Resolving FTICR

An added consideration is that the TOF instruments are easily and quickly calibrated. As the mass range increases again (m/z 5,000-50,000), magnetic-sector instruments (with added electric sector) and ion cyclotron resonance instruments are very effective, but their prices tend to match the increases in resolving powers. At the top end of these ranges, masses of several million have been analyzed by using Fourier-transform ion cyclotron resonance (FTICR) instruments, but such measurements tend to be isolated rather than targets that can be achieved in everyday use. 

Figure 6.17 Temperature-resolved in-source pyrolysis FTICR-MS of flame-retarded polystyrene (56 spectra with a sampling interval of 1.1 s) from 300 K to 1200K. After Heeren and Boon [224], Reprinted from International Journal of Mass Spectrometry and Ion Processes, 157/158, R.M.A. Heeren and J.J. Boon, 391-403, Copyright (1996), with permission from Elsevier...

FTICR-MS is capable of powerful mixture analysis, due to its high mass range and ultrahigh mass resolving power. However, in many cases it is still desirable to couple a chromatographic interface to the mass spectrometer for sample purification, preconcentration, and mixture separation. In the example given above, DTMS under HRMS conditions provides the elementary composition. Apart from DTMS, PyGC-MS can be performed to preseparate the mixture of molecules and to obtain the MS spectrum of a purified unknown. Direct comparison with the pure reference compound remains the best approach to obtain final proof. 

Mass analyzers interrogate and resolve ions produced by an ion source based on their m/z ratios. Several types of mass analyzers are utilized for proteomic analysis including time-of-flight (TOF) quadrupoles, ion traps, and Fourier transform ion cyclotron resonance (FTICR). Mass analyzers may be assembled in hybrid configurations. MS instruments such as quadrupole TOF and quadra-pole ion trap-FTICR facilitate diversified applications and achieved great success. 

The m/z values of peptide ions are mathematically derived from the sine wave profile by the performance of a fast Fourier transform operation. Thus, the detection of ions by FTICR is distinct from results from other MS approaches because the peptide ions are detected by their oscillation near the detection plate rather than by collision with a detector. Consequently, masses are resolved only by cyclotron frequency and not in space (sector instruments) or time (TOF analyzers). The magnetic field strength measured in Tesla correlates with the performance properties of FTICR. The instruments are very powerful and provide exquisitely high mass accuracy, mass resolution, and sensitivity—desirable properties in the analysis of complex protein mixtures. FTICR instruments are especially compatible with ESI29 but may also be used with MALDI as an ionization source.30 FTICR requires sophisticated expertise. Nevertheless, this technique is increasingly employed successfully in proteomics studies. 

Performance Parameters. Typical resolving power for the commercial instrument is up to about 130,000 (FWHM) for m/z 400 Th. The mass resolving power is m/z dependent it decreases with fmfz (see the FTICR, described in Section 2.2.6, which decreases linearly with m/z). 

The acquisition speed is, as for the FTICR, resolution dependent. With Thermo Fischer Scientific s orbitrap the desired mass resolving power can be selected. With the lowest setting (7500 FWHM) the acquisition time for one ion injection is 0.3 s and with the highest setting (100,000 FWHM) it is 1.9 s. 

A mass calibration for FTICR analyzers with superconducting magnets is very stable and is valid for many days for normal applications. Mass accuracy < 1 ppm can be obtained over a fairly wide mass range. Unique elemental composition can be determined for masses over 800 Da [262]. Recently, 0.1 ppm mass accuracy, which required a mass resolving power >300,000, has been achieved for several thousand peaks by a 14.5 T instrument [263] and commercial instruments with mass accuracy <0.2 ppm are available. As with the orbitrap (see Section 2.2.5) the frequency is... 

The FTICR analyzer is relatively slow. In a low resolution mode (<25,000 FWHM) scans can be performed in substantially less than a second. A high resolution scan is more time demanding, and more than 1 s is often required for mass resolving powers of 100,000 or more. 

The modem sector instruments have resolving power about 60,000 to 80,000, and in some cases up to 150,000. There are reports of resolution about 70,000 in the case of ion traps. The newly developed (2005) Orbitrap instruments demonstrated resolving power up to 200,000. Super high resolution (dozens of millions) may be achieved using FTMS (FTICR). 

Instrnments combining several analyzers in sequential order are very common. This combination allows mass spectrometry and mass spectrometry experiments (MS/MS) to be carried out. Modern MS/MS includes many different experiments designed to generate substructural information or to qnantitate componnds at trace levels. A triple quadru-pole mass spectrometer allows one to obtain a daughter ion mass spec-trnm resnlting from the decomposition of a parent ion selected in the first qnadrnpole. The MS/MS experiments using an FTICR or ion trap, however, are carried ont in a time-resolved manner rather than by spatial resolntion. 

Orbitrap The newest of the major mass analyzers, the Orbitrap is a hybrid MS consisting of a LIT mass analyzer, or transmission quadmpoles connected to the high-resolution Orbitrap mass analyzer. The Orbitrap utilizes electrical fields between sections of a roughly egg-shaped outer electrode and an inner (spindle) electrode (Chapter 5). Ions orbit between the inner and outer electrodes and their oscillation is recorded on detector plates (Hardman and Makarov, 2003 Hu et al., 2005). As with the FTICR, fast Fourier transform of the raw data is used to convert the data for mass analysis, making the Orbitrap the second major type of FTMS instrument. The resolving power of the Orbitrap is intermediate... 

Hertkorn et al. (2006) analyzed DOM that was isolated by ultrafiltration from surface and deep waters. They used ll and 13C NMR spectra and FTICR mass spectra to resolve DOM into three normative components heteropolysaccharides, carboxyl-rich alicyclic molecules (CRAM), and peptides (accounting for all N).They reported that surface DOM contains 60.1% heteropolysacharides, 22.9% CRAM, and 23.6% peptides and that deep DOM contains 27.1% heteropolysaccharides, 50.9% CRAM, and 20.7% peptides. Unlike Sannigrahi et al. (2005), who gave empirical formulae of the endmembers of the three-component model that was used to analyze their 13C NMR data, Hertkorn et al. (2006) did not provide empirical formulae for heteropolysaccharides, CRAM, and peptides. It is unclear how to calculate average chemical formulae of surface and deep DOM from their results. 

Ion-molecular reactions are used to resolve isobaric interferences, as discussed, in ICP-MS with a collision/reaction cell or by utilizing ion traps. The mass spectra of Sr, Y and Zr (Fig. 6.10a) without O2 admitted into the collision cell and (Fig. 6.10b) with 10 Pa Oj are different. By introducing oxygen, selective formation of YO and ZrO, but not SrO, is observed. This behaviour of different oxide formation is relevant for an interference free determination of Sr. Ultrahigh mass resolving power ICP mass spectrometry (at m/Am 260 000) selectively removes unwanted ions prior to transfer to the FTICR analyzer cell by gas-phase chemical reactions, e.g., for separation of Ca from " Ar+ obtained with a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a 3 tesla superconducting magnet. ... 

With a high mass range of biopharmaceuticals, the need to detect small mass differences and small amounts of sample materials in the case of impurities, apart from high-resolution extreme sensitivity, is required. Sophisticated techniques such as combined ESI and Eourier transform ion cyclotron resonance (FTICR) MS show superb resolving properties with p.p.m. mass accuracy and mass resolution above 10. ... 

The higher resolution of FTICR-MS resolves many of these interference peaks so that they do not generate sufficient signal intensity for detection. 

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