Ionization techniques MALDI

The liquid volume of a sample required for analysis depends on the ionization technique, MALDI or ESI, and the introduction technique (see Table 4.1). The following statements assume that we are analyzing a sample near the detection limit of the analyte in a specific mass spectrometer. For MALDI-MS, the researcher typically spots 0.1 to 1 jL onto the MALDI sample plate. Thus, a minimum starting volume of 1 of 5 jL of sample is recommended. For ESI, the required sample volume is primarily dependent on the sample introduction technique. If the researcher uses a nanoflow electrospray technique, capillary EC, or capillary electrophoresis, then typically a l-pL voliune is required. However, larger sample volumes are recommended for ease of handhng. If the voliune is small, then the analysis may be limited to one experiment when additional MS or MS-MS experiments are desired. For higher flow rate ESI sources, the researcher should supply 50 pL or more for direct infusion experiments or for loading 5 to 20 pL onto an analytical EC column. 

In this article, focus is on the soft ionization technique MALDI, which is mainly in use for synthetic polymers. ESI is briefly discussed, although the electrospray method has been generally conflned to polar polymers, which are water-soluble. Other ionization methods that have been used on synthetic polymers, mainly laser desorption (LD) and fleld desorption (FD), are confined to low... 

Both ionization techniques, MALDI and ESI, deliver best results if the samples are pure and concentrated. In both cases, peptide and protein samples usually enter the sample preparation as aqueous solutions, which can contain varying amounts of an organic solvent, most often acetonitrile, methanol, or ethanol, and are usually acidified (pH 1-3). Acidification is, with only a few exceptions, a prerequisite for MALDI but not for ESI, which is far more flexible regarding the pH of the sample solution if appropriate (compatible) buffer systems and ion-pairing agents are used. 

The growing interest for the identification and characterization of polar and large compounds caused the development and the introduction of new ionization techniques, such as electrospray ionization (ESI)[4], and matrix assisted laser desorption ionization (MALDI),[5] and their more recent improvements, thus establishing new MS based approaches for studying large molecules, polymers and biopolymers, such as proteins, carbohydrates, nucleic acids. 

The introduction and development of more recent ionization techniques, such as ESI, APCI and MALDI, have allowed the study of nonvolatile and thermally unstable compounds. 

Over the years, a lot of desorption ionization techniques have been introduced to MS, such as plasma desorption, field desorption, laser desorption, secondary ion mass spectrometry, fast atom bombardment, matrix assisted laser desorption and desorption electrospray ionization. Most of them are actually no longer used. In the following paragraphs, both matrix assisted laser desorption (MALDI) and desorption electrospray ionization (DESI) will be discussed. 

Sequencing peptides with tandem mass spectrometry was carried out in the early 1980s (Biemann, 1986 Hunt et al., 1986 Hall et al., 1993). Usually the sensitivity and the lengths of sequences achievable were not sufficient to compete with Edman sequencing techniques. In 1988 and 1989, two efficient cold ionization techniques for large molecules were discovered MALDI (Karas and Hillenkamp, 1988) and the electrospray... 

With soft ionization techniques such as MALDI, ions of m/z 200000 can be routinely detected. The mass range is mainly limited by the fact that with the detector the response decreases with increasing m/z of the ions. The mass resolution of a TOF mass analyzer is relatively poor (unit mass resolution and less) and is affected by factors that create a distribution in the flight time of ions with the same m/z. The simplest way to increase the mass resolution is to increase the length of flight tube or to reduce the kinetic energy spread of the ions leaving the source. 

Mass spectrometry is also used for nanoparticles investigations. Two ionization techniques often used with liquid and solid biological samples include electrospray ionization (ESI) and matrix-assisted laser desorption/ioiuzation (MALDI). Inductively coupled plasma (ICP) sources are mainly used for metal analysis. In general,... 

Mass spectrometry methods based on soft ionization techniques, 59,61,88,89 matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF), have been successfully applied for the direct analysis of grape and wine extracts and for monitoring flavonoid reactions in model solution studies. They give access to the molecular weights of the different species present in a fraction or extract and, through fragmentation patterns, provide important information on their constitutive units. Description of the various MS techniques can be found in Chapters 1 and 2. 

There are many different ionization techniques available to produce charged molecules in the gas phase, ranging from simple electron (impact) ionization (El) and chemical ionization (Cl) to a variety of desorption ionization techniques with acronyms such as fast atom bombardment (FAB), plasma desorption (PD), electrospray (ES), and matrix-assisted laser desorption ionization (MALDI) (Mano and Goto 2003). 

Over the last decade or so, new ionization techniques such as ES and MALDI have been introduced and have increased still further the use of mass spectrometry in biology. Identification of proteins and characterization of their primary structure is a rapidly growing field in the postgenomic era. ES ionization was the first method to extend the useful mass range of instruments to well over 50 kDa (Mano and Goto... 

The sample is usually dissolved in a mixture of water and organic solvent, commonly methanol, isopropanol, or acetonitrile. It can be directly infused, or injected into a continuous flow of this mixture, or be contained in the effluent of an HPLC column or CE capillary. First introduced in late 1980s, MALDI is a soft ionization technique that allows the analysis of intact molecules of high masses. It allows determination of the molecular mass of macromolecules such as peptides and proteins more than 300 kDa in size. 

In 1974, Comarisov and Marshall60 developed Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). This technique allows mass spectrometric measurements at ultrahigh mass resolution (R = 100000-1000000), which is higher than that of any other type of mass spectrometer and has the highest mass accuracy at attomole detection limits. FTICR-MS is applied today together with soft ionization techniques, such as nano ESI (electrospray ionization) or MALDI (matrix assisted laser/desorption ionization) sources. 

Two recently developed mass spectrometric techniques have had a major impact on the analysis of large biomolecules matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS). MALDI-MS was first introduced by Karas and Hillenkamp66 and Tanaka et al.61 in 1988 and has experienced an exponential development. It has become a widespread soft ionization technique for bioorganic samples, especially large biomolecules. Fenn and co-workers68 first published the successful soft ionization technique for... 

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