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Fourier Transform Mass Spectrometry FTMS

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1 Fourier Transform Mass Spectrometry FTMS
The International Proteomics Tutorial Program HUPOEuPA Michaela Scigelova, Martin Hornshaw, Anastassios Giannakopulos, Alexander Makarov (Thermo Fisher Scientific)

2 Overview Key performance characteristics of FTMS
Mass accuracy Resolution Fourier Transform in mass spectrometry – FTMS Fourier Transform ion cyclotron resonance – FTICR Discussion FTICR Factors impacting on the resolution/accuracy of the measurement Fragmentation techniques Orbitrap analyzer Discussion and comparisons – FTICR and Orbitrap analyzer

3 Key Performance Characteristics of FT Mass Spectrometers
Mass Accuracy Mass Resolution measures of the ability to distinguish two peaks of slightly different mass-to-charge ratios Δm, in a mass spectrum Defined as a full width of a peak at half of the maximum peak height (FWHM). The peaks of the measured compounds must be sufficiently well resolved in order to be able to determine their mass accurately A larger resolution indicates a better separation of peaks. In real-life samples (e.i. complex mixtures) the mass of a peak can be determined accurately only if measured at adequate resolution.

4 MASS ACCURACY Accurate mass measurement can be used to determine the elemental composition of an analyte* This acts as a powerful ‘filter’ enabling Confirmation of target compound identification Elimination of false positive identifications Identification of unknowns *Kind T, Fiehn O: Metabolomic database annotations via query of elemental compositions: Mass accuracy is insufficient even at less than 1 ppm. Bioinformatics 7, (2006)

5 Example: Accurate Mass As a Powerful Filter
H = N = O = S = C = Mass measured Tolerance [Da] Suggestions Calc Mass 32.0 +/- 0.2 O2 CH3OH N2H4 S 32.02 +/- 0.02 +/ But here is the interesting thing – only the mass of 12C is a nice round number (12). All other elements are either a tiny bit larger (H, N) or a tiny bit smaller (O, S). They show a so called ‘mass defect’. If we can measure with enough accuracy, then this mass defect can effectively exclude some of the compound suggestions as only limited number of combinations of elements would fit the measured mass within certain limits (maximum allowed mass deviation). Mass accuracy is thus a powerful filter. Assuming better than 1 ppm mass deviation, generally, a unique elemental composition can be obtained for compounds < 300 Da* But with an additional information from Isotopic Abundance Ratios, unique elemental composition can be obtained for compounds up to 2200 Da. *Kind T, Fiehn O: Metabolomic database annotations via query of elemental compositions: Mass accuracy is insufficient even at less than 1 ppm. Bioinformatics 7, (2006) Accurate mass makes life easier…

6 Quercetin fragmentation spectrum interpretation
Example: Structural elucidation aided by accurate mass measurement of fragments in MS/MS (or MSn) spectra Quercetin fragmentation spectrum interpretation This example shows the MS/MS spectrum of a flavonoid Quercetin (structure circled in red), acquired using the LTQ Orbitrap instrument. Quercetin parent ion mass is recorded in the spectrum (i.e., there is some parent ion still left after the fragmentation step; m/z 303) together with the masses of many resulting fragments. All of the recorded fragments are measured with mass deviation of less than 3 ppm (no internal calibration/lock mass used). Such rich spectrum plus accurate mass of the measured fragments provide useful information that confirms not only the elemental composition of the starting compound, but also its structure. The assignment of the peaks can be a non-trivial matter requiring the knowledge of fragmentation mechanisms. Some advanced software packages can take these into consideration together with the accurate mass information and produce a well annotated spectrum as seen here. Software used for spectrum annotation: Mass FrontierTM from HighChem

7 Example: Peptide Identification – Effect of Mass Accuracy
Peptides of human database: This slide considers a proteomics example. Let’s take a human protein database and all tryptic peptides therein. The graph A shows how many peptides would match a search criteria based on the accurate mass of the peptide alone considering mass deviation of 50, 20, 10, 5, 1, 0.1 and 0.01 ppm, respectively. Graph B is a zoom into the lower section of the graph A. These two graphs show clearly, that with the improved mass accuracy (lower mass deviation) the number of possible peptide candidates drops significantly. This translates to a much higher confidence for peptide identifications (as the accurate mass as a ‘filter’ reduces significantly the number of potential false positive identifications). The other observation is that even with a really low mass deviation (at present not attainable on any routinely used mass spectrometer, e.i., 0.1 or ppm) one will still encounter many cases in the human database where more than 1 peptide will fit in the bin of a selected mass +/- a respective deviation. This is summarized on the histograms C. Here, the x-axis shows the number of peptide matches in a bin while the y-axis denotes the frequency of occurrence. Thus, for the tolerance +/- 5 ppm one can observe that a majority of peptide mass bins will have more than one peptide in it, while for the tolerance +/- 0.1 a large proportion of peptide mass bins (but by far not all) has only one peptide in it. Note, this calculation does not include peptide modifications which will add significant amount of candidates. In conclusion, some other information is needed to support the peptide identification than just its accurately measured mass – for instance, a part of its sequence from an MS/MS spectrum… Courtesy of David Fenyo, Rockefeller University

8 RESOLUTION High resolution ensures that ions of only one exact mass contribute to a particular peak. Implications for: Accurate mass measurement of compounds in mixtures Hence providing a certain confidence interval for elemental composition/identification of measured compounds Reliable and accurate quantitation In real world samples, there is no ‘accurate mass’ without an adequate resolution

9 Number of elemental composition suggestions*
Example: Effect of mass resolution on the confidence of mass accuracy determination Resolution used translates to a ‘confidence interval’ (tolerance) for accurate mass measurement Knowing such a tolerance (+/- mmu) is important when used for generating elemental composition suggestions Example of Pirimicarb m/z 239 Resolution Mass tolerance (mmu) Number of elemental composition suggestions* 15,000 +/- 9 14 80,000 +/- 1.7 1 Pesticide Pirimicarb was measured in a mixture of other 115 pesticides and food toxins in a horse feed matrix. In this example, one can consider that 14 elemental formulas can be calculated for the measurement at 15,000 resolution (mass tolerance +/- 9 mmu) assuming CHNO elements while only 1 formula is applicable for resolution 80,000 (mass tolerance +/- 1.7 mmu). Conclusion: High resolution enables more confident elemental composition suggestion. *Assuming CHNO elements

10 Example: Effect of mass resolution on accurate mass measurement
Accurate mass determination of Pirimicarb enabled when sufficiently high mass resolving power separates its peak from that of a co-eluting interference of nearly same mass. This is an example of a complex mixture where many matrix components and other analytes could co-elute with our pesticide of interest and some of them could have a very similar mass. Pesticide Pirimicarb (C11H18N4O2; [M+H]+ = ) was measured in a mixture of other 115 pesticides and food toxins in a horse feed matrix. First spectrum was acquired by LC/MS with resolution settings 15,000. The coeluting interferences might remain unresolved at resolution settings 15,000. This results to the measured mass of the peak being skewed by the presence of the impurity towards a slightly higher value (here, mass deviation 6.5 ppm). By resolving the interference using higher resolution settings (here 80,000) a much improved accuracy of mass measurement is achieved, here sub ppm mass deviation. Conclusion: increasing resolution resulted in improved accuracy of mass measurement. Courtesy of Markus Kellmann, Thermo Fisher Scientific

11 Example: Effect of mass resolution on compound detection
The presence of an interfering compound causes a mass deviation for the compound of interest (Pirimicarb; mass deviation 6.5 ppm) Performing a screening experiment and setting the mass tolerance to +/-5 ppm, Pirimicarb would escape detection altogether resulting in a false negative result The concept of high resolution/accurate mass has implications for screening and quantitation in real-life samples. If one would try to detect Pirimicarb from the dataset acquired at 15,000 resolution using mass of Pirimicarb plus/minus mass deviation less than 6.5 ppm as a filter, there would be no detected peak. One would obtain a false negative result. Courtesy of Markus Kellmann, Thermo Fisher Scientific

12 Example: Effect of mass resolution on compound quantitation
Example: a background component (‘matrix’) can not be separated from the analyte at resolution 15,000 and contributes to peak area determination (black trace). Quantitation of the analyte is not impaired at resolution 80,000 (orange trace). R = 80,000 The concept of high resolution/accurate mass has huge implications for screening and quantitation in real-life samples. Following the same example of Pirimicarb, when one would try to detect/quantify Pirimicarb from this data set considering mass deviation of 6.5 ppm or higher, the peak area acquired with resolution 15,000 is largely inflated. ‘Hidden’ impurities contribute to the peak area of Pirimicarb, resulting in over-quantitation. Courtesy of Markus Kellmann, Thermo Fisher Scientific

13 Fourier Transform in Mass Spectrometry FTMS

14 Fourier Transform In FTMS masses are represented by frequencies
Frequencies can be measured very accurately  FTMS offers high resolution/accuracy The signal complexity in FTMS can be considerable as illustrated by this example: In Fourier transform mass spectrometers masses are represented by frequencies and since frequencies can be measured very accurately Fourier transform mass spectrometers (FTMS) can offer potentially high measurement accuracy. To illustrate the signal complexity that one encounters in FTMS, let’s consider four frequencies representing four masses. Waves with frequency “v” and intensity 1, frequency “2v” and intensity 0.5, frequency “5v” and intensity 1.5 and frequency “8v” and intensity 0.2 (figure a) have waveforms that are represented individually superimposed in figure b. An FTMS detector will see all these four waveforms combined into the signal looking something like that in figure c. The real picture is far more complicated, as it is not unusual in a proteomic experiment to have hundreds or even thousands of molecular species present in a single spectrum. In addition, each molecular species has also its isotopomers. One can easily see that many thousands of frequencies at intensities which correspond to the amount of the molecular species in the sample can be combined to make a “time domain” spectrum.

15 Fourier Transform FT decomposes a function into a spectrum of its ‘frequency components’ Time domain Frequency domain / mass In FTMS the ions are observed (image current is induced on detection plates, 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 intensities corresponding to the amount of the molecular species in the sample is treated with the mathematical tool of the Fourier transform. The diagram shows the transformation of time domain signal to frequency domain which reveals the masses of the detected ions. It is because each mass has a characteristic frequency of oscillation. The Fourier transform decomposes a function into a spectrum of its frequency components. As a musical analogy would be to decompose chords into the note components.

16 Fourier Transform By using again an analogy, it is like trying to decompose the view of the five boats onto components projected to both shores (view 1 and view 2). The projection of each boat changes with time as the boats rotate due to the current/wind. Everything would have been much easier if all boats were always facing the same direction (same phase), or at least the initial direction for each boat was known and the rate of their rotation was known. The magnitude mode is the equivalent of the widest image the boats have ever presented on one of the axis as they rotate due to the current and the wind. A positive identification of each boat can be achieved. But there is a potential problem with this ‘wide view’ magnitude mode: as the number of boats on the lake increases some boats will not be visible any more because they will be obscured by others.

17 FT: Effect of a phase shift of the time domain signal on the spectrum
FT decomposes the frequency spectrum into a complex (in the mathematical sense, i.e., containing a real and an imaginary part) spectrum. At zero phase shift the spectrum is the absorption mode in real part (see case (a) on the figure) – the only instance of practical utility for us At all other instances (non-zero phase shift) the data can not be used for deriving a spectrum In a) the signal starts out along x and so the spectrum is an absorption mode in the real part and a dispersion mode in the imaginary part. In b) there is a phase shift of 45 degrees. The real and imaginary parts of the spectrum are now mixtures of absorption and dispersion. In c) the phase shift is 90 degrees; now the absorption mode appears in the imaginary part of the spectrum. Finally, in d) the phase shift is 180 degrees giving a negative absorption line in the real part of the spectrum. The vector diagrams illustrate the position of the signal at time zero. Ref.: James Keeler, "Understanding NMR Spectroscopy", 2nd Edition, Wiley 2009.

18 FT: ‘Magnitude’ spectrum
This operation eliminates the phase dependency at non-zero phase shift Drawback: the resolution of ‘magnitude’ spectrum is about 2x lower The Fourier transform operation decomposes the frequency spectrum into a complex (in the mathematical sense, i.e., containing a real and an imaginary part) spectrum. When the phase is zero the real part of the frequency domain spectrum shows what we call an absorption mode line, and in the case of the exponentially decaying signal it is known as absorption mode 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 negative parts. Figure illustrates the real and imaginary parts of the solution with their position and height being phase dependent. To eliminate the phase dependency, a square root of the sum of the square of the real and imaginary parts is used, known as the “magnitude spectrum”. The drawback is that the magnitude spectrum has only about half the resolution obtainable from that particular data.

19 Apodization Figure courtesy of Robert Malek, Thermo Fisher Scientific
Apodization and zero filling are two mathematical approaches for improving the appearance of FTMS mass spectra. Although they will rarely interfere with the proteomics analysis because they are taken care of by the instrument automatically in most cases, it is good to have an idea what they are. Apodization literally means “removing the foot”. It is used to remove artifact peaks due to the FT from the base of spectrum adjacent to the real peaks. It is a mathematical function which is zero-valued outside of a chosen interval (a width of the peak in our case). In order to transform the time domain signal (detected by the FTICR or Orbitrap and digitized) accurately into the constituent frequencies, long periods of observation are required (long transients). In practice, transients can not be very long (maximum a few seconds) due to time restrictions (eg eluting chromatographic peaks) or, more importantly, collisions of the ions under observation with residual gas. Weighing functions, referred to as windows, are applied to the data to reduce the artifact smaller peaks introduced (spectral leakage) due to the finite observation intervals The most commonly used window in FTMS is the “raised cosine” from Hann apodisation which has the advantage of low aliasing but decreases slightly the resolution (approximately by a factor of 2). One can observe this effect on the black (apodized magnitude spectrum) and blue (unapodized magnitude spectrum) trace. Zero filling is a data processing technique where zero points are added at the end of the time domain signal before Fourier transformation. The effect of zero filling is to increase the number of points in the spectrum (which then works out smoother in appearance) and an apparent increase in resolution. Since the zeros contain no further frequency information, there is no higher resolution or mass accuracy achieved and the result is purely cosmetic. Figure courtesy of Robert Malek, Thermo Fisher Scientific

20 Ion Cyclotron Resonance
Fourier Transform Ion Cyclotron Resonance FTICR

21 FTICR - Theory A charged particle in a magnetic field
A charged particle in a magnetic field with velocity at 90 deg 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 “Lorentz force” induces a change in the direction of the velocity, but not its magnitude. A minute fraction of time later the force is still normal to the new direction of the velocity vector. Following the vector of velocity we see that it prescribes a circle and as a result the ion is trapped by the magnetic field on a circular trajectory. The frequency of this rotation is characteristic for each mass and magnetic field and is well defined. An instrument such as this one where ions are excited to a larger trajectory in the presence of the magnetic field is called “ion cyclotron resonance” (ICR) mass spectrometer. This technique is old and used predominately for ion molecule reactions [“Ion Cyclotron Resonance Spectrometry”, Darold Wobschall, Rev. Sci. Instrum. 36, 466 (1965), doi: / ]. Adding the ‘Fourier Transform’ to ‘ion cyclotron resonance’ offers the advantage of detecting all frequencies simultaneously rather than detecting one frequency at a time. The theory of cyclotron resonance was developed by Lawrence in the 1930s and he obtained for that reason the Nobel prize in Other designs followed over the following decades, producing instruments that were used principally to study ion-molecule reactions. In 1974, Comisarow and Marshall adapted Fourier transform methods to ICR spectrometry and built the first FTICR instrument. (“Fourier transform ion cyclotron resonance spectroscopy”. Melvin B. Comisarow and Alan G. Marshall, Chemical Physics Letters, Volume 25, Issue 2, 15 March 1974, Pages , doi: / (74) ) A charged particle in a magnetic field with velocity vector at 90 deg to the magnetic field experiences a force normal to the plane defined by the velocity and the magnetic field. When the vector of velocity is at any other angle then the component at 90 deg to the magnetic field (radial) will play a role in trapping the particle, while the component parallel to the field (axial) will offer no confinement, thus resulting to a helical path.

22 FTICR – Ion Trapping For the FTICR mass spectrometer to operate, a few more components are required apart from the magnetic field and the ions. In the radial direction the ions are confined by the magnetic field so not much more needs to be done. In the axial direction, though, the magnetic field offers no confinement. In order to prevent ions from escaping a static electric potential, typically a few volts, is applied at the “trapping” electrodes (Blue). Yet two other essential components for the operation of the FTICR instrument are: a) means to detect the image current produced by the circling ions. The closer the ions circulate to the detection plates (red) the stronger image current signal they induce. b) means for exciting the ions onto a larger radius so that they can be detected by the detection plates. That is achieved by using excitation plates (green) where an excitation waveform is applied. Along the magnetic field lines (axial direction) ions are trapped by an applied electric potential, while on the plane perpendicular to the magnetic field lines (radial direction) ions are trapped by the magnetic field.

23 FTICR – Motion of Trapped Ions
B v in radians/sec qv x B in Hz ωc ωC : “unperturbed by trapping fields” angular cyclotron frequency ω+ : “reduced” cyclotron frequency ω- : “magnetron” frequency ωz : trapping oscillatory frequency q = z (i.e., charge) B The movement of the ions trapped in the ICR cell can be decomposed into several motions. In addition to the ‘cyclotron’ frequency (which is the one we are interested in as it can provide us with the information about the ion’s m/z) there is a precession motion within the trap characterised by so called ‘magnetron’ frequency. In addition, the ion packet also moves in axial direction from one trapping electrode to another with so called ‘trapping’ oscillatory frequency. We try to minimize the ‘not-so-useful’ magnetron motion. By cooling and careful excitation of the ion packet inside the ICR cell most of the magnetron motion can be converted into the cyclotron motion. The latitude of the axial trapping motion can be minimized by applying higher voltage onto the trapping electrodes (restricting the ions’ motion in axial direction), however, strong trapping potentials interfere with the cyclotron motion. ωz ω- ω+

24 FTICR - Theory B ωz ω- ω+ in SI units
a: in m, m: in u z: in multiple charge elements in SI units From these three formulas one can estimate the deviation from the unperturbed cyclotron frequency for different trapping potentials. If one wanted to evaluate the change of the cyclotron frequency, one could just evaluate the following formula for (omega)z and substitute it to the reduced cyclotron ((omega)+) and magnetron formulas ((omega)-) : characteristic of the trap geometry a: is the trapping electrode (end-cap) separation for cell geometries: cube = cylinder= open = The stronger the trapping potential, the greater the deviation from the unperturbed ICR frequency

25 FTICR - Excitation t w t w t w frequency sweep (chirp)
Stored Waveform Inverse Fourier Transform (SWIFT) t w Stored Waveform Inverse Fourier Transform (SWIFT), excitation and ejection of part of the spectrum The diameter of the ion cyclotron motion as the ions are introduced in to the ICR cell is relatively small. This is not convenient for ion detection. Therefore, an excitation voltage is applied to increase the kinetic energy of the ions and hence the diameter of the cyclotron motion. The cyclotron frequency is not affected by the excitation process because it is independent of ion’s kinetic energy, just the radius of the cyclotron motion increases. m/z calculations based on the cyclotron frequency thus remain unaffected. One could excite one mass (frequency) at a time, but this would defeat the purpose of using FT for detection. Alternatively, one could generate a waveform which excites all ions simultaneously, and if possible, provides the same amount of excitation for each mass (frequency). Frequency sweep and stored waveform inverse Fourier transform waveforms try to excite equally the ions within a mass range. Frequency sweep achieves that by scanning a range of frequencies and stored waveform inverse Fourier transform waveforms by performing a reverse Fourier transform to the theoretical mass range that needs to be excited, then recording this transformation, and finally, “playing” this time domain signal to the ions in the ICR cell. Stored waveform inverse Fourier transform (SWIFT) can be also used in order to excite a bit more a particular part of the mass spectrum and eject these ions from the ICR cell. Further reading: “FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY: A PRIMER” A.G. Marshall, C.L. Hendrickson, and G.S. Jackson, Mass Spectrometry Reviews, 1998, 17, 1–35 “Fourier transform ion cyclotron resonance mass spectrometry: the teenage years”, A.G. Marshall and P.B. Grosshans, Anal. Chem. 1991, 63(4) 215A time domain frequency domain “FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY: A PRIMER” A.G. Marshall, C.L. Hendrickson, and G.S. Jackson, Mass Spectrometry Reviews, 1998, 17, 1–35

26 FTICR Spectrum Example: peptide MRFA, cluster of isotopes around m/z 526. Bottom pane: isotope simulation at a resolving power of 1,000,000. FTICR spectrum of the peptide MRFA (SIM of the isotopes around m/z 526) using a 7T magnet at a transient length of 12.3 s (16 Msamples) in comparison with an isotope simulation at a resolving power of 1,000,000. Note that the contributions of isotopes of 34S, 2x 13C, 18O, 13C+15N can be clearly distinguished in this spectrum. For a comparison, Da is the mass of an electron The use of FT-ICR MS for complex mixture analysis has also been demonstrated in experiments with petrochemicals. Electrospray ionisation FT-ICR MS of South American crude oil gave a spectrum of more than 11,100 resolved peaks, of which more than 75% could be assigned to a unique elemental composition [Hughey C, Rodgers R, Marshall A.; Anal. Chem. 15; 74(16), (2002).]. The high resolution is required to resolve between –32SH4 and –12C3, as there are only 3.4 mDa difference between these two moieties. Mass of an electron ( Da) is marked in the spectrum to convey the right perspective. Mass of an electron Da 1e

27 FTICR - Non-Ideal Conditions
FTICR requires that ions are trapped within a finite volume by the electrodes. But these electrodes produce DC and RF electric fields in the trap. This has undesirable consequences: The relationship between ICR orbital frequency and m/z becomes non-linear making calibration difficult ICR signal strength no longer varies linearly with rf excitation magnitude and duration Coulomb forces between ions broaden (i.e., resolution suffers) and shift (i.e., mass accuracy suffers) the mass spectral peaks The spatially non-uniform excitation field may eject ions axially before they can be detected (loss of signal which means shorter detection times which means lower resolution) REFERENCES Discussed well in: “Fourier transform ion cyclotron resonance mass spectrometry: the teenage years”, Alan G. Marshall and Peter B. Grosshans, Anal. Chem., 1991, 63(4), pp 215A-229A 1 (“Exact mass measurement by Fourier transform mass spectrometry”, E. B. Ledford Jr., Sahba Ghaderi, R. L. White, R. B. Spencer, P. S. Kulkarni, C. L. Wilkins, M. L. Gross; Anal. Chem., 1980, 52 (3), pp 463–468, DOI: /ac50053a021 “Space charge effects in Fourier transform mass spectrometry. II. Mass calibration”, Edward B. Ledford Jr., Don L. Rempel, M. L. Gross, Anal. Chem., 1984, 56 (14), pp 2744–2748, DOI: /ac00278a027 “Parametric mode operation of a hyperbolic Penning trap for Fourier transform mass spectrometry”, D. L. Rempel, E. B. Ledford Jr., S. K. Huang, M. L. Gross, Anal. Chem., 1987, 59 (20), pp 2527–2532, DOI: /ac00147a018) 2 (“Mass-dependent z-excitation of ions in cubic traps used in FTMS”, S. K. Huang, D. L. Rempel and M. L. Gross, Int. J. of Mass Spectrom. and Ion Proc., 1986, 72 (1-2), pp , doi: / (86) “Relation of signal sensitivity and ion z-motion in cubic cells. Theory and implication for ion kinetic studies”, D. L. Rempel, S. K. Huang and M. L. Gross, Int. J. of Mass Spectrom. and Ion Proc, (2), 30 June 1986, Pages « Theory of ion cyclotron resonance mass spectrometry: resonant excitation and radial ejection in orthorhombic and cylindrical ion traps”, Peter B. Grosshansa and Alan G. Marshall, Int. J. of Mass Spectrom. and Ion Proc, 1990, 100, pp ) 3 (“Space charge effects in Fourier transform mass spectrometry. II. Mass calibration”, Edward B. Ledford Jr., Don L. Rempel, M. L. Gross, Anal. Chem., 1984, 56 (14), pp 2744–2748, DOI: /ac00278a027 “Theory of space-charge shift of ion cyclotron resonance frequencies “, J.B. Jeffries, a, S.E. Barlowa and G.H. Dunn, Int. J. Mass Spectrom. Ion Proc. 1983, 54 (1-2), pp , doi: / (83) ) 4 (“Excitation of the z-motion of ions in a cubic ICR cell”, W.J. Van Der Hart and W.J. Van De Guchte, Int. J. Mass Spectrom. Ion Proc. , 1988, 82 (1-2), pp 17-31 “z-axis oscillation sidebands in FT/ICR mass spectra ”, Stephen E. Delong, Dale W. Mitchell, Daniele J. Cherniak and T. Mark Harrison , Int. J. Mass Spectrom. Ion Proc., (3) pp , doi: / (89)    “Coupling of axial and radial motions in ICR cells during excitation”, P. Kofel, M. Allemann and Hp. Kellerhals, K. P. Wanczek , Int. J. Mass Spectrom. Ion Proc., 1986, 74(1), pp 1-12, doi: / (86)   “Ejection of low-mass charged particles in high magnetic field ICR spectrometers ”, M. Allemann, P. Kofel and Hp. Kellerhals, K. -P. Wanczek, Int. J. Mass Spectrom. Ion Proc., 1987, 75(1), pp 47-54, doi: / (87) “Mass-dependent z-excitation of ions in cubic traps used in FTMS”, S. K. Huang, D. L. Rempel and M. L. Gross, Int. J. Mass Spectrom. Ion Proc., 1986, 72 (1-2), 15-31, doi: / (86)   )

28 Factors impacting on the resolution/accuracy of the measurement
FTICR Discussion Factors impacting on the resolution/accuracy of the measurement

29 FTICR – Factors impacting on the resolution/accuracy of the measurement
Magnetic field strength Acquisition duration (transient) Strength and accuracy of the DC electric field used to confine the ions axially Strength and accuracy of the RF electric field used to excite the ions in a coherent ion cyclotron motion Homogeneity of the magnetic field Ion-ion coulomb interactions Although the increased strength of the magnetic field plays an important role in increasing resolution, an FTICR mass spectral peak can be broadened, shifted and distorted (which in turn affects mass accuracy and resolution) according to other instrumental parameters which are: strength and homogeneity of the magnetic field strength and accuracy of the DC electric field used to confine the ions axially strength and accuracy of the RF electric field used to excite the ions in a coherent cyclotron ion motion ion-ion coulomb interactions FTICR glossary Broadband excitation/detection: Excitation or detection covering a wide range of frequencies/masses Direct mode ICR detection Amplification and analogue-to-digital conversion of the signal induced on the detection electrodes Heterodyne mode ICR detection As in direct mode, but the signal is mixed with a frequency near to the mass that needs to be detected. As a result a lower frequency signal is produced which can be sampled at lower rate and create smaller files when long time domain signals are acquired. Only a very narrow mass range can be detected this way. Fellgett advantage: The advantage in speed for a given resolution, and S/N for a given time transient, when simultaneous detection takes place. For an N-point spectrum the Fellgett advantage is a factor N for speed and square root of N for S/N

30 FTICR – Example: Effect of Magnetic Field Strength on Resolution
To obtain higher resolution (within a given time duration) one can simply increase the magnet strength. The main difference between a 9.4T magnet for NMR and for FTICR is the volume where the magnetic field has to have very good qualities in terms of stability and homogeneity. While a couple of cm diameter of a magnet bore might be enough for NMR, 10 cm or more diameter might be required for FTICR. Thus, the cost of larger magnets for FTICR is significant. There are instruments with magnetic field strength of 20 T in operation. (“Fourier transform ion cyclotron resonance mass spectrometry in a 20 T resistive magnet.” Hendrickson CL; Drader JJ; Laude DA; Guan S; Marshall AG, Rapid Commun Mass Spectrom.  1996; 10(14):  ). Please remember that the apodized resolution will be almost half the resolution displayed in this figure. It is not easy to quote directly the apodized resolution because each manufacturer will use different apodization algorithms with a different effect on the resulting resolution Please remember that the apodized resolution will be about half the resolution displayed in this figure. It is not easy to quote directly the apodized resolution because each manufacturer will use different apodization algorithms with a different effect on the resulting resolution Note: logarithmic scale both on mass and resolution; resolution defined as FWHM

31 FTICR – Benefits of High Field Magnets
Higher mass resolving power ( m/Δm resolving power will increase linearly with increasing magnetic field) Higher mass accuracy as a consequence of increased resolving power Data acquisition speed (time needed to acquire a time domain signal of a given mass resolving power varies as 1/B) Higher maximum ion kinetic energy (useful for CID, as an example at 3T an ion of 1000 Da and argon collision gas has centre of mass kinetic energy (CMCE) of 1.67eV where at 9.4T has 16.4 eV) Upper mass limit increases quadraticaly with magnetic field (B) Ion trapping duration (The length of time required for the ion magnetron radius to expand to the radius of the trap increases quadraticaly with B) Number of trapped ions (increases quadraticaly with B) Quadrupolar axialisation efficiency (the rate of conversion of magnetron to cyclotron motion increases linearly with B) Peak coalescence (varies as 1/B2) Further reading: “ Advantages of High magnetic Field for FTICR MS”,Alan G. Marshall and Shenheng Guan, Rapid Commun. Mass Spectrom., 1996, 10, pp

32 FTICR – Benefits of High Field Magnets
Further reading: “ Advantages of High magnetic Field for FTICR MS”,Alan G. Marshall and Shenheng Guan, Rapid Commun. Mass Spectrom., 1996, 10, pp 14.5 T FTICR at the National High Magnetic Field Laboratory, Florida State University, USA

33 FTICR – Example: Effect of Acquisition Duration on Resolution
Another way to increase the resolution without going for a higher field strength is to allow the transient acquisition for a longer time. Of course, there are limits because: long acquisition times might not be always practical, particularly in the case of LC-MS coupling; 2) the transient can be recorded only for a limited time before it dies out due to collisions with residual gas. While all mass spectrometers require vacuum for the analysis and detection of ions, the performance of the FTMS instruments is more sensitive to pressure than other instruments. High vacuum is required to achieve high resolution. A vacuum of 10(power)-9 to 10(power)-10 Torr (1 Torr = 1.33 mbar = Pa) is required. Please remember that the apodized resolution will be almost half the resolution displayed in this figure. It is not easy to quote directly the apodized resolution because each manufacturer will use different apodization algorithms with a different effect on the resulting resolution Note: logarithmic scale both on mass and resolution; resolution defined as FWHM

34 FTICR – Example: Increasing performance by better controlling the excitation electric field
In a cell with central excitation electrodes only, all isopotential lines meet at the gap between excitation and trapping electrode. Therefore, ions are heavily exposed to axial components of the excitation field. standard open ICR cell excitation In an improved version the axial components inside the trapping region are reduced by applying the excitation waveforms also to the outer electrodes, positioned adjacent to the trapping rings, and by using a grid with the excitation field applied inside the ICR cell. FTICR – increase in performance by controlling electric fields better. Controlling the electric fields used for trapping and excitation has been an ongoing struggle since the early years of FTICR with greater or lesser success. References “Elimination of z-Ejection in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry by Radio Frequency Electric Field Shimming”, M. Wang, A. G. Marshall, Anal. Chem. (1990), 62, “Field-Corrected Ion Cell for Ion Cyclotron Resonance”, C. D. Hanson, M. E. Castro, E. L. Kerley and D. H. Russel, Anal. Chem., (1990), 62, “The Infinity Cell: a New Trapped-ion Cell With Radiofrequency Covered Trapping Electrodes for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry”, P. Caravatti, M. Allemann, Org. Mass Spectrom. (1991), 26, “Elimination of Axial Ejection during Excitation with a Capacitively Coupled Open Trapped-Ion Cell for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry”, S. C. Beu, D. A. Laude, Jr., Anal. Chem. (1992), 64, “A novel high-performance Fourier transform ion cyclotron resonance cell for improved biopolymer characterization“, J. E. Bruce, G. A. Anderson, C. Y. Lin, M. Gorshkov, A. L. Rockwood, R. D. Smith, J. Mass Spectrom. (2000)35, 85-94 improved excitation (Finnigan LTQ FT)

35 FTICR – Example: Effect of electric field homogeneity on mass measurement accuracy
External calibration mass accuracy is limited mainly by the variation of ion numbers in the cell Mass assignment error given by: wherein DB is the error of the electric field-dependent calibration parameter B The grid cell (see previous and next slides) reduces DB by a factor of 4 Same effect on Dm could be achieved by increasing the frequency by a factor of 2, i.e. by exchanging the 7 T magnet with a 14 T one. This is exemplified on the example below: measured mass deviations for a population of 1e6 ions (+/- 100 ions) at m/z 1000 The relation between observed ICR orbital frequency ω+ and z/m is not linear which leads to a mass calibration equation which has two adjustable parameters. The parameter A depends on magnetic field, while the parameter B depends on applied and induced electric field. In FTICR mass spectrometry the external calibration mass accuracy is mainly limited by the variation of ion numbers in the cell. This causes an error (Delta)B of the calibration electric field dependent parameter B in the calibration formula resulting in a mass assignment error (Delta)m which is given by the formula on the top. Thus to increase the mass accuracy one has to reduce (Delta)B or increase the cyclotron frequency f by means of a higher magnetic field. The improved ICR cell reduces (Delta)B and thus (Delta)m by a factor of ca. 4. The same effect on (Delta)m could be achieved by increasing the frequency by a factor of 2, i.e. by exchanging the 7 Tesla magnet with a 14 Tesla magnet. Normal cell 15 T : 0.39 ppm 7 T : 1.77 ppm Improved cell 7 T : ppm

36 Grid Cell Used in LTQ FT Instrument
Grid cell used to minimize the excitation field perturbations. Graphical rendering of the ICR cell of the LTQ FT Ultra instrument depicting the grids placed inside the cylindrical electrodes over the entire length of the ICR cell. The excitation waveforms are supplied to these grids so that the excitation field extends well past the trapping region. The trapping rings are segmented because the potentials applied to the segment behind the grids have to be 4.6 fold higher than those applied to the grid-free segments in order to establish the same trapping potential.

37 FTICR – Example: Effect of the electric field homogeneity on mass measurement accuracy
A homogenous electric field with reduced axial components of the excitation field allows use of higher excitation amplitude This results in a significantly higher ion signal Figures show the mass deviation at m/z 524 (peptide MRFA) measured for two different excitation amplitudes target 1e6, excitation amplitude 0.25 -8.0 -4.0 0.0 4.0 8.0 20 40 60 80 100 scan number deviation (ppm) target 1e6, excitation amplitude 0.50 -8.0 -4.0 0.0 4.0 8.0 20 40 60 80 100 scan number deviation (ppm) A homogenous electric field with reduced axial components of the excitation field allows use of higher excitation amplitudes resulting in a significantly higher ion signal. In other words, excited ions are moving on a trajectory with a larger radius, thus they are closer to the detection electrodes and as a result, signal is better. The beneficial effect on the signal-to-noise increases with decreasing m/z because the cyclotron energy and thus also the z-ejection varies inversely with m/z. The overall improvement of the detection sensitivity averages out at a factor of approximately 2.

38 FTICR – Detection, phase correction
mixed mode Re Im response FFT excitation FFT phased Fourier deconvolution-based phase correction consists of a complex division of the time domain ICR signal by the spectrum of the time domain excitation waveform to yield a phased broadband response. The critical requirement for implementing this process is that the detection event must incorporate the excitation interval, and the excitation and detection spectra must be temporarily synchronised. In practice, this simultaneous excitation and detection is very difficult due to detector saturation. “Broadband Phase Correction of FT-ICR Mass Spectra via Simultaneous Excitation and Detection” Steven C. Beu, Greg T. Blakney, John P. Quinn, Christopher L. Hendrickson, and Alan G. Marshall, Anal. Chem. 2004, 76, pp Work in Marshall’s lab, shows how phase correction can be achieved and magnitude and absorption spectra of electrospray-ionized ubiquitin, [M + 10H]10+ (at 9.4 T) derived from the same time-domain data are shown. Their approach uses a variable capacitor added between each excitation and detection electrode pair, and the resulting bridge was manually tuned such that the coupling of the two opposite-phase components of the differential excitation largely cancels at the preamplifier input. Nulling is increasingly difficult for larger high-field instruments because of greater coupling capacitance with a large cell assembly and the required use of a higher amplitude excitation waveform. “Broadband Phase Correction of FT-ICR Mass Spectra via Simultaneous Excitation and Detection” Steven C. Beu, Greg T. Blakney, John P. Quinn, Christopher L. Hendrickson, and Alan G. Marshall, Anal. Chem. 2004, 76, pp

39 Fragmentation techniques
FTICR Discussion Fragmentation techniques

40 FTICR Fragmentation Techniques
FTICR has been used with a wide variety of fragmentation techniques CID IRMPD ECD ECD method has some remarkable advantages: Fragmentation not directed by peptide bond protonation It ‘preserves’ post-translational modifications Wide choice of applicable fragmentation techniques plus the high resolution/mass accuracy of the detected fragments make FTICR very powerful for analysis of large peptides/proteins By ‘preserving’ post-translational modification we understand that a particular moiety (for instance a phosphate) remains bound to its original amino acid during the fragmentation step. One can thus have a ‘positive proof’ of the modification location within the peptide as the modified amino acid has a corresponding difference in mass. Some PTMs are more stable than others, but mostly, they will be lost during the collisional fragmentation used in ion traps or quadrupoles; it might be then really difficult to figure out where within a peptide the modification was originally present unless one does more substantial experiment (such as MS3 if one has an ion trap). ETD/ECD are thus fragmentation methods that offer unique advantages in this respect. R.A. Zubarev, D.M. Horn, E.K. Fridriksson, N.L. Kelleher, N.A. Kruger, M.A. Lewis, B.K. Carpenter, and F.W. McLafferty, “Characterization of Multiply Charged Protein Cations”, Anal. Chem. 2000, 72,

41 FTICR – CID and ECD fragmentation spectra
A single scan ECD MS/MS spectrum of the doubly charged precursor of substance P at m/z The spectrum exhibits intense ECD fragment ion peaks. Bearing in mind that the cyclic structure of proline does not allow formation of c- and z-type fragments, all possible N-Ca bonds are cleaved, allowing even de novo sequencing of peptides with unknown amino acid sequences. Electron capture dissociation (ECD) has recently evolved as an alternate activation method, especially for peptide and protein sequencing with Fourier-transform ion cyclotron resonance-mass spectrometry (FTICR-MS). With ECD, multiply charged cations are irradiated with low energy electrons produced by an emitter cathode behind the ICR cell. Electron capture produces a radical cation [M+nH](n-1)+• which can dissociate by a rapid, facile fragmentation of the N-Ca bond of the peptide chain, producing mainly c- and z•-type fragment ions.[1] In contrast to ECD, the most commonly used activation methods such as collision induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) induce dissociation by vibrational excitation of the precursor. Since these activation methods induce “ergodic” processes, i.e. they add internal energy to the precursor slower than the rate of energy randomization, usually cleavage of the weakest bonds within the precursor is observed. Within peptides, the backbone amide bond has the lowest energy barrier to dissociation and predominantly b- and y-type fragment ions are formed. However, substituents added in co- and post-translational modifications often have lower energy barriers than those of backbone cleavage. This can result in more complex tandem mass spectra and potentially the loss of the information on the attachment site of these substituents. Due to the non-ergodic nature of ECD, co- and post-translational modifications are preserved. ECD allows site specific analysis of phosphorylation,[2,3] O- and N-linked glycosylation,[4-7] and sulfation.[8] ECD holds much promise as a supplementary dissociation technique to CID for unambiguous protein identification, de novo sequencing[9] and detailed protein characterization.[10]. ECD shows higher degrees of fragmentation, allowing the distinction of leucine and isoleucine residues[11-13] and even between D- and L-amino acids.[14] ECD can also provide additional information of the amino acid composition by careful examination of side chain losses.[15-17] However, the overall efficiency of ECD is typically lower than that obtained with CAD. Long ion accumulation, activation, and detection times in the ICR cell together with the need for the addition of multiple spectra makes the use of ECD for on-line separation of complex peptide or protein mixtures quite challenging. References: [1] Zubarev, R.A., Kelleher, N.L., and McLafferty, F.W., Electron capture dissociation of multiply charged protein cations. A nonergodic process, J. Am. Chem. Soc. 1998, 120, [2] Stensballe, A., Jensen, O.N., Olsen, J.V., Haselmann, K.F., and Zubarev, R.A., Electron capture dissociation of singly and multiply phosphorylated peptides, Rapid Commun. Mass Spectrom. 2000, 14, [3] Shi, S.D., Hemling, M.E., Carr, S.A., Horn, D.M., Lindh, I., and McLafferty, F.W., Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry, Anal. Chem. 2001, 73, [4] Mirgorodskaya, E., Roepstorff, P., and Zubarev, R.A., Localization of O-glycosylation sites in peptides by electron capture dissociation in a Fourier transform mass spectrometer, Anal. Chem. 1999, 71, [5] Hakansson, K., Cooper, H.J., Emmett, M.R., Costello, C.E., Marshall, A.G., and Nilsson, C.L., Electron capture dissociation and infrared multiphoton dissociation MS/MS of an N-glycosylated tryptic peptide to yield complementary sequence information, Anal. Chem. 2001, 73, [6] Budnik, B.A., Haselmann, K.F., Elkin, Y.N., Gorbach, V.I., and Zubarev, R. A., Applications of electron-ion dissociation reactions for analysis of polycationic chitooligosaccharides in fourier transform mass spectrometry, Anal. Chem. 2003, 75, [7] Mormann, M., Macek, B., de Peredo, A.G., Hofsteenge, J., and Peter- Katalinic, J., Structural studies on protein O-fucosylation by electron capture dissociation, Int. J. Mass Spectrom. 2004, 234, [8] Kelleher, N.L., Zubarev, R.A., Bush, K., Furie, B., Furie, B.C., McLafferty, F.W., and Walsh, C.T., Localization of labile posttranslational modifications by electron capture dissociation: the case of gamma-carboxyglutamic acid, Anal. Chem. 1999, 71, [9] Budnik, B.A., Olsen, J.V., Egorov, T.A., Anisimova, V.E., Galkina, T.G., Musolyamov, A.K., Grishin, E.V., and Zubarev, R.A., De novo sequencing of antimicrobial peptides isolated from the venom glands of the wolf spider Lycosa singoriensis, J. Mass Spectrom. 2004, 39, [10] Kjeldsen, F., Haselmann, K.F., Budnik, B.A., Sorensen, E.S., and Zubarev, R.A., Complete characterization of posttranslational modification sites in the bovine milk protein PP3 by tandem mass spectrometry with electron capture dissociation as the last stage, Anal. Chem. 2003, 75, [11] Kjeldsen, F., Haselmann, K.F., Budnik, B.A., Jensen, F., and Zubarev, R. A., Dissociative capture of hot (3-13 eV) electrons by polypeptide polycations: an efficient process accompanied by secondary fragmentation, Chem. Phys. Lett. 2002, 356, [12] Kjeldsen, F., Haselmann, K.F., Sorensen, E.S., and Zubarev, R.A., Distinguishing of Ile/Leu amino acid residues in the PP3 protein by (hot) electron capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry, Anal. Chem. 2003, 75, [13] Kjeldsen, F., and Zubarev, R., Secondary losses via gamma-lactam formation in hot electron capture dissociation: A missing link to complete de novo sequencing of proteins?, J. Am. Chem. Soc. 2003, 125, [14] Adams, C.M., Kjeldsen, F., Zubarev, R.A., Budnik, B.A., and Haselmann, K.F., Electron capture dissociation distinguishes a single D-amino acid in a protein and probes the tertiary structure, J. Am. Soc. Mass Spectrom. 2004, 15, [15] Cooper, H.J., Hudgins, R.R., Hakansson, K., and Marshall, A.G., Characterization of amino acid side chain losses in electron capture dissociation., J. Am. Soc. Mass Spectrom. 2002, 13, [16] Haselmann, K.F., Budnik, B.A., Kjeldsen, F., Polfer, N.C., and Zubarev, R.A., Can the (Mo - X) region in electron capture dissociation provide reliable information on amino acid composition of polypeptides?, Eur. J. Mass Spectrom. 2002, 8, [17] Cooper, H.J., Håkansson, K., Marshall, A.G., Hudgins, R.R., Haselmann, K.F., Kjeldsen, F., Budnik, B.A., Polfer, N.C., and Zubarev, R.A., Letter: The diagnostic value of amino acid side-chain losses in electron capture dissociation of polypeptides. Comment on: “Can the (M·-X) region in electron capture dissociation provide reliable information on amino acid composition of polypeptides?”, Eur. J. Mass Spectrom. 8, (2002), Eur. J. Mass Spectrom. 2003, 9, The CID MS/MS spectrum of substance P. The doubly charged peptide precursor ions were subjected to CID in the linear ion trap and the fragment ions were transferred into the ICR cell and detected. The spectrum looks somewhat more complex compared to the ECD spectrum. The fragment ion peaks of this spectrum are sufficient to identify substance P in a database search, but de novo sequencing would be a challenge.

42 FTICR – ECD fragmentation spectra of phosphopeptides
Peptide PKKKKYAKEAWPGKKPTPSLLI Phosphorylation on serine S(19) Diagnostic c/z fragments highlighted in the spectrum In ECD fragmentation spectra, PTM remain bound to the respective amino acids which makes it easier to confirm their location within the peptide sequence. The following set of slides shows ECD fragmentation spectra of peptide phosphorylated at different locations Courtesy of Etienne Waelkens, University of Leuven, Belgium, and Martin Zeller, Thermo Fisher Scientific

43 FTICR – ECD fragmentation spectra of phosphopeptides
Peptide PKKKKYAKEAWPGKKPTPSLLI Phosphorylation on theronine T(17) Diagnostic c/z fragments highlighted in the spectrum In ECD fragmentation spectra, PTM remain bound to the respective amino acids which makes it easier to confirm their location within the peptide sequence The following set of slides shows ECD fragmentation spectra of peptide phosphorylated at different locations Courtesy of Etienne Waelkens, University of Leuven, Belgium, and Martin Zeller, Thermo Fisher Scientific

44 FTICR – ECD fragmentation spectra of phosphopeptides
Peptide PKKKKYAKEAWPGKKPTPSLLI Phosphorylation on tyrosine Y(6) Diagnostic c/z fragments highlighted in the spectrum In ECD fragmentation spectra, PTM remain bound to the respective amino acids which makes it easier to confirm their location within the peptide sequence The following set of slides shows ECD fragmentation spectra of peptide phosphorylated at different locations Courtesy of Etienne Waelkens, University of Leuven, Belgium, and Martin Zeller, Thermo Fisher Scientific

45 FTICR – Intact protein measurement
Analysis of intact proteins benefits from ultra-high resolution Ubiquitin (MW 8560), a detail of charge state 8+ 1071.0 1071.5 1072.0 1072.5 m/z 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 120000 Intensity R=545300 z=8 R=537804 R=528104 R=495804 R=553704 R=467004 R=480004 R=600804 R=351604 R=700304 Corresponds to resolution 1,000,000 at m/z 400 Spectra courtesy of E. Damoc, Thermo Fisher Scientific

46 FTICR – Fragmentation of Intact Protein
ECD fragmentation spectrum of Ubiquitin (12+) Spectra courtesy of E. Damoc, Thermo Fisher Scientific

47 FTICR – Fragmentation of Intact Protein
Sequence coverage within ECD fragmentation spectrum of Ubiquitin (12+) 71 out of 72) possible bonds were cleaved obtaining 147 fragment ions (bonds next to Pro not cleavable) Courtesy of M. Zeller, Thermo Fisher Scientific

48 Orbitrap FTMS Analyzer

49 Orbitrap Analyzer – Electrostatic Field
Orbitrap mass analyzer. Ions are captured in a quadro-logarithmic electrostatic field (see the equation insert). An outer electrode enclosing a central spindle electrode consists of two halves separated by a dielectric material. The image current of ions moving as concentric rings along the central electrode (oscillations in axial direction denoted as z in the drawing) is picked up by the outer electrode sections. The potential distribution of the field can be represented as a combination of quadrupole and logarithmic potentials. In the absence of any magnetic or rf fields, ion stability is achieved only due to ions orbiting around an axial electrode. Orbiting ions also perform harmonic oscillations along the electrode with frequency proportional to (m/z)(power)(-1/2). These oscillations are detected using image current detection and are transformed into mass spectra using FT, similarly to FTICR. Copyright: Thermo Fisher Scientific

50 Orbitrap Analyzer – Trapping Ions
The Orbitrap analyzer is an ion trap Moving ions are trapped around an electrode Electrostatic attraction is compensated by centrifugal force arising from the initial tangential velocity Potential barriers created by end-electrodes confine the ions axially One can control the frequencies of oscillations (especially the axial ones) by shaping the electrodes appropriately Thus we arrive at … This slide is animated so view in the ‘presentation mode’. Electrostatic Axially Harmonic Orbital Trapping: A High-Performance Technique of Mass Analysis, Alexander Makarov, Anal. Chem. 2000, 72, Orbital trapping was first implemented by Kingdon in 1923 [Kingdon, K. H. Phys. Rev. 1923, 21, ]. In its classical shape, the Kingdon trap contains a wire stretched along the axis of an outer cylinder with flanges enclosing the trapping volume. When a voltage is applied between the wire and the cylinder, the strong field attracts ions to the wire. Only ions that have enough tangential velocity miss the wire and survive. In some way, their orbits are similar to orbits of planets or asteroids in the Solar system, the wire playing the role of the Sun. Motion along the wire is restrained by the field curvature caused by the flanges of the outer cylinder. Further variations of the Kingdon trap are known, for example, with two parallel wires [McIlraith, A. H. Nature 1966, 212, ] or more elaborate electrode shapes (“ideal Kingdon trap” [Knight, R. D. Appl. Phys. Lett. 1981, 38, ]). Orbital trapping has been widely used in experiments on the spectroscopy of ions [Knight, R. D. Appl. Phys. Lett. 1981, 38, ; Lewis, R. R. J. Appl. Phys. 1982, 53, ; Yang, L.; Church, D. A. Nucl. Instrum. Methods Nucl. Res. 1991, B56/57, ; Sekioka, T.; Terasama, M.; Awaya, Y. Rad. Effects Defects Solids 1991, 117, ]. In these applications, ions have been formed within the trap or injected tangentially prior to switching on the field of the trap. Orbital traps Kingdon (1923)

51 Ion Injection and Formation of Ion Rings
A short ion packet of a particular m/z enters the field Increasing the voltage on the central electrode squeezes ions to a curved trajectory around the central electrode Voltage stabilizes and ion trajectories are also stabilized Angular spreading forms a ROTATING RING High charge capacity can be achieved due to the shielding effect of the central electrode (e.g., can not see the ions on the other side of the electrode) (r,φ) (r,z) This slide is animated so view in the ‘presentation mode’.

52 Orbitrap Analyzer – Detection
Image current detected on outer electrodes Frequency dependence on ions’ m/z Frequencies pertaining to ion populations of a particular m/z obtained using Fourier Transform This slide is animated so view in the ‘presentation mode’. Electrostatic Axially Harmonic Orbital Trapping: A High-Performance Technique of Mass Analysis, Alexander Makarov, Anal. Chem. 2000, 72,

53 Intact Protein Analysis – Depth of Information
Myoglobin infusion Orbitrap detection RP 100,000 . Intact protein analysis with high resolution mass spectrometry. Top panel shows the charge state envelope of myoglobin. A section of the spectrum with a species carrying 15 charges is presented on middle panel, and it is further enlarged to show the individual isotopomers on bottom panel.

54 Intact Protein Enolase ~46 kDa
LTQ Orbitrap XL RP 100,000 at 400 m/z Deconvolved Monoisotopic Mass 1.0 ppm

55 Fragmentation of intact protein - Enolase
LTQ Orbitrap XL RP 100,000 at 400 m/z 24 kDa fragments y221 0.63 ppm (mono) y222 0.95 ppm (mono)

56 High Masses: IgG (~147,000 Da) analyzed by LC/MS with the Orbitrap detection
Resolution used: 15,000 „Mass measurement and top-down HPLC/MS anakysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer“ P.Bondarenko et al., JASMS 2009, 20, P.Bondarenko et al., Mass measurement and top-down HPLC/MS anakysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer JASMS 2009, 20,

57 Orbitrap analyzer – Fragmentation techniques
As implemented within a hybrid linear ion trap–Orbitrap instrument, the Orbitrap device is used solely as a mass analyzer Fragmentation of peptides is carried out in an ion trap or a multipole, i.e., outside the Orbitrap analyzer CID used for a vast majority of experiments aiming at peptide identification/quantitation ETD (similar to ECD on FTICR) applied to PTM and large peptide/protein analysis* CID and/or ETD can be engaged based on the analyzed peptide characteristics. Decisions are taken automatically by the instrument on-the-fly** *McAlister, G.C., Phanstiel, D., Good, D.M., Berggren, W.T. and Coon, J.J. Implementation of electron-transfer dissociation on a hybrid linear ion trap/orbitrap mass spectrometer. Anal. Chem. 79, 3525–3534 (2007). Danielle L. Swaney, Graeme C. McAlister and Joshua J. Coon. Decision tree–driven tandem mass spectrometry for shotgun proteomics. Nature Methods 5, (2008).

58 Orbitrap ETD fragmentation: Top-down Analysis of Proteins
* Example: α-Defensin 5 with intact disulfide links ETD on 4+ precursor ions with Orbitrap detection c142+ / c142+ top-down and middle-down analysis of proteins benefits from ETD. Mass accuracy and resolving power are vital here in order to identify fragments. Example: high resolution enabled to identify 3 different fragments in a small section of m/z range of the MS/MS spectrum. z213+ / z213+ z61+ 58

59 Orbitrap Analyzer – Detection, Phase Correction
phase correction is much simpler in Orbitrap analyzer since there is no excitation step and the t=0 is the ejection from the c-trap All ions are ejected at moment t=0 from the C-trap along lines converging on the Orbitrap entrance. Ions enter Orbitrap analyzer as a short packet at the maximum Z The moment of entry is Injection at the maximum Z automatically initiates axial oscillations detected as image current at frequency CE OE-1 OE-2 C-trap Lenses Deflector Leff Z

60 Orbitrap Analyzer – Implementing Phase Correction
Resolving power improvement: Phase correction ON Intact yeast enolase (46.64 kDa), 47+ ion, 760 ms transients 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 separation. b) The same experiment without the phase correction achieves isotopic separation at FWHM. The phase correction results in fold improvement in resolution. Both spectra have been through different apodization Phase correction OFF Figures courtesy of E. Damoc, Thermo Fisher Scientific

61 FTICR and Orbitrap Analyzers
Discussion FTICR and Orbitrap Analyzers

62 Use of FTICR as part of a hybrid instrument

63 Use of Orbitrap analyzer as part of a hybrid instrument
1. Ions are stored in the Linear Trap 2. …. are axially ejected 3. …. and trapped in the C-trap 4. …. they are squeezed into a small cloud and injected into the Orbitrap analyzer 5. …. where they are electrostatically trapped, while rotating around the central electrode and performing axial oscillation The oscillating ions induce an image current into the two halves of the Orbitrap outer electrode, which can be detected using a differential amplifier This slide is animated so view in the ‘presentation mode’. Ions of only one mass generate a sine wave signal

64 Use of FTICR or Orbitrap analyzer as part of a hybrid instrument
Parallel acquisition delivers accurate mass on the precursor ion together with ion trap MS/MS spectra of selected precursor ions Full Scan MS MS/MS Ion 1 MS/MS Ion 2 MS/MS Ion 3 1 High resolution full scan detected in the FTMS 3 Unit resolution MS/MS scans detected in the LTQ ion trap (up to 10 MS/MS spectra detected in the LTQ Velos ion trap)

65 Use of FTICR or Orbitrap analyzer as part of a hybrid instrument
Up to 10 MS/MS spectra

66 Use of FTICR or Orbitrap Analyzer as part of a hybrid instrument
Combination of various fragmentation and detection modes Example: phosphopeptide analysis Full Scan MS MS/MS MS/MS MS3 MS3 High resolution full scan detected in the FTICR / Orbitrap High resolution MS/MS scans detected in the FTICR / Orbitrap Unit resolution MS3 detected in the ion trap  These two scans provide information on possible neutral loss of phosphate group from the precursor Provides information about the phosphate location within the peptide

67 Resolution vs Mass Dependence Comparison of FTICR and Orbitrap Analyzer
Please note that the resolution quoted for the FTICR is non-apodized while the resolution of the Orbitrap is apodized, which means that the apodized resolution for the FTICR would be almost half the value displayed in this graph. Note: logarithmic scale both on mass and resolution; resolution defined as FWHM

68 Relative Instrument Sizes
FTICR Cell Benchtop Orbitrap MS Superconducting Magnet

69 Relevant topics on videos
Mass spectrometry basics FTICR Fourier Transform Recommended reading: August issue of JASMS 2009 dedicated to Orbitrap analyzer and its applications


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