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FAQ about Fourier Transform Ion Cyclotron Resonance (FTICR) Mass Spectrometry*

Contributed by: Richard Harkewicz, Ph.D.; Consultant, YCC/Keck MS Resource; Senior Project Scientist, Department of Chemistry and Biochemistry, University of California, San Diego

Frequently Asked Questions

Why are FTICR instruments so expensive?

The main cost (as for FT-NMR's) is the superconducting magnet, which accounts for as much as 1/2 of the total instrument cost.

What is the magnet needed for and why does it need to be so large?

FTICR performance parameters all improve in proportion to the strength of the magnet, "B" or "B2" (see: Marshall, A. G.; Guan, S. (1996) "Advantages of High Magnetic Field for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry", Rapid Commun. Mass. Spectrom. 10, 1819-1823). Basically, ICR frequency is proportional to B, so as B increases, all of the ICR frequencies also increase, as does the DIFFERENCE between any two ICR frequencies. That's why resolving power is proportional to B. The magnet also serves to confine ions in a plane perpendicular to B at the most homogeneous spot located at the center of the magnet. The magnet needs to be large because ICR dynamic range and upper mass limit also scale as the diameter of the magnet bore, and analysis/experiments which are very difficult or impossible at lower magnet size, can be routine at larger magnet size. Currently commercially available magnet sizes produced for FT-ICR MS instruments are 4T, 7T, 9.4T, 12T, 15T, 18T, and 21T.

What is Fourier Transform analysis and why is it needed?

Fourier transform (FT) analysis consists of determining the ion cyclotron resonances (which refers to the number of orbits completed/sec), not by scanning slowly through the spectrum as a function of frequency, but by recording the oscillating charge induced in a detector plate by cyclotron motion of ions as they approach and recede from that plate. FT of that time-domain data (i.e. decay of signal as in NMR) then yields ALL of the cyclotron frequencies at once, for a time saving by a factor of ~10,000.

How does ICR measure the mass of a peptide?

Electrospray produces protonated peptides that are present within and on the surface of microdroplets. As the droplets pass through a heated metal/glass capillary, the solvent evaporates, and the droplets become over-charged and fission into ever-smaller droplets until only single, usually multiply charged peptide ions (e.g., 2+, 3+, 4+ for tryptic peptides) are left. During an FTICR experiment ions are accumulated continuously in an octopole (or hexapole) ion trap which is external to the magnet. Then the ions are suddenly pulsed out of that trap (by simultaneously raising the dc voltage at the far end and dropping the front end potential), and then guided through a long octopole (or hexapole), into the ICR ion trap located at the center of the magnet. After pulsing the ions into a high-radius cyclotron orbit, we measure the time-domain signal and FT (as noted above) to generate a spectrum of ICR frequencies. We then convert from ICR frequency to ion mass-to-charge ratio. Finally, we determine ion charge as the reciprocal of the m/z spacing between isotopic peaks within a nominal mass unit (i.e., one more carbon-13 instead of carbon-12), to convert from m/z to mass (Da or amu).

How can you trap an ion for minutes/hours?

Ions are confined in a plane perpendicular to the magnetic field by their (circular) cyclotron motion. In other words, for "B" (magnetic field strength) along the z-axis, ions rotate in the x-y plane and thus cannot escape along the x- or y-direction. To prevent escape along the z-direction, we apply a small d.c. voltage (~1 Volt) to each of two "end cap" electrodes placed perpendicular to the z-axis, and located at z = +2 inches and z = -2 inches from the center of the trap. Those electrodes create an approximately parabolic potential well, so that ions oscillate linearly along the z-axis like a weight on a spring, and thus can't escape in the z-direction. There is a hole in the center of each end cap electrode to admit the ions in the first place, and to allow for introduction of photons or electrons to fragment the ions after they are trapped. In the absence of rf excitation, ions can be trapped for hours; after excitation, ions can be trapped for minutes.

Why do you need such a good vacuum?

ICR resolving power is simply the number of ion cyclotron orbits during the data acquisition period. For ions of 1,000 Da at 9.4 T, the cyclotron frequency is ~150 kHz. Ergo, ions need to be trapped (without colliding with unrelated neutral molecules like hydrogen or oxygen in the air) for ~4 s in order to achieve mass resolving power of ~500,000 (i.e., m/Δm = ν/Δν = 500,000 = 150,000/(1.2/T) see Marshall, A. G.; Comisarow, M. B.; Parisod, G. (1979) "Relaxation and Spectral Line Shape in Fourier Transform Ion Cyclotron Resonance Spectroscopy", J. Chem. Phys. 71, 4434-4444.) Ion-neutral collision frequency is about 1 per second at 10-8 torr (and is proportional to pressure). Hence, the pressure should be less than 10-8 torr to avoid collisions during the acquisition time (which otherwise would cause broadening of the ICR peaks).

Why is ICR inherently able to provide better resolution and mass accuracy than conventional mass spectrometers?

The main reason is that a superconducting magnet has a stability of a few parts per billion (ppb) per hour. Quadrupole and TOF instruments each require a voltage of ~1-10 keV, and it is (so far) not possible to stabilize such a voltage to better than ~1 part in 10,000. Ergo, FT-ICR has 10-100X higher resolving power.

What are reasonable FTICR technical capabilities to expect for peptides/proteins?

We routinely achieve ~5-15 ppm mass accuracy for peptides of m/z ~1000-2000 with external mass calibration, and 1 ppm or better with internal calibration. Generally speaking, mass resolving power (and mass accuracy) decrease as 1/(m/z) (i.e., mass accuracy is worse at higher m/z). There is a brief discussion of mass resolving power, mass resolution, and mass accuracy in: Marshall, A. G.; Hendrickson, C. L.; Shi. S. D.-H. (2002) "Scaling MS Plateaus with FTICR MS," Anal. Chem. 74, 252A-259A.

How do you get peptide sequence data from an ICR instrument?

If the idea is to search for a segment of a known protein, then the higher mass accuracy of FTICR narrows the search considerably (e.g., we found myoglobin uniquely (including the correct biological species) from the accurate mass of as few as one peptide). If, however, the idea is to determine the peptide sequence, then one needs MS/MS. Briefly, electron capture dissociation (ECD) will generate abundant fragments at just about every peptide linkage except proline, without detaching phosphate or sugar. Hence ECD is a good approach for finding "sequence tags" and for identifying the site(s) of posttranslational modification(s). Infrared multiphoton dissociation also cleaves the peptide backbone, but is especially efficient at cleaving sugar-sugar linkages. Ergo, it is the best way to determine the branching pattern in the glycan portion of a glycopeptide.


* We are indebted to Dr. Alan G. Marshall, Professor of Chemistry at Florida State University and Director of The National High Magnetic Field Laboratory ICR Program, for his earlier contributions to this website.