Matrix-assisted laser-desorption/ionization

After introduction of the target into the vacuum, an UV laser pulse is used to desorb and ionize the sample. Nitrogen laser emitting at 337 nm and Nd YAG laser emitting at 355 nm are the most widely used. MALDI is a very powerful technique for the analysis of synthetics and natural biopolymers. It has completely replaced former techniques such as fast atom bombardment (FAB). In 

The high throughput capability of MALDI and the different ionization mechanisms make this technique also an attractive alternative to electrospray ionization for the analysis of low relative molecular mass compounds (LRMM) [44]. However, interferences of matrix ions and the ionization of the low relative molecular mass compounds are the challenges of this technique [45, 46]. 

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. 

MALDI shares many features with FAB in that it employs a matrix which transfers energy to an analyte molecule to facilitate the ionization of polar and thermally labile, high-molecular-weight molecules. In this case, however, the energy is provided by a pulsed laser at a wavelength which may be absorbed by a matrix material such as nicotinic or sinapinic acids. The ability to obtain mass 

A major difference between MALDI and FAB is that a solid rather than a liquid matrix is used and a mixture of the analyte and matrix is dried on the laser target. For this reason, the effective combination of HPLC with MALDI is not as readily achieved although, since MALDI is largely free of the suppression effects experienced with FAB, it is able to provide useful analytical data directly from mixtures. 

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. 

Matrix-assisted laser desorption ionization (MALDI) was simnltaneously introduced in 1988 by two research groups [23, 24]. In 2002, Koichi Tanaka, together with John Fenn (see Sect. 7.2.6) and the NMR spectroscopist Knit Wiithrich, received the Nobel Prize for Chemistiy for his ground-breaking woik in the identification and stracture elucidation of biological macromolecules. 

In a typical MALDI experiment (see Fig. 7.3), e.g., for peptide or protein analysis, 0.3-1 pi of an aqueous analyte solution is mixed with 0.5-1 pi of a 5 mM so-Intion of an appropriate matrix, e.g., 2, 5-dihydroxybenzoic add (DHB), sinapinic acid, or a-cyano-4-hydroxycinnaniic acid (CHCA), in 50% aqueous acetonitrile containing 0.1% trifluoroacetic add, and then deposited onto a metal taiget. Other 

The ionization process is not fully rmderstood [29, 30]. Generally, a two-step process is assumed to take place. The laser energy is absorbed by the matrix molecules, which are desorbed and ionized by protonation. In the hot plume generated in this ablation step, proton transfer between rrratrix ions and analyte molecules leads to protonated analytes. Instead of by protonation, cationization by for instance Alkali -ions present in the sample preparation may take place. 

The shock waves resulting from a bombardment of a few thousand fragments per second induce the desorption of neutrals and ions. This technique has allowed the observation of ions above 10 000 Da [16]. However, nowadays it is of limited use and has been replaced mainly by matrix-assisted laser desorption ionization. 

Laser desorption (LD) is an efficient method for producing gaseous ions. Generally, laser pulses yielding from 106 to 1010 W cm 2 are focused on a sample surface of about 10 3-l() 4cm2, most often a solid. These laser pulses ablate material from the surface, and create a microplasma of ions and neutral molecules which may react among themselves in the dense vapour phase near the sample surface. The laser pulse realizes both the vaporization and the ionization of the sample. 

This technique is used in the study of surfaces and in the analysis of the local composition of samples, such as inclusions in minerals or in cell organelles. It normally allows selective ionization by adjusting the laser wavelength. However, in most conventional infrared LD modes, the laser creates a thermal spike, and thus it is not necessary to match the laser wavelength with the sample. 

Since the signals are very short, simultaneous detection analysers or time-of-flight analysers are required. The probability of obtaining a useful mass spectrum depends critically on the specific physical proprieties of the analyte (e.g. photoabsorption, volatility, etc.). Furthermore, the produced ions are almost always fragmentation products of the original molecule if its mass is above approximately 500 Da. This situation changed dramatically with the development of matrix-assisted laser desorption ionization (MALDI) [17,18]. 

This was introduced in 1988 principally by Karas and Hillenkamp [19-21], It has since become a widespread and powerful source for the production of intact gas-phase ions from a broad range of large, non-volatile and thermally labile compounds such as proteins, oligonucleotides, synthetic polymers and large inorganic compounds. The use of a MALDI matrix, which provides for both desorption and ionization, is the crucial factor for the success of this ionization method. The method is characterized by easy sample preparation and has a large tolerance to contaminantion by salts, buffers, detergents, and so on [22,23], 

Two series of ions are evident, representing monoglucosides (1-5) and acetylglucosides (6-10) of the following aglycones cyanidin (1, 6 1.40), peonidin (2, 7 1.41), delphinidin (3, 8 1.42), petunidin (4, 9 1.43) and malvidin (5, 10 1.44). These results were obtained with a first-generation (continuous mode) MALDI mass spectrometer that did not have delayed 

MALDI is considered the most sensitive of these techniques, with detection limits in the femtomoles/microliter range being relatively behind these other two, and this probably explains the general lack of continued interest in them in recent years. Of course statements concerning the relative sensitivities of the four ionization techniques have to take into account the chemical differences between compound classes, which plays a major part in the ionization of a given compound by a given ionization technique. 

The IMS had a resolving power of about 25 in this design. One primary advantage of MALDI over DART and DESI is that the last two ion sources are continuous, and 

FIGURE 3.17 IM-MS 2D plot of a short oligonucleotide, apeptide, and a lipid. Note the various trend lines along which these compounds and their fragments are aligned. (From Woods et al., Lipid/peptide/nucleotide separation with MALDI-ion mohility-TOF MS, Anal. Chem. 2004, 76(8), 2187-2195. With permission.) 

Mobility-mass trend lines were first demonstrated to be specific for classes of compounds by Karasek, Kim, and Rokushika in 1978 in a study of reduced mobility as a function of compound class. In this study, they demonstrated that Him trend lines (they reported them as KJni) were larger in the following order primary alkylamines secondary alkylamines n-alkanes tertiary alkylamines benzene derivatives. These samples were introduced into the IMS as vapors. Thus, the use of Him for class identification appears to be useful for all types of sample introduction. 

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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). 

FigureBl.7.2. Schematic representations of alternative ionization methods to El and PI (a) fast-atom bombardment in which a beam of keV atoms desorbs solute from a matrix (b) matrix-assisted laser desorption ionization and (c) electrospray ionization.

Until about the 1990s, visible light played little intrinsic part in the development of mainstream mass spectrometry for analysis, but, more recently, lasers have become very important as ionization and ablation sources, particularly for polar organic substances (matrix-assisted laser desorption ionization, MALDI) and intractable solids (isotope analysis), respectively. 

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. 

El = electron ionization Cl = chemical ionization ES = electrospray APCI = atmospheric-pressure chemical ionization MALDI = matrix-assisted laser desorption ionization PT = plasma torch (isotope ratios) TI = thermal (surface) ionization (isotope ratios). 

Ionization can be improved in many cases by placing the sample in a matrix formed from sinapic acid, nicotinic acid, or other materials. This variant of laser desorption is known as matrix-assisted laser desorption ionization (MALDI). The vaporized acids transfer protons to sample molecules (M) to produce protonated ions [M + H]+. 

The ablated vapors constitute an aerosol that can be examined using a secondary ionization source. Thus, passing the aerosol into a plasma torch provides an excellent means of ionization, and by such methods isotope patterns or ratios are readily measurable from otherwise intractable materials such as bone or ceramics. If the sample examined is dissolved as a solid solution in a matrix, the rapid expansion of the matrix, often an organic acid, covolatilizes the entrained sample. Proton transfer from the matrix occurs to give protonated molecular ions of the sample. Normally thermally unstable, polar biomolecules such as proteins give good yields of protonated ions. This is the basis of matrix-assisted laser desorption ionization (MALDI). 

Laser-desorption mass spectrometry (LDMS) or matrix-assisted laser desorption ionization (MALDI) coupled to a time-of-flight analyzer produces protonated or deprotonated molecular ion clusters for peptides and proteins up to masses of several thousand. 

MALDI. matrix-assisted laser desorption ionization... 

Matrix-assisted laser desorption/ionization (MALDI) is widely used for the detection of organic molecules. One of the limitations of the method is a strong matrix background in low-mass (up to 500-700 Da) range. In present work an alternative approach based on the application of rough matrix-less surfaces and known as surface-assisted laser desoi ption/ionization (SALDI), has been applied. 

Electron impact (El), or Efectrospray ionization (ESI), or Matrix-assisted laser desorption ionization (MALDI)... 

Most biochemical analyses by MS use either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALD1), typically linked to a time-of-flight (TOF) mass analyzer. Both ESI and MALDl are "soft" ionization methods that produce charged molecules with little fragmentation, even with biological samples of very high molecular weight. 

MALDI (Section 12.4) Matrix-assisted laser desorption ionization a mild method for ionizing a molecule so that fragmentation is minimized during mass spectrometry. 

Matrix-assisted Laser Desorption/ Ionization Mass Spectrometry... 

Tandem mass spectrometry (MS/MS) is a method for obtaining sequence and structural information by measurement of the mass-to-charge ratios of ionized molecules before and after dissociation reactions within a mass spectrometer which consists essentially of two mass spectrometers in tandem. In the first step, precursor ions are selected for further fragmentation by energy impact and interaction with a collision gas. The generated product ions can be analyzed by a second scan step. MS/MS measurements of peptides can be performed using electrospray or matrix-assisted laser desorption/ionization in combination with triple quadruple, ion trap, quadrupole-TOF (time-of-flight), TOF-TOF or ion cyclotron resonance MS. Tandem... 

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. 

The molecular weights and molecular weight distributions (MWD) of phenolic oligomers have been evaluated using gel permeation chromatography (GPC),23,24 NMR spectroscopy,25 vapor pressure osmometry (VPO),26 intrinsic viscosity,27 and more recently matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).28... 

Maleamic acid, cyclization of, 293 Maleic anhydride, 59 Maleimido azine, 307 Manganese diacetate catalysts, 71 Mark-Houwink-Sakurada equation, 57 Material safety data sheets (MSDSs), 246 Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), 385, 388 McGrath, J. E., 327 MDI isomers, 210 MDIs. See Methylene diphenyl diisocyanates (MDIs)... 

Matrix-assisted laser desorption ionization post-source-decay mass spectrometry... 

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. 

Experimentation showed that the protein was not glycosylated and that the sequence at the iV-amino acid terminus corresponded to that expected. The C-terminus sequence, however, did not correspond to that predicted and these data were interpreted in terms of the presence of a heterogeneous, truncated, protein. A study of the tryptic digest fragments from this protein with matrix-assisted laser desorption ionization (MALDI) with post-source decay enabled the authors to suggest the positions at which the parent protein had been truncated. 

The unseparated digest mixture was studied directly by mass spectrometry using matrix-assisted laser desorption ionization (MALDI) and this showed six of the polypeptides detected by LC-MS and three of the expected polypeptides that had not been detected by LC-MS. In contrast, MALDI did not show three polypeptides observed by LC-MS. 

Matrix-assisted laser desorption ionization (MALDI) A method used for the ionization of high-molecular-weight compounds. In this approach, the analyte is crystallized with a solid matrix and then bombarded with a laser of a frequency which is absorbed by the matrix material. 

Matrix material A material used in fast-atom bombardment and matrix-assisted laser desorption ionization to transfer energy to an analyte molecule to bring about its ionization. 

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