Mass-spektrometriya

Furye o'zgarishining massa spektrometriyasi (FTMS)
FTMS is based on the principle of monitoring a charged particle's orbiting motion in a magnetic field (Figure 2.13-14). While the ions are orbiting, a pulsed radio frequency (RF) signal is used to excite them. This RF excitation allows the ions to produce a detectable image current by bringing them into coherent motion and enlarging the radius of the orbit. The image current generated by all of the ions can then be Fourier-transformed to obtain the component frequencies of the different ions, which correspond to their m/z. Because the frequencies can be obtained with high accuracy, their corresponding m/z can also be calculated with high accuracy. It is important to note that a signal is generated only by the coherent motion of ions under ultra-high vacuum conditions (10^-11 - 10^-9 Torr). This signal has to be measured for a minimum time (typically 500 ms to 1 second) to provide high resolution. As pressure increases, signal decays faster due to loss of coherent motion due to collisions (e.g. in ~ <150 ms) and does not allow for high resolution measurements (Figure 2.14).
Figure 2.13: A side view of an FTMS instrument with ESI source. The ESI ions are formed and guided into the analyzer cell using a single stage quadrupole rod assembly. The analyzer cell rests in the superconducting magnet (diagram courtesy IonSpec Corporation).
Ions undergoing coherent cyclotron motion between two electrodes are illustrated in Figure 2.13. As the positively charged ions move away from the top electrode and closer to the bottom electrode, the electric field of the ions induces electrons in the external circuit to flow and accumulate on the bottom electrode. On the other half of the cyclotron orbit, the electrons leave the bottom electrode and accumulate on the top electrode as the ions approach. The oscillating flow of electrons in the external circuit is called an image current. When a mixture of ions with different m/z values are all simultaneously accelerated, the image current signal at the output of the amplifier is a composite transient signal with frequency components representing each m/z value. In short, all of the ions trapped in the analyzer cell are excited into a higher cyclotron orbit, using a radio frequency pulse. The composite transient image current signal of the ions as they relax is acquired by a computer and a Fourier transform is used to separate out the individual cyclotron frequencies. The effect of pressure on the signal and resolution is demonstrated in Figure 2.14.
Figure 2.14: ESI FTMS data generated on multiple proteins, the sinusoidal composite image current for all m/z ions can be Fourier transformed to measure frequencies (and therefore m/z) accurately.
In addition to high resolution, FTMS also offers the ability to perform multiple collision experiments (MSⁿ). FTMS is capable of ejecting all but the ion of interest. The selected ion is then subjected to a collision gas (or another form of excitation such as laser light or electron capture) to induce fragmentation. Mass analysis can then be carried out on the fragments to generate a fragmentation spectrum. The high resolution of FTMS/MS also yields high-accuracy fragment masses.
Figure 2.15: Pressure effect on transient signal and resolution.
FTMS is a relative neophyte to biomolecular analysis, yet many of its advantages are generating more and more interest. It is now becoming more common to couple ultrahigh resolution (>10⁵) FTMS to a wide variety of ionization sources, including MALDI, ESI, APCI, and EI. The result of an FTMS analyzer's high resolving power is high accuracy (often at the part per million level) as illustrated for a protein in Figure 2.15 where individual isotopes can be observed. The Fourier transform of the ICR signal greatly enhances the utility of ICR by simultaneously measuring all the overlaying frequencies produced by the ions within the ICR cell. The individual frequencies can then be easily and accurately translated into the ion's m/z.
Figure 2.16: A demonstration of deconvolution from an FTMS mass spectrum of a 10 KDa protein at a resolution of 30,000. The cluster of peaks represents the isotope distribution of a protein and the 0.2 m/z isotope spacing indicates a 5+ charge state.
In general, increasing magnetic field (B) has a favorable effect on performance. The Fourier transform of the ICR signal, by measuring overlaying frequencies simultaneously, allows for high resolution and high mass accuracy without compromising sensitivity. This is in sharp contrast to double sector instruments that suffer from a loss in sensitivity at the highest resolution and accuracy. The high resolution capabilities of FTMS are directly related to the magnetic field of the FTMS superconducting magnet, with the resolution increasing as a linear function of the field. The ion capacity as well as MS/MS kinetic energy experiments increases as a square of the magnetic therefore improving dynamic range and fragmentation data. One challenge in increasing B is the magnetic mirror effect where ion transmission to the inside of magnetic field becomes more difficult due to magnetic field lines. Also, manufacturing high field magnets with larger bores and excellent field homogeneity (in the ICR housing) becomes technically more difficult.
FTMS instrumentation is affected by the magnetic field in the following ways: