MALDI time-of-flight mass spectrometer

Fig. 1 Biomolecular interaction analysis/mass spectrometry (BIA/MS). Biosensor chips are derivatized with affinant (or used with an affinant of streptavidin) and used in the BIA analysis of biological fluids. The chips are then introduced into a MALDI time-of-flight mass spectrometer and retained ligands analyzed by virtue of molecular weight.

C. MALDI Time-of-Flight Mass Spectrometer (MALDI-TOF-MS)... 

FIGURE 4.5 The five mass spectrometers commonly used for proteomic research, (a) ESI triple-stage quadrupole mass spectrometer (b) ESI quadrupole ion trap (QIT) mass spectrometer (c) MALDI time-of-flight mass spectrometer. 

Development of Miniaturized MALDI Time-of-Flight Mass Spectrometers for Homeland Security and Clinical Diagnostics... 

Figure 2.5 Combined linear/reflectron MALDI time-of-flight mass spectrometer.

Time-of-flight mass spectrometers are interfaced with MALDI and electrospray ion sources. For proteomic applications they are most often interfaced with MALDI (Fig. 3). All ions from the ion source are accelerated to the same energy and then are left to drift in a field-free region. 

The MALDI procedure has been used recently in several variations to determine the molecular weight of large protein molecules—up to several hundred kDa. The combination of a pulsed laser beam and a time-of-flight mass spectrometer (Section 2.2.5.) is particularly effective. 

Major methods for introducing proteins and other macromolecules into mass spectrometers are electrospray and matrix-assisted laser desorption/ionization (MALDI).18-27 Most often, MALDI is used with a time-of-flight mass spectrometer, which can measure mlz up to 106. Typically, 1 p,L of a 10 jxM solution of analyte is mixed with 1 p,L of a 1-100 mM solution of an ultraviolet-absorbing compound such as 2,5-dihydroxybenzoic acid (the matrix) directly on a probe that fits into the source of the spectrometer. Evaporation of the liquid leaves an intimate mixture of fine crystals of matrix plus analyte. 

Matrix-assisted laser desorption/ionizadon time-of-flight mass spectrometer (MALDI-TOF-MS) with UV laser (i.e., 337-nm nitrogen laser), MALDI probe, and delayed extraction or reflectron... 

Griffin, T. J. Gygi, S. P. Rist, B. Aebersold, R. 2001. Quantitative pro-teomic analysis using a MALDI quadrupole time-of-flight mass spectrometer. Anal. Chem, 73,978-986. 

Kaufmann, R., Kirsch, D., and Spengler, B. (1994). Sequencing of peptides in a time-of flight mass spectrometer Evaluation of post source decay following matrix-assisted laser desorption ionization (MALDI). Int. ]. Mass. Spectrom. Ion Processes 131, 355-385. 

Often soft ionization techniqnes such as fast-atom bombardment (FAB) and matrix-assisted laser desorption (MALDI) greatly increase the utility of mass spectrometry in the analysis of fluorinated compounds. Using a commercial matrix-assisted laser desorption time-of-flight mass spectrometer, molecular ions of a polyelectrolyte,... 

Mass spectrometry has assumed great importance in determinations of the molar masses of biological macromolecules, even quite large ones. This is due to developments such as electrospray ionisation (ESI) and matrix assisted laser desorption/ ionisation (MALDI), which have made it possible to determine the molar masses of biopolymers up to several 100 kDa (Pitt 1996 Kellner et al. 1999 Snyder 2000). The combination of MALDI techniques with time-of-flight mass spectrometers (MALDI-TOF) is of particular significance for determination of the molar masses of proteins with high sensitivity (typically pmol quantities, although exceptionally fmol) and precision (proteins up to 100 kDa with precision of about 0.01 %). Mass spectrometry can provide very accurate measurements of protein molar mass that can yield information about even minor structural modifications not readily accessible by other means. 

All mass spectra were acquired on a Kratos Kompact MALDI III time of flight mass spectrometer with a 337 nm N2 laser and a 20 kV extraction potential in the linear mode. Membranes were affixed to the Kratos sample slide with tape. Every spectrum was the average of 50 laser shots. Spectra were calibrated from external standards desorbed from the same membrane being tested. 

In MALDI, the sample is deposited on a target and co-ciystallized with a solid matrix [14-15]. The target is transferred to vacuum and bombarded by photon pulses from a laser, in most cases a nitrogen laser (337 nm) nowadays. The ionization results from efficient electronic excitation of the matrix and subsequent transfer of the energy to the dissolved analyte molecules, which are desorbed and analysed as protonated or cationized molecules [7]. The ionization process is not fully understood. Extremely high molecular-mass compounds, e.g., in excess of 200 kDa, can be analysed using the MALDI, if performed on a time-of-flight mass spectrometer (Ch. 2.4.3). 

Mass spectrometry is a powerful qualitative and quantitative analytical tool that is used to assess the molecular mass and primary amino acid sequence of peptides and proteins. Technical advancements in mass spectrometry have resulted in the development of matrix-assisted laser desorption/ion-ization (MALDI) and electrospray ionization techniques that allow sequencing and mass determination of picomole quantities of proteins with masses greater than 100kDa (see Chapter 7). A time-of flight mass spectrometer is used to detect the small quantities of ions that are produced by MALDI. In this type of spectrometer, ions are accelerated in an electrical field and allowed to drift to a detector. The mass of the ion is calculated from the time it takes to reach the detector. To measure the masses of proteins in a mixture or to produce a peptide map of a proteolytic digest, from 0.5 to 2.0 p.L of sample is dried on the tip of tlie sample probe, which is then introduced into tire spectrometer for analysis. With this technique, proteins located on the surfaces of cells are selectively ionized and analyzed. 

Primer extension reactions can be analyzed using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDl-TOF MS). Specially prepared single-base primer extension products are deposited on MALDI targets, evaporated by a laser beam, and directed into the time of flight mass spectrometer. This sensitive, label-free method can discriminate which base (A, C, G, or T) was added to the primer at the very SNP position, and affords excellent error tracking. 

The mass spectrometer most widely used with MALDl is the TOF-type (time of flight) mass spectrometer, mainly due to its large mass range. MALDI-TOF is equipped with an ion mirror to reflect ions, which uses an electric field, doubling the ion flight path and increasing thus the resolution [33, 34]. 

Further performance improvements in analysing nucleic acids could be achieved by the introduction of 3-hydroxypicolinic add as matrix [8] and the introduction of delayed extraction in a linear time-of-flight mass spectrometer [9]. If, for MALDI Fourier transform mass spectrometry, the molecular weight range in analysing nucleic add fragments could be extended further this type of MALDI MS would become of significant value due to the extraordinary resolution possible [10, 11]. In order to reach the sensitivity level necessary for MALDI-TOF MS analysis an amplification step has to be incorporated into the sample preparation process for... 

---