Though its name is a mouthful, Fourier Transform-Ion Cyclotron Resonance (FT-ICR) mass spectrometry is one of the most powerful, and most expensive, tools available for biomarker analysis and bottom-up proteomics. Although commercially produced FT-ICR instruments were introduced only recently, the primary technology behind them has been around for many decades; the cyclotron, which accelerates charged particles in the presence of a high-frequency AC voltage and a magnetic field, was invented by Nobel laureate EO Lawrence in 1929. ICR mass spectrometry was first described in the late 1940s by Hipple, Sommer, and Thomas, but it was not until Alan Marshall and Melvin Comisarow invented the FT-ICR mass spectrometer in 1974 that the technique could be used to detect multiple ions simultaneously.1
Unlike methods such as time-of-flight that destroy the ions during analysis, FT-ICR is a nondestructive method that both allows users to run further experiments on the ions and permits longer data collection times, thus increasing sensitivity. “The longer we can detect, the better the certainty and precision,” notes Paul Speir, assistant vice president in charge of FT-MS, Bruker Daltonics.
In an FT-ICR instrument, ions are first generated at the source (ESI, APCI, APPI, or MALDI), and then injected into an ion trap mass analyzer cell in the center of a magnetic field (instruments are currently available with 7, 9.4, and 12 Tesla superconducting magnets). The ions move in a circular orbit in a plane perpendicular to the magnetic field, and are constrained to the region by a set of trapping plates perpendicular to the magnetic field axis, to which a voltage is applied. As the ions spin, they move about the center of the magnetic field with a rotational frequency inversely proportional to the ion’s mass-to charge-ratio; light ions spin faster than heavy ions of the same charge.
Ions are excited by sweeping a radio frequency (RF) pulse across a set of excitation plates parallel to the magnetic field axis; ions of a specific mass-to-charge ratio will absorb radio frequencies that resonate with their cyclotron frequency (the frequency at which they spin around the center of the magnetic field). This accelerates them to a larger radius. When the ions pass the detector plates, the ions generate an image current that can be detected and measured. The resulting spectrum is a superposition of every m/z signal, which can be deconvoluted by Fourier Transform to produce a frequency spectrum, and then calibrated to produce a mass spectrum.
According to Bernard Delanghe, product manager for FT-MS, Thermo Electron, the main advantage of the instruments is that they provide accurate mass and high resolution, and have a high dynamic range—qualities critical for resolving individual peptides from complex samples. “You have extremely accurate mass—up to 0.5 ppm—and resolution of more than 100,000 fwhm. So it means if you have … lots of peptides eluting at the same time [from an LC system], you can resolve them all spectrally, and sequence them at the same time in the Linear Trap,” Delanghe says. Researchers involved in bottom-up proteomics, in which a digest of a complex sample is analyzed to identify peptide components and their parent proteins, have been particularly attracted to FT-ICR instruments. Other applications include structural proteomics, small molecule identification and characterization, metabolomics, and biomarker and drug discovery.
Additionally, FT-ICR can be used to identify and locate post-translational modifications when combined with a fragmentation technique called Electron Capture Dissociation (ECD), in which multiply protonated ions interact with, and capture, low-energy electrons to produce a radical cation that readily fragments. Unlike other so-called ergodic dissociation methods that induce fragmentation by vibrationally exciting precursor ions, thereby cleaving weakly bound modifications, ECD leaves these modifications intact, allowing researchers to pinpoint their precise location on the peptide.2 First described in 1998 by Roman Zubarev, Neil Kelleher, and Fred McLafferty,3 ECD, which can be performed only on an FT-ICR instrument, can be combined with other fragmentation methods such as infrared multiphoton dissociation (IRMPD) and collision-induced dissociation (CID) to provide a more complete picture of the analyte.
The high performance and versatility of FT-ICR systems comes at a steep price—a “routine” system with a 7 T superconducting magnet costs in the vicinity of $800,000; the price skyrockets to $2 million for an instrument equipped with a 12 T magnet. But both Delanghe and Speir note that Thermo and Bruker each have sold hundreds of instruments in the past two years to academic proteomics facilities and large pharmaceutical companies. They add that demand continues to grow, in particular because both companies have developed hybrid instruments that take advantage of other complementary mass spectrometry technologies. Thermo’s LTQ-FT, for instance, is a hybrid linear ion trap/FT-ICR, and Bruker’s APEX-Qe is a triple quadrupole mass analyzer interfaced to an FT-MS. These technologies allow the user to dissect proteins of interest from complex mixtures prior to FT-MS analysis, or to enrich for low-abundance proteins by selectively accumulating specific ions, thus increasing instrument throughput.4 Further advancements include improvements in software ease-of-use, which are meant to make FT-ICR mass spectrometry even more routine and flexible, says Speir.
Intrigued by the magnetic appeal of FT-ICR instruments? Take a look at the product descriptions below for more information about these powerful mass spectrometers.
1. S. Borman, H. Russell, G. Siuzdak, “A Mass Spec Timeline,” Today’s Chemist at Work, Sept. 2003, pp. 47-49 (www.tcawonline.org).
2. R. Malek et al., “Electron Capture Dissociation on the Finnigan LTQ-FT: Preserving Post-translational Modifications during Peptide Fragmentation,” Thermo Electron Application Note #30081, www.thermo.com/eThermo/CMA/PDFs/Articles/articlesFile_27531.pdf.
3. R.A. Zubarev, N.L. Kelleher, F.W. McLafferty, “Electron capture dissociation of multiply charged protein cations. A nonergodic process,” J. Am. Chem. Soc., 120:3265-66, 1998.
4. A. Constans, “Putting a new spin on FT-MS,” The Scientist, 17[9]:46, 2003.