Lasers for MALDI

The aim of this chapter is to provide an overview of the instrumentation currently available for the mass analysis of MALDI ions. It is not intended as an exhaustive treahse on each instrument, and consequently if more detail is required the reader should examine the Usted reference material in detail. In the first sections of the chapter, the use of lasers for MALDI is discussed, and how they are coupled to the mass spectrometer, together with details of vibrational cooling and tandem mass spectrometry of MALDI ions. This is followed by a description of the different mass analyzer designs, with emphasis placed on commercially available instrument configurations. Definitions of aU acronyms, as well as technical descriptions of terms (such as peak centroid and resolving power) are included at the end of the chapter. 

Most often proteins are the bacterial biopolymers studied using MALDI MS either from fractions or whole cells. They are not the only isolated cellular biopolymers studied by MALDI, nor the first. Very soon after the introduction of MALDI there were a few reports of the analysis of bacterial RNA or DNA from bacterial fractions. One of the first applications of MALDI to bacteria fractions involved analysis of RNA isolated from E. coli,4 Other studies included analysis of PCR-amplified DNA,5 6 DNA related to repair mechanisms7 and posttranscriptional modification of bacterial RNA.8 While most MALDI studies involve the use of UV lasers, IR MALDI has been reported for the analysis of double stranded DNA from restriction enzyme digested DNA plasmids, also isolated from E. coli.9... 

A lot of features of MALDI are conveyed by its name it is a desorption ionization, produced by a laser beam, and assisted by a matrix (Figure 2.5). The analyte (1 pmol or less) is mixed with a suitable matrix in a 1 1000 or higher molar ratio. The matrix is composed of a compound with a strong absorption at the wavelength of the laser used. These two factors, matrix excess and its strong absorption, ensure that the energy from the laser pulse is absorbed by the matrix and not by the analyte, thus avoiding its decomposition. Nicotinic acid, sinapinic acid (SA), 2,5-dihydroxy benzoic acid (2,5-DHB) and 2-(4-hydroxyphenylazo)benzoic acid (HABA) are some of the most commonly used matrices for MALDI. 

Also for MALDI, there is a special case worth mentioning. Surface-enhanced laser desorption/ionization (SELDI) is a technique that utilizes special sample plates [196, 197]. These have different modified surfaces, for example, hydrophobic, anionic, or antibody treated. Which type of surface to select depends on the application. After application of analyte the surface is washed according to a protocol leaving only the desired components on the target. Finally, a MALDI matrix is applied before analysis in the spectrometer. See Chapter 12 for an application example of SELDI. 

The development of mass spectroscopic techniques such as matrix assisted laser desorption (MALDI) and electrospray mass spectrometry has allowed the absolute determination of dendrimer perfection [7,8], For divergent dendrimers such as PAMAM and PPI, single flaws in the chemical structure can be measured as a function of generation to genealogically define an unreacted site of or a side reaction producing a loop at a particular generation level. Mass spectromet-ric results on dendrimers, not only demonstrate the extreme sensitivity of the technique, but also demonstrate the uniformity of the molecular mass. The polydispersity index of Mw/Mn for a G6 PAMAM dendrimer can be 1.0006 which is substantially narrower than that of living polymers of the same molecular mass [7],... 

In the MALDI technique a pulsed laser beam strikes a solid sample and heats, vaporizes, and ionizes compounds with little decomposition.201-209 Proteins or other biopolymers are mixed with a "matrix" Fiat absorbs the heat of Fie laser beam. The protein sample together with Fie matrix is dried. Most proteins form crystals and Fie laser beam is directed toward individual protein crystals or aggregates. Various materials are used for the matrix. Compounds as simple as glycerol, succinic acid, or urea can be used with an infrared laser. For proteins an ultraviolet nitrogen laser tuned to 337 nm is usually employed with an ultraviolet light-absorbing matrix such as hydroxy-benzoic acid, 2,5-dihydroxybenzoic acid, a-hydroxy-... 

MALDI is considered a surface analysis technique and a relatively nondestructive ionization technique (Page and Sweedler, 2002). Typically, a sample spot on the target can be assayed multiple times because only a small fraction is vaporized for each laser pulse. However, no further spectra are detected after a completed ablation by the laser in MALDI. The depleted amounts were observed to be associated with the sample identity, sample spot size, and MALDI matrices (Page and Sweedler, 2002). 

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... 

A laser beam serves as the source of desorption and ionization. Many different types of laser light have been studied for MALDI-TOF-MS. The most used lasers include pulsed N2 laser with a wavelength of 337 nm and Nd-YAG solid-state laser with a wavelength of 355 nm. The ideal laser should deliver an efficient and controllable quantity of energy to the samples, and in order to avoid thermal decomposition this energy must be transferred quickly. The samples have absorption of the laser energy radiation and ionization. 

It is important to note that each of these ionization sources, either a laser in MALDI-TOF or the high voltage ionization of droplets in ESI-MS/MS or LC-MS/ MS, will each produce a different spectrum of detectable ions and intensities because the effectiveness and nature of peptide ionization is quite different for each source. In addition, the presence of multiple peptides that influence each other s ionization potential notably through ion suppression makes most peptide ion measurements only semiquantitative. 

Table 1.1 Some common lasers used for MALDI.

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