Ionization methods matrix-assisted laser desorption

The development of soft ionization methods (electrospray ionization and matrix-assisted laser desorption ionization, and others not discussed here) has contributed to the remarkable progress seen in mass spectrometry applied to biochemistry and molecular biology research progress, and is beginning to find applications in archaeology. 

Although electrospray ionization and matrix-assisted laser desorption ionization allow to transfer much larger ions into the gas phase, it is FD that can be regarded the softest ionization method in mass spectrometry. [27,74] This is mainly because the ionization process itself puts no excess energy into the incipient ions. Problems normally arise above 3000 u molecular weight when significant heating of the emitter causes thermal decomposition of the sample. 

Analytes must be liberated from their associated solvent molecules as well as be ionized to allow mass separation. Several ionization methods enable ion production from the condensed phase and have been used for the coupling of CE to MS. Among them, atmospheric pressure ionization (API) methods, matrix-assisted laser desorption/ionization (MALDI), and inductively coupled plasma (ICP) ionization are mainly used. API techniques are undoubtedly the most widespread ionization sources and cover different analyte polarity ranges. 

The fifth category of ionization includes the laser ionization mass spectrometry (LIMS) methods. Matrix-assisted laser desorption ionization (MALDI) uses... 

Moreover, the typical tools of supramolecular chemistry, such as NMR spectrometry, require concentrations usually in excess of 10 " mol/1. and other favorite methods such as mass spectroscopy [fast-atom bombardment (FAB), electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI)], and vapor-pressure os-moinetry do not directly provide information about supramolecular behavior in solution. The most favorite method, x-ray analysis, suffers from the limitation posed by the ultimate requirement of being able to grow single crystals. While this is. in numerous instances, possible in the case of pure molecular entities, supramolecules, being mixed molecular objects by nature, are usually difficult to grow in the form of a single crystal. 

Waters Corporation Synapt. The samples are solutions of biological material, and the ion sources are uniformly EIS, nanospray ionization, or matrix-assisted laser desorption and ionization as described in Chapters 4,9,15, and 18. Studies with the Synapt traveling wave instrument have revealed details of biomolecular ions in the gas phase that are not available by MS alone or by other methods. The full meaning of such studies and relevance for in vivo biomolecular activity is currently under discussion and debate " nonetheless, IM-MS for explorations of biomolecules certainly has affected the visibility of mobility as a measurement method and the level of technology that has been advanced through pharmaceutical and medical concerns. 

Numerous methods of ionization exist, including electron impact (El), fast-atom bombardment (FAB), chemical ionization (Cl), matrix-assisted laser desorption ionization (MALDl), and electrospray ionization (ESI). 

Four of the most commonly used desorption/ionization methods for MSI are secondary ion mass spectrometry (SIMS), desorption electrospray ionization (DESI), matrix-assisted laser desorption/ionization (MALDI), and laser ablation (LA) with post-ionization. Other desorption/ionization approaches such as laser desorption/ionization (LDI) see Chapter 9, (12)), desorption/ionization on silicon (DIOS) (13), electrospray ionization (ESI) (14), and nanostructure-initiator mass spectrometry (NIMS) (15, 16) also have great potential in MSI. Importantly, many mass spectrometers equipped with a MALDI ion source can be used with related ionization processes such as LDI, DIOS, LA, and laser-NIMS. Erequently, a specific ion source arrangement is optimized for a specific mass analyzer for example, MALDI is often interfaced to a time-of-flight (TOF) mass analyzer (described below) although it can also be used with ICR-based instruments. 

Thermogravimetric analysis Differential thermal analysis Differential scanning calorimetry Thermal volatiUszation analysis Evolved gas analysis Mass spectroscopy methods Matrix-assisted laser desorption/ionization Imaging chemiluminescence... 

After presenting an overview chapter and the fundamentals, the book focuses on instrumentation and ionization sources. It describes an ion-mobility-capable quadrupole time-of-flight mass spectrometer, the differential mobility analyzer, a cryogenic-temperature ion mobility mass spectrometer, the atmospheric solids analysis probe method, and laserspray ionization. In the final applications-oriented chapters, the contributors explore how homebuilt and commercial instruments using electrospray ionization and matrix-assisted laser desorption/ionization (MALDl) methods are employed to solve biological and synthetic issues. 

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. 

The state-of-the-art biological mass spectrometric soft-ionization technologies (matrix-assisted laser desorption/ionization (MALDI) electrospray ionization (ESI)) facilitate the routine characterization of proteins. Those two ionization methods are integrated with several differention analyzers to form a variety of mass spectrometers. 

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. 

Laser Desorption/Ablation Ionization Methods. Matrix-assisted laser desoiption ionization (MALDI) relies on the use of a solid chromophQric matrix, chosen to absorb laser light, which is co-mixed with the analyte (4). Typically, a solution of a few picomoles of analyte is mixed widi a lOO-to-5000 fold excess of the matrix in solution. A few microliters of the resulting solution are deposited on a mass spectrometer solids probe, and the solvent is allowed to evaporate before inserting the probe into the mass spectrometer. When the pulsed laser beam strikes the sample surface in the spectrometer, the sample molecules are desorbedAonized at high efficiency. The various mechanisms for matrix and non-matrix assisted laser desorption have been discussed (5,6). 

Ionization of large biomolecules, such as proteins and DNA, with LDI or any of the modified post-ionization methods was limited to very small fragments, typically under 1000 Da due to decomposition of the relatively fragile larger molecules upon ionization. Fragmentation during ionization isn t always a problem, but it s extremely useful to know the mass of the precursor species first, and these ions weren t usually observed. In 1985, two significant developments were made which led directly to the development of electrospray ionization and matrix-assisted laser desorption/ionization (MALDI), for which the Nobel prize was (jointly) awarded in 2002. Electrospray ionization is covered extensively elsewhere in this book, but the development of MALDI is to be addressed here. 

The development of electrospray ionization and matrix-assisted laser desorption/ ionization as soft mass spectrometric ionization methods occurred virtually in parallel. The sudden growth of each began at almost the same time and was spurred primarily by the new possibilities that their advent provided for analyses of biopolymers, especially proteins. The impact of the two approaches on analytical chemistry—and in particular, biomolecule analysis— was both major and complementary. The overlapping histories of development of electrospray and MALDI over the past quarter century are like two sides of the same coin for this reason, both have been included in this new volume. 

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.

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. 

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). 

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. 

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 mass spectrometry (MALDI-MS) is, after electrospray ionization (ESI), the second most commonly used method for ionization of biomolecules in mass spectrometry. Samples are mixed with a UV-absorbing matrix substance and are air-dried on a metal target. Ionization and desorption of intact molecular ions are performed using a UV laser pulse. 

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... 

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