Mass spectrometers MALDI

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... 

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. 

Mass spectrometer (MALDI TOF Voyager-DE STR, Applied Biosystems, USA). 

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

Imniunoaffinity separations performed on a matrix-assisted laser desorption ionization-mass spectrometer (MALDI-MS) probe tip have given rise to the term PAMS (probe affinity MS) [134]. Antibiotin covalently bound to the surface of the probe allowed MS discrimination of bovine insulin with different degrees of biotinylation. The method has also been used to detect lysozyme in human tears by binding antilysozyme to the MS probe. Based on the same PAMS principle, modifications aimed to obtain faster and improved retention of Abs have been carried out by immobilizing the Abs on the probe via a nitrocellulose film instead of binding them directly to the MALDI probe surface [135]. The procedure allowed determination of a therapeutic peptide and one metabolite. 

A connnon feature of all mass spectrometers is the need to generate ions. Over the years a variety of ion sources have been developed. The physical chemistry and chemical physics communities have generally worked on gaseous and/or relatively volatile samples and thus have relied extensively on the two traditional ionization methods, electron ionization (El) and photoionization (PI). Other ionization sources, developed principally for analytical work, have recently started to be used in physical chemistry research. These include fast-atom bombardment (FAB), matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ES). 

Other methods of sample introduction that are commonly coupled to TOP mass spectrometers are MALDI, SIMS/PAB and molecular beams (see section (Bl.7.2)). In many ways, the ablation of sample from a surface simplifies the TOP mass spectrometer since all ions originate in a narrow space above the sample surface. 

When a pulsed laser is used, ions are only produced for the duration of the pulse, i.e. they are not produced continuously and the mass spectrometer used must be capable of producing a mass spectrum from these pulses of ions. As discussed below in Section 3.3.4, the time-of-flight (ToF) mass analyser is the most appropriate for this purpose and has the added advantage of being able to measure very high m/z ratios. Indeed, the recent dramatic developments in the performance of the ToF mass analyser have largely been occasioned by the requirement to produce useful spectra from MALDI. 

However, because of the high price, MALDI-TOF mass spectrometers have not come into wide use. Vapor pressure osmometry (VPO), an old and traditional method for estimating molecular weight, is useful in the field of CPO chemistry. The experimental error of this measurement is approximately 10% however, the obtained data are sufficiently useful to estimate the number of porphyrins in a molecule. 

Medzihradszky, K.F., Campbell, J.M., Baldwin, M.A., Falick, A.M., Juhasz, P., Vestal, M.L., and Burlingame, A.L., The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer, Anal. Chem., 72, 552, 2000. 

The mass spectrometer is a mass-flow sensitive device, which means that the signal is proportional to the mass flow dm/dl of the analyte, i.e. the concentration times the flow-rate. It is only now possible to realise the high (theoretically unlimited) mass range and the high-sensitivity multichannel recording capabilities that were anticipated many years ago. Of considerable interest to the problem of polymer/additive deformulation are some of the latest developments in mass spectrometry, namely atmospheric pressure ionisation (API), and the revival of time-of-flight spectrometers (allowing GC-ToFMS, MALDI-ToFMS, etc.). 

Lasers have advanced the analytical use of mass spectrometers to characterise additives in polymers, and routine application of MALDI is no longer limited to high molecular masses only. MALDI can now clearly produce isotopically resolved mass spectra of small molecules (<800 Da) in an L-ToF instrument, which can be used successfully for the characterisation of molecules of different chemical classes. High mass resolution with an improvement of mass accuracy to... 

The direct whole-cell method of Holland et al. was extremely rapid, even in comparison to Lubman s MALDI analysis of fractions collected after bacterial sonnication. With the whole-cell approach bacteria were simply sampled from colonies on an agar plate, mixed with the matrix, air-dried, and introduced in batches into the mass spectrometer for analysis. In all of the spectra obtained in these and later experiments, each bacterial strain showed a few characteristic high-mass ions that were attributed to bacterial proteins. Studies demonstrating the whole cell methodology for strain-level differentiation were reported independently by Claydon et al. at almost the same time.18 Shortly thereafter a third study on whole-cell MALDI included bacteria from pathogenic and nonpathogenic strains appeared.19... 

Figure 6.2 Whole-cell MALDI-TOF spectra of two Vibrio species taken using a modern TOF mass spectrometer. Even in the linear mode V. parahaemolyticus can be easily differentiated from V. vulnificus.

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