for a signal on the detector (64).

FIG. 16. Spectrum of a monoclonal

antibody acquired during an LC/MS
analysis with Orbitrap detection. Mol-
ecules of immunoglobulin G (approx.
147 kDa) accept a range of different
charges during electrospray ionization
process, which are then represented as
different charge states in the spectrum.
The most intense species here carries 55
charges while increasing charge states
appear to the left and decreasing to the
right thereof. Courtesy of Zhongqi Zhang
and Pavel Bondarenko, Amgen.
FIG. 17. Intact protein analysis with

high resolution mass spectrometry.
Top panel shows the charge state enve-
lope of myoglobin. A section of the spec-
trum with a species carrying 15 charges
is presented on middle panel, and it is
further enlarged to show the individual
isotopomers on bottom panel.
Improving Performance Characteristics of FTMS—Develop- the gap between the inner and outer electrodes. This provides
ment is focused on obtaining higher resolving power (and higher field strength for a given voltage leading to increased
hence better mass accuracy) over a fixed acquisition time. frequency of oscillations. Resolving power in excess of
One way to achieve this is to increase the frequency of ion 600,000 at m/z 195 and isotopic resolution of medium-size
oscillations in the analyzer so that more oscillations are sam- (approx. 40 kDa) proteins has been reported (65). For FTICR,
pled for each frequency or mass in the given time transient. the impact of larger field strength (a bigger magnet), improved
For the Orbitrap analyzer, this can be achieved via decreasing homogeneity of electric fields (both RF and DC) in the ICR cell,

effect of the ICR cell design, and a requirement for a far more An improvement in resolution almost by a factor of two can
precise control of the ion population within the ICR cell than result from the implementation of phase correction for FTMS
for the Orbitrap analyzer (see end of Section Image Current data. Fourier deconvolution-based phase correction consists
Detection) have been discussed in the Sections Mass Calibra- of a complex division of the time domain ICR signal by the
tion, Problems with Potential Field, Effect of Magnetic Field on spectrum of the time domain excitation waveform to yield a
Resolution (High Field Magnets), and Effect of Transient Time phased broadband response. The critical requirement for im-
Duration on Resolution above. plementing this process is that the detection event must
incorporate the excitation interval, and the excitation and
detection spectra must be temporarily synchronized.
In practice, this simultaneous excitation and detection is
very difficult because of detector saturation. Work in Mar-
shall’s lab showed how phase correction in FTICR could be
achieved, and magnitude and absorption spectra of electro-
spray-ionized ubiquitin, [M⫹10H]10⫹ (at 9.4 T) derived from
the same time-domain data were shown (66). Their approach
used a variable capacitor added between each excitation and
detection electrode pair, and the resulting bridge was manu-
ally tuned so that the coupling of the two opposite-phase
components of the differential excitation largely canceled at
the preamplifier input. Such “nulling” is increasingly difficult
for larger high-field instruments because of greater coupling
FIG. 18. Phase correction in the Orbitrap analyzer. Diagram capacitance of a large cell assembly and the required use of
showing three different ion signals (frequencies) that can be traced
a higher amplitude excitation waveform.
back to the point in time when they have an identical phase. This
“time ⫽ 0” corresponds to the time the ions leave the C-trap to enter Recently, another approach has been implemented (67).
the Orbitrap field. During the frequency sweep when the frequency which cor-
FIG. 19. Resolution improvement

using phase correction in the Orbitrap
analyzer. Charge state 47⫹ of intact
yeast enolase (46.64 kDa) was detected
in the standard Orbitrap analyzer (760
ms transients). A, The phase corrected
spectrum shows baseline isotopic sepa-
ration. B, The same experiment without
the phase correction achieves isotopic
separation at FWHM. The phase correc-
tion results in 1.6- to 1.7-fold improve-
ment in resolution. Both spectra have
been through different apodization.

responds to a particular m/z is reached, these ions become development. For example, portable high resolution instru-
excited. As a result the time between excitation and detection mentation or novel geometry hybrid systems are just a few
is different for each m/z. The phase of every m/z needs to be lines of thought to mention.
calculated:
Acknowledgments—We would like to thank scientists from Thermo
冋冉冊册
Fisher Scientific who provided data, spectra and graphics used in
共␻e ⫺ ␻i兲 many figures within this article and accompanying slide set, namely
␸i共␻i兲 ⫽ ␸0 ⫹ ␻i共ti ⫹ tdelay兲 ⫽ ␸0 ⫹ ␻i ⫹ tdelay
共sweeprate兲 Eugen Damoc, Robert Malek, Martin Zeller, and Marcus Kellmann.
Many thanks also to David Fenyo, Rockefeller University, USA, Eti-
where ␸i and ␻i are the phase angle and excitation frequency, enne Waelkens, University of Leuven, Belgium, and Pavel Bond-
respectively, for a particular m/z, ␸0 is the initial phase angle, arenko and Zhongqi Zhang from Amgen for allowing the use of their
␻e is the frequency at the end of the frequency sweep, and material in some of the figures.
tdelay is the time between the end of the frequency sweep and ¶ To whom correspondence should be addressed: Thermo Fisher
the detection. Scientific, Hanna-Kunath-Str. 11, 28199 Bremen, Germany. Tel: ⫹49
It should be noted that the accumulated cyclotron phase is 172 61 37 660; Fax: ⫹49 421 5493 396; E-mail: Michaela.
the sum of the phase accumulation during the frequency [email protected].
sweep plus a temporally increase between the instant of
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Fourier Transform Mass Spectrometry

Molecular & Cellular Proteomics 10.7 10.1074/mcp.M111.009431–1

FIG. 1. Fragmentation (MS/MS)

FIG. 2. Effect of mass accuracy on

10.1074/mcp.M111.009431–2 Molecular & Cellular Proteomics 10.7

deviation. This is summarized in the histograms in Fig. 2C

Molecular & Cellular Proteomics 10.7 10.1074/mcp.M111.009431–3

Fourier Transform Mass Spectrometry

10.1074/mcp.M111.009431–4 Molecular & Cellular Proteomics 10.7

FIG. 7. Ion motion within an ICR cell. A, Force affecting a charged

Fourier Transform Ion Cyclotron Resonance

Molecular & Cellular Proteomics 10.7 10.1074/mcp.M111.009431–5

and one revolution is equal to 2␲ radian

wherein “␯“ is the frequency of revolutions measured in Hz,

wherein “Vtrap” is the trapping potential in the axial direction,

10.1074/mcp.M111.009431–6 Molecular & Cellular Proteomics 10.7

masses or ranges of masses that need to be excited, and

wherein “A” and “B” are constants that can be obtained by

Molecular & Cellular Proteomics 10.7 10.1074/mcp.M111.009431–7

FIG. 10. Effect of magnetic field

10.1074/mcp.M111.009431–8 Molecular & Cellular Proteomics 10.7

FIG. 11. Effect of transient duration

Molecular & Cellular Proteomics 10.7 10.1074/mcp.M111.009431–9

10.1074/mcp.M111.009431–10 Molecular & Cellular Proteomics 10.7

peptide bonds in total, but the presence of three proline

Molecular & Cellular Proteomics 10.7 10.1074/mcp.M111.009431–11

FIG. 14. Resolution achievable with

10.1074/mcp.M111.009431–12 Molecular & Cellular Proteomics 10.7

Molecular & Cellular Proteomics 10.7 10.1074/mcp.M111.009431–13

FIG. 15. Schematic representation

10.1074/mcp.M111.009431–14 Molecular & Cellular Proteomics 10.7

FIG. 16. Spectrum of a monoclonal

FIG. 17. Intact protein analysis with

Molecular & Cellular Proteomics 10.7 10.1074/mcp.M111.009431–15

FIG. 19. Resolution improvement

10.1074/mcp.M111.009431–16 Molecular & Cellular Proteomics 10.7

Molecular & Cellular Proteomics 10.7 10.1074/mcp.M111.009431–17

Soc. Mass Spectrom. 6, 533–535 Spectrom. 1, 288 –294

10.1074/mcp.M111.009431–18 Molecular & Cellular Proteomics 10.7

Molecular & Cellular Proteomics 10.7 10.1074/mcp.M111.009431–19

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