MALDI-qTOF

The ability to resolve and characterize complicated protein mixtures by the combination of 2DLC and online mass spectrometry permits the combination of sample fractionation/simplification, top-down protein mass information, and bottom-up peptide level studies. In our lab, the simplified fractions generated by 2D(IEX-RP)LC are digested and analyzed using common peptide-level analysis approaches, including peptide mass fingerprinting (Henzel et al., 1993 Mann et al., 1993), matrix-assisted laser desorption/ionization (MALDI) QTOF MS/MS (Millea et al., 2006), and various capillary LC/MS/MS methodologies (e.g., Ducret et al., 1998). 

MALDI-QTOF instraments basically follow an ion optical arrangement similar to that shown in Figure 2.16. The first quadrupole, Qo, is simply used to cool the ions kinetic energy and focus them onto the quadrupole axis so that they enter Qi with a well-defined initial trajectory, and is always used in RF-only mode. Qi is a mass-filtering quadrupole which can either be used in RF-only mode to pass a wide range of mass/charge values, or in mass-filtering mode to select particular... 

However, when TLC is coupled to a MALDI-qTOF " or MALDI-FTMS mass spectrometer, the height variance does not greatly influence the resulting spectra because (1) the extraction potentials are a few volts, rather than 10+ kilovolts, and (2) the ions are trapped or focused before analysis, which cools and sharpens the kinetic energy distribution. 

The abihty to desorb and cool ions even from such a hot matrix as a-cyano-4-hydroxycinnamic acid suggests that another approach may also work. While only a half dozen matrix preparation methods are routinely used in MALDl, all of these techniques have been developed initially for MALDl-TOF instruments. Since MALDl-TOFMS requires very flat desorption surfaces for the best resolving power, many potential matrices have not been explored. However, systems where the external ion source is decoupled from the mass analyzer, as is the case with MALDI-QTOF, MALDI-QIT, and MALDl-FTMS instruments, do not have this limitation. Indeed, some of the best signals from MALDl-FTMS systems are generated from large, irregular DHB crystals. Thus, there is a high probability that exploration of matrices with the decoupled MALDl sources will identify new matrices with favorable properties, such as improved ion abundance or reduced matrix adduction. 

QToF-MS >20 000 ESI, APCI, MALDI Mass accuracy <5 ppm (m/z 150-900) resolution 5000 nanoflow... 

Figure 4.1 Schematic representations of a time-of-fl ight/time-of-fl ight (TOF/TOF) instrument with a matrix-assisted laser desorption/ionization (MALDI) source (a), and a quadrupole time-of-flight (qTOF) instrument which can be interchangeably...

For general purpose instruments (scenario 2), the most important parameters are resolution and accurate mass measurement for the identification and characterization of unknowns. Typical are MS/MS systems based on TOE or FT (orbitrap or ICR). The major categories here are TOF/TOF, QTOF, LT-TOF, LT-orbitrap, and LT-FT-ICR. For analytes of >1 kDa, TOF/TOF systems in combination with MALDI sources are straightforward and should be evaluated where multiple users are involved. QTOF, LT-TOF, and LT-orbitrap are all suited to LC operation at regular or nanoflow rates. The resolution of orbitraps is much higher (250,000) than that of TOF instruments (20,000 to 60,000), although available resolution on the former is subject to duty cycle limitations. Table 3.13 lists representative examples of the types of instruments one might consider for common applications. 

Background Since its introduction 15 years ago, imaging of tissues by MALDI has rapidly expanded to include peptides, proteins, multiple classes of lipids, as well as small molecules such as metabolites and xenobiotics. Tissues examined have ranged from frozen tissue sections to formalin-fixed paraffin-embedded sections, and have been obtained from a wide variety of sources, including from whole animals to specific tissues, e.g., kidney and eye lens, as well as normal and diseased states. While TOF (and TOF/TOF) remains the most frequently used technique, other approaches, including MS/MS using QTOF, trap-TOF, ion mobility, and FT-trap formats, have also been reported. This paper describes analysis of tissue sections from the whole body to subsections of tissues, down to the cellular level. 

Mott TM, Everley RA, Wyatt SA, Toney DM, Croley TR. Comparison of MALDI-TOE/MS and LC-QTOF/MS methods for the identification of enteric bacteria. Int J Mass Spectrom. 

Both ionization methods, MALDI and ESI, are adequate for the analysis of low-and high-molecular-weight molecular ions. Data provided by mass-to-charge ratio (m/z) determination can in some cases directly depict a compositional assignment of the molecule under investigation if the resolution and mass accuracy are sufficient, as in quadrupole TOF (QTOF) analyzers, or high, as in ion cyclotron resonance (ICR) and Orbitrap analyzers. 

Since the use of a QMF is not practical for a pulsed MALDI technique and because ESI often produces multiply charged ions, thus decreasing the ion s m/z, there has been little attempt commercially to increase the mass range of the QMF beyond miz 4000. However, several researchers have increased the QMF to mIz 9000 and 45,000. ° Sobott and co-work-ers ° showed that it was possible to analyze miz 22,000 within an isolation window of 22 Th on a quadrupole time-of-flight (QTOF) tandem mass spectrometer by lowering the QMF to 300 kHz. 

Figure 18.13. Molecular weight measurements of the human major vault protein (MVP). MALDI-TOF-MS (A) and ESl-QTOF-MS (B) of the denatured MVP protein (pH < 3) show the molecular weight of the MVP chain to be 96.8 kDa, (C) ESI-GEMMA of native MVP (pH 6.8) demonstrates the assembly of the MVP vault to a 96-mer, 9.4-MDa complex.

Heller, M. Mattou, H. Menzel, C. Yao, X. Trypsin catalyzed O-to- RQ exchange for comparative proteomics Tandem mass spectrometry comparison using MALDI-TOF, ESl-QTOF, and ESI-ion trap mass spectrometers. J. Am. Soc. Mass Spectrom. 2003, 14, 704-718. 

---