Matrix-assisted laser desorption/ionization techniques

In recent years, TLC was successfully combined with different ionization techniques, matrix-assisted laser desorption/ionization (MALDI), ESI, atmospheric pressure chemical ionization (APCI), desorption electrospray ionization (DESI), electrospray-assisted laser desorption ionization (ELDI), and LDI for identification and quantification of organic and biomolecules. In this section, the interfacing of TLC techniques with MALDI-ESI/MS, DESI-MS, ELSI-MS, and LDI-MS will be described, performance will be discussed, and selected applications in the separation and identification of lipids, gangliosides, dyes, drugs, and medicinal compounds will be presented. 

The three techniques — laser desorption ionization, laser ablation with secondary ionization, and matrix-assisted laser desorption — are all used for mass spectrometry of a wide variety of substances from rock, ceramics, and bone to proteins, peptides, and oligonucleotides. 

Two relatively new techniques, matrix assisted laser desorption ionization-lime of flight mass spectrometry (MALDI-TOF) and electrospray ionization (FS1), offer new possibilities for analysis of polymers with molecular weights in the tens of thousands. PS molecular weights as high as 1.5 million have been determined by MALDI-TOF. Recent reviews on the application of these techniques to synthetic polymers include those by Ilantoif54 and Nielen.555 The methods have been much used to provide evidence for initiation and termination mechanisms in various forms of living and controlled radical polymerization.550 Some examples of the application of MALDI-TOF and ESI in end group determination are provided in Table 3.12. The table is not intended to be a comprehensive survey. 

There are no reports currently available of an on-line application of the very powerful MALDI technique (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). However, this technique has become increasingly important for the analysis of complex molecules and quality control, for example, for glycosylated material [177] or peptides [104] or nucleotides [324],... 

The dynamic development of mass spectrometry has had a huge impact on lipid analysis. Currently, a variety of suitable mass spectrometers is available. In principal, a mass spectrometer consists of an ion source, a mass analyzer, and an ion detector. The typical features of each instrument (Fig. 2) result mostly from the types of ion source and mass analyzer. To date, the ionization techniques apphed to lipid analysis include Electrospray Ionization (ESI or nano-ESI), Atmospheric Pressure Chemical Ionization (APCI), Matrix-Assisted Laser Desorption/Ionization... 

Soft ionization techniques such as electrospray ionization and matrix assisted laser desorption are now routinely used to determine the mass of large hydrophilic polymers like proteins (27). However, as is usual for the ionization process, the presence of sails and detergents, which is common for biological samples, can affect the process significantly. The use of the on-line capillary reversed-phase HPLC in combination of the electrospray mass spectrometer (LC/MS) has made it possible to analyze such samples directly (10,16, 28). When GAP-43 isolated from the membrane fractions of bovine brain was analyzed, a single major peak with a minor peak corresponding to a phosphorylated species was observed (Fig. la). To study the posttranslational modifications in detail, the protein was digested with specific proteases such as lysyl... 

Recent advances in protein analysis by MS are due to the introduction of electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), MSN scan modes, as well as improvements in instrument sensitivity, resolution, and mass accuracy. With these improved techniques, researchers will continue to use MS to help elucidate primary, secondary, and to a lesser extent, tertiary structure of proteins. 

Mass spectrometry This is often used for analysis of plastics. Thermal degradation can be carried out using an ion source, or in a pyrolyzer connected directly to a mass spectrometer or via a chromatographic column. Two soft ionization techniques, electrospray ionization and matrix-assisted laser desorption ionization (MALDI), are the most common in the analyses of large molecules. MALDI-MS is usually used offline but online connection with a... 

MS was first successfully applied to analysis of intact microor nisms more than 40 years ago (Anhalt and Fenselau 1975). These efforts have expanded and have been particularly significant after the introduction of the soft ionization MS techniques—matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) (Perm et al. 1989 Tanaka 2003 Karas andHillenkamp 1988). Both techniques (recognized by the Nobel Prize in Chemistry in 2002) allow the ionization and transfer into vacuum of large, intact, nonvolatile biomolecules, such as proteins. Various types of mass analyzers—quadrupole, ion trap, time-of-flight (TOF)— have been coupled to both MALDI and ESI ion sources, allowing multiple stages (tandem) MS to be performed for structure elucidation of analytes of interest. All these instrumental developments have allowed MS to become a well-established... 

To this end, both fundamentals and applications are presented. Accordingly, the book begins with an overview chapter and fundamentals (Chapters 1 to 3) followed by sections emphasizing instrumentation (Chapters 4 to 7) and ionization sources (Chapters 8 and 9). In the subsequent applications (Chapters 10 to 16), homebuilt and commercial instrumentation using electrospray ionization and matrix-assisted laser desorption/ionization methods are employed to solve biological and synthetic motivated questions. In this way, it is the intent of the editors to cover the current status of IMS-MS in such a way as to make it conveuieut for those readers unacquainted with this technique to understand its fundamental theory and practical applications. As a consequence, it is expected that this volume could serve as a useful specialized textbook for an advanced course on IMS-MS. 

To perform MS, one must make ions from neutral molecules. Ionization methods have advanced from the classic electron ionization (El), through chemical ionization (CI), field desorption (FD), fast atom bombardment (FAB) and thermospray to the atmospheric pressure ionization (API) techniques currently favored. El is classic, but is restricted in its applicability to thermally stable, volatilizable compounds. ED was always a specialized niche technique applicable to some larger compounds. EAB enjoyed a meteoric rise and fall in use first reported in 1981, but has all but disappeared now, being replaced by the API techniques atmospheric pressure Cl (APCI) °° and electrospray ionization (ESI). ° Matrix-assisted laser desorption ionization (MALDI) has shown significant utility for characterizing larger proteins, approximately 100 kDa and larger. 

Of these MSI techniques, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is a soft ionization technique which does not produce fragments, or, at least, produces fewer ions fragments. In addition, its high sensitivity, accuracy, and spatial resolution made it suitable to measure the composition of compounds or biomacromolecules. So it can determine the distribution of peptides, proteins, or other chemicals and so on within thin slices of biological samples. Robinson et al. used MALDI-MS to measure the localization of water-soluble carbohydrates in wheat stems in situ and their method (Figure 10.26) may be useful for researchers of metallomics to carry out the study between metal or non metal and organism. 

Some solid materials are very intractable to analysis by standard methods and cannot be easily vaporized or dissolved in common solvents. Glass, bone, dried paint, and archaeological samples are common examples. These materials would now be examined by laser ablation, a technique that produces an aerosol of particulate matter. The laser can be used in its defocused mode for surface profiling or in its focused mode for depth profiling. Interestingly, lasers can be used to vaporize even thermally labile materials through use of the matrix-assisted laser desorption ionization (MALDI) method variant. 

Mass spectroscopy is a useful technique for the characterization of dendrimers because it can be used to determine relative molar mass. Also, from the fragmentation pattern, the details of the monomer assembly in the branches can be confirmed. A variety of mass spectroscopic techniques have been used for this, including electron impact, fast atom bombardment and matrix-assisted laser desorption ionization (MALDI) mass spectroscopy. 

Matrix-assisted laser desorption ionization (MALDI) is not yet a technique that has been used extensively for LC-MS applications. It is included here because it often provides analytical information complementary to that obtained from LC-MS with electrospray ionization, as illustrated later in Chapter 5. 

Due to the high mass, low volatility, and thermal instability of chlorophylls and derivatives, molecular weight determination by electron impact (El) MS is not recommended. Desorption-ionization MS techniques such as chemical ionization, secondary ion MS, fast-atom bombardment (FAB), field, plasma- and matrix-assisted laser desorption have been very effective for molecular ion detection in the characterization of tetrapyrroles. These techniques do not require sample vaporization prior to ionization and they are effective tools for allomerization studies. 

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... 

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