MALDI mass spectrometry principles

Sauer, S. Typing of single nucleotide polymorphisms by MALDI mass spectrometry Principles and... 

In principle, mass spectrometry is not suitable to differentiate enantiomers. However, mass spectrometry is able to distinguish between diastereomers and has been applied to stereochemical problems in different areas of chemistry. In the field of chiral cluster chemistry, mass spectrometry, sometimes in combination with chiral chromatography, has been extensively applied to studies of proton- and metal-bound clusters, self-recognition processes, cyclodextrin and crown ethers inclusion complexes, carbohydrate complexes, and others. Several excellent reviews on this topic are nowadays available. A survey of the most relevant examples will be given in this section. Most of the studies was based on ion abundance analysis, often coupled with MIKE and CID ion fragmentation on MS " and FT-ICR mass spectrometric instruments, using Cl, MALDI, FAB, and ESI, and atmospheric pressure ionization (API) methods. 

Recent detection methods for glycan array include fluorescent assay, SPR, MALDI-TOF mass spectrometry, and nanoparticle assay. Fluorescence-based measurement is the prevalent principle for detecting binding to glycan microarrays. Rhodamine [9],... 

To benefit general readers, the discussion has been limited to methodologies that are accessible to nonspecialists and that can be carried out on commercially available spectrometers without special modifications. The chapter illustrates the principles of mass spectrometry by demonstrating how various techniques [MALDI, ESI, Fourier transform ion cyclotron resonance (FT-ICR), ion traps, and tandem mass spectrometry (MS-MS)] work. It also provides examples of utilizing mass spectrometry to solve biological and biochemical problems in the field of protein analysis, protein folding, and noncovalent interactions of protein-DNA complexes. 

The advent of both ESI and MALDI revolutionized the analysis of large biomolecules of low volatility such as peptides and proteins by their capability to form stable ions with little excess energy, enabling the determination of molecular weights even in protein mixtures. To obtain information specific to the primary structure of proteins, however, principles such as the activation of molecules via collisions with small neutral molecules, which have been used in the study of gaseous ion chemistry for decades, had to be adapted and helped to propel mass spectrometry to being of the most important tools in the field of proteomics. 

The promising use of various MALDI- and ESI-MS approaches in proteome research has accelerated many developments in the instrumental design and concepts of mass spectrometry. Novel MS techniques and intriguing combinations of existing instruments have emerged. Nonetheless, the basic principle of measuring the mass-to-charge ratios of analytes remains. 

A recently introduced technique for the separation of larger molecules is matrix-assisted faser Resorption-ionisation mass spectrometry (MALDI-MS). Developed by Karas et al. [4, 5] in 1988, it has been successfully used to determine the mass of biomolecules up to 500.000 Da. This method is based on the principle that the dissolved specimen is mixed with a matrix, and then crystallizes. After drying, a laser pulse is directed onto the solid matrix to photo-excite the matrix material,resulting in desorption and soft ionisation of the analyte.The molar mass is then determined by the lime ef ilight (TOF). 

While the principles of time-of-flight (TOF) mass spectrometry were well established for many years, significant breakthroughs in TOF technology and application were made in the 1990 s [57-58], This can be attributed to the emergence of MALDI as an ionization technique. TOF is considered as the ideal mass analyser for MALDI. This stimulated developments and research in TOF analysers, which in turn led to the rediscovery of the orthogonal-acceleration TOF (oaTOF). 

Techniques for the Ionization of Molecules The measurability of molecules by MSI is enabled through the local desorption and ionization of the molecules from a surface. In theory, all types of molecules that can undergo these two chemical processes can be measured. Many techniques have been developed or adapted to achieve desorption and ionization of molecules from surfaces, but three different desorption/ioniza-tion techniques made their way to commercially available products. The acronyms of these technologies are desorption electrospray ionization (DESI), MALDI, and secondary ion mass spectrometry (SIMS). The principles of these three methods and a comparison of their possibilities and limitations are outlined throughout this section and summarized in Figure 1 and Table 2, respectively. 

The general principle of mass spectrometry (MS) is to produce, separate and detect gas phase ions. Traditionally, thermal vaporization methods are used to transfer molecules into the gas phase. The classical methods for ionization are electron impact (El) and chemical ionization (Cl). Most biomolecules, however, undergo severe decomposition and fragmentation under the conditions of both methods. Consequently, the capabilities of mass spectrometry have been limited to molecules the size of dinucleotides [1]. Analysis of oligonucleotides with a mass range of up to 3000 Da became feasible with the development of plasma desorption (PD) methods [2]. However, until the invention of soft ionization techniques such as ESI- and MALDI MS, mass spectrometric tools were not widely considered for routine applications in biological sciences. 

There is no doubt that for the analysis of all these compounds, mass spectrometry is the first choice to obtain information about the molecular masses of the investigated compounds. Mass spectrometry offers high information content with extremely low sample consumption. In principle, any mass spectrometric method can be employed. From the diverse ionization techniques which are available for the analysis of combinatorially sythesized compounds, especially the ESI-technique but also the MALDI-technique have gained most common acceptance. The reasons for this development are various and are discussed in the following sections. 

The introduction of Matrix-Assisted Laser Desorption/Ionization (MALDI) and Electrospray Ionization (ESI) (Chapter 1) has dramatically increased the mass range for molar mass analyses by mass spectrometry. In principle, both techniques are able to produce intact quasi-molecular ions of polymers with high molar mass (>100,000 Da). 

Figure 2.10. Schematic diagram of MALDI-TOF-MS instrumentation. (Reproduced from C. Dass, Principles and Practice of Biological Mass Spectrometry, Wiley-Interscience, 2001.)...

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