Matrix-assisted laser desorption/ionization MALDI TOF

With the identities and amounts of amino acids known, the peptide is sequenced to find out in what order the amino acids are linked together. Much peptide sequencing is now done by mass spectrometry, using either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) linked to a time-of-flight (TOF) mass analyzer, as described in Section 12.4. Also in common use is a chemical method of peptide sequencing called the Edman degradation. 

The focus of this chapter is the development of a technique often called wholecell matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) or whole-cell MALDI-TOF MS. Some groups prefer to use terms such as intact or unprocessed rather than whole, but the intended meaning is the same regardless of which word is used. As noted in the first chapter of this book, there are many different methods for the analysis of bacteria. However, for the analysis of intact or unprocessed bacteria, whole-cell MALDI-TOF MS is the most commonly used approach. This method is very rapid. MALDI-TOF MS analysis of whole cells takes only minutes because the samples can be analyzed directly after collection from a bacterial culture suspension. Direct MALDI MS analysis of fungi or viruses is similar in approach1,2 but is not covered in this chapter. MALDI-TOF MS of whole cells was developed with very rapid identification or differentiation of bacteria in mind. The name (whole cell) should not be taken to imply that the cells are literally intact or whole. Rather, it should be taken to mean that the cells that have not been treated or processed in any way specifically for the removal or isolation of any cellular components from any others. In whole-cell analysis the cells have been manipulated only as necessary to... 

Samples collected during CE analysis can be used for off-line mass analysis by matrix-assisted laser desorption ionization (MALDI) and time-of-flight (TOF) MS. 

ToF mass spectrometers as dynamic instruments gained popularity with the introduction of matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) as effective pulsed ion sources for the soft ionization of large biomolecules (up to 10s dalton) due to their high ion transmission.38 ToF mass spectrometers, quadrupole analyzers and/or magnetic sector fields can be combined in tandem mass spectrometers (MS/MS) for the analysis of organic compounds. 

Electrospray ionization and matrix-assisted desorption ionization were both introduced around the same time, in the late 1980s. In fact matrix-assisted laser desorption ionization (MALDI) was first mentioned in the literature in 1987 (Karas et al., 1987). In the years prior to that, there were limited reports of the application of laser desorption MS. Early developments in MALDI focused primarily on macromolecules, particularly peptides and proteins. Historically, MALDI ion sources have predominantly been coupled to time-of-flight (TOF) instruments. TOF requires precise timed ionization events, and since ions are generated in MALDI by a pulsed desorption, this combination is complementary. Mass spectra generated by MALDI can be relatively simple, containing predominantly singly charged ions. The importance of both ESI and MALDI are well proven in the analysis of biomolecules, and both techniques were awarded the Nobel Prize for chemistry in 2002 (Chapter 1). 

Mass spectrometers are used not only to detect the masses of proteins and peptides, but also to identify the proteins, to compare patterns of proteins and peptides, and to scan tissue sections for specific masses. MS is able to do this by giving the mass-to-charge ratio of an ionized species as well as its relative abundance. For biological sample analysis, mass spectrometers are connected to an ionizing source, which is usually matrix-assisted laser desorption ionization (MALDI) [14], surface-enhanced laser desorption/ioni-zation (SELDI, a modified form of MALDI) [15], or electrospray ionization [16]. These interfaces enable the transfer of the peptides or proteins from the solid or liquid phase, respectively, to the gas (vacuum) phase inside the mass spectrometer. Both MALDI and electrospray ionization can be connected to different types of mass analyzers, such as quadrupole, quadruple-ion-traps, time of flight (TOF), or hybrid instruments such as quadrupole-TOF or Fourier transform-ion cyclotron resonance. Each of these instruments can... 

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-stain-ing, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method. 

Developments in mass spectrometry technology, together with the availability of extensive DNA and protein sequence databases and software tools for data mining, has made possible rapid and sensitive mass spectrometry-based procedures for protein identification. Two basic types of mass spectrometers are commonly used for this purpose Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) and electrospray ionization (ESI)-MS. MALDI-TOF instruments are now quite common in biochemistry laboratories and are very simple to use, requiring no special training. ESI instruments, usually coupled to capillary/nanoLC systems, are more complex and require expert operators. We will therefore focus on the use of MALDI-... 

Fig. 1. Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) spectrum of a trypsin-digested one-dimensional gel band. Peaks are labeled with their monoisotopic masses. Note that these are not the masses of the peptides, but of the peptide (pseudo)molecular ions. In MALDI spectra, peptide molecular ions arise predominantly through the addition of a proton to the peptide, giving a mass increase of 1.007 Da. The molecular ions are usually denoted as MH+ or [M+H]+.

Matrix-assisted laser desorption ionization (MALDI) and surface-enhanced laser desorption ionization (SELDI) have been used online with TOF-MS for protein differential profiles of intact or hydrolyzed biological matrices in proteomics. The potential use of affinity chips, grafted with specific Ab towards the drug compound for MALDI or SELDI, will bring sensitive and selective tools for macromolecules. Specific Ab towards either the intact protein or several signature peptides... 

Most of the mass spectrometry applications for combinatorial chemistry will be described in the following sections of this chapter. Here we will give a short overview of MS techniques utilized for the characterization of resin-bound molecules. The majority of publications in this field describe applications of matrix-assisted laser desorption ionization (MALDI), combined with time-of-flight (TOF) detection. The major difference of MS application for analysis of resin-bound molecules from the above-described NMR and IR applications is that analyte should not be covalently bound to solid support prior to mass measurement. Detachment of compound molecules from resin can be done chemically (for example, by bead exposure to TFA vapors) [30,31] or photochemically, such that cleavage, desorption, and ionization of molecules occur simultaneously upon stimulation by laser radiation [32], Since the... 

Another approach to selenized yeast is to characterize Se-containing proteins. The application of polyacrylamide gel electrophoresis (PAGE) has been proposed with the introduction of the protein spot to ICP-MS via LA or ET atomization [62, 93]. The feasibility of matrix-assisted laser desorption ionization (MALDI)D timeDof-Bight mass spectrometry (TOF-MS) prior to ES tandem MS as applied to tryptic protein digests has also been explored [135, 136]. 

At the beginning of the 1990s, two new ionization methods, electrospray ionization (ESI) [9] and matrix-assisted laser desorption/ionization (MALDI) coupled to time-of-flight (TOF) analysers [10] that avoided such inconveniences, were developed and continue to revolutionize the role of mass spectrometry in biological research. These methods allow the high-precision analysis of biomolecules of very high molecular weight. 

Elaboration of nerve agent toxicokinetics requires sophisticated analytical tools to detect and, if possible, to quantify the free toxicants as well as adducts with proteins and enzymes. Analysis of OP nerve agents has been performed by capillary electrophoresis (CE), biosensors, matrix-assisted laser desorption/ionization (MALDI) MS, desorption electrospray ionization MS (DESI MS), ion mobility time-of-flight MS (IM-TOF MS), nuclear magnetic resonance spectroscopy (NMR), LC-UV, gas chromatography (GC), and many more techniques (Hooijschuur et al, 2002 John et al, 2008). 

These interface techniques for connecting the HPLC with the MS-system are very sensitive for most of the substances of interest to the flavour industry. Therefore, HPLC-MS coupling techniques have become an increasingly powerful tool for quality control of flavourings, especially for the analysis of complex mixtures like process flavourings or contaminants present in such complex mixtures. New developments in the area of mass detection systems, such as time-of-flight (ToF) mass analysers and tandem mass spectrometry systems or the features of matrix-assisted laser desorption ionization (MALDI) techniques, may enhance the analytical capabilities of these systems in the near future [16, 17, 28-31 ]. 

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