MALDI peptide ionization techniques

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... 

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. 

FAB and PD have been replaced by electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) in the analytical mass spectrometry laboratory, because both of these newer techniques have a wider mass range of analysis and have lower detection limits. ESI and MALDI have become invaluable ionization techniques for nonvolatile components. This is particularly true for a wide range of biological molecules including proteins, peptides, nucleic acids, etc. Samples can be analyzed by ESI using either direct injection or introduction through liquid chromatography. 

The use and development of high-resolving separation techniques as well as highly accurate mass spectrometers is nowadays essential to solve the proteome complexity. Currently, more than a single electrophoretic or chromatographic step is used to separate the thousands of proteins found in a biological sample. This separation step is followed by analysis of the isolated proteins (or peptides) by mass spectrometry (MS) via the so-called soft ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) combined with the everyday more powerful mass spectrometers. Two fundamental analytical strategies can be employed the bottom-up and the top-down approach. 

MALDI and ESI represent the predominant ionization techniques in mass spectrometry-based proteomics, as recognized by the Nobel Prize in chemistry in 2002. MALDI is mainly used to volatize and ionize simple polypeptide samples for mass spectrometric (MS) analysis at high speed. The analysis of more complex peptide mixtures is usually conducted via ESI mass spectrometry (ESI MS) coupled online with a high-pressure liquid chromatography (HPLC) system to concentrate and separate peptides prior to MS analysis. 

With the advent of very sensitive ionization techniques such as matrix assisted laser desorption (MALDI) coupled with time-of-flight (TOF) mass analysis, measurement of the intact mass of peptides at sub-pmol levels has become a reality (2) of which we have taken advantage for the systematic screening of HPLC fractions. Partial sequence information can be obtained by carrying out enzymatic hydrolysis with exoproteinases (e.g. carboxypeptidases and aminopeptidases) (3, 4). More recently, MALDI has been used to measure metastable decomposition occurring in the first field free region of a reflectron TOF instrument (referred to as post source decay (PSD)) with only marginally more sample (5-7). 

Mass spectrometry is a powerful qualitative and quantitative analytical tool that is used to assess the molecular mass and primary amino acid sequence of peptides and proteins. Technical advancements in mass spectrometry have resulted in the development of matrix-assisted laser desorption/ion-ization (MALDI) and electrospray ionization techniques that allow sequencing and mass determination of picomole quantities of proteins with masses greater than 100kDa (see Chapter 7). A time-of flight mass spectrometer is used to detect the small quantities of ions that are produced by MALDI. In this type of spectrometer, ions are accelerated in an electrical field and allowed to drift to a detector. The mass of the ion is calculated from the time it takes to reach the detector. To measure the masses of proteins in a mixture or to produce a peptide map of a proteolytic digest, from 0.5 to 2.0 p.L of sample is dried on the tip of tlie sample probe, which is then introduced into tire spectrometer for analysis. With this technique, proteins located on the surfaces of cells are selectively ionized and analyzed. 

For high-molecular-weight samples, most commonly proteins and peptides, but also polysaccharides and synthetic polymers, the choice of an ionization method will be limited to ESI (Section 9.10.2.2.4) and/or MALDI (Section 9.10.2.2.7) (see also Table 5 for a comparison of ESI and MALDI). As mention above, MALDI is a solid-phase-based ionization technique and ESI is a flow-based liquid technique. Both readily generate... 

More success was obtained using on-line solid phase extraction before offline analysis by capillary electrophoresis with matrix-assisted laser-desorption ionization (MALDI) MS [18]. Because microdialysis is a method used for analysis of small molecules and peptides, MALDI is not used frequently with microdia lysis sampling. Another disadvantage is that MALDI cannot be used on-line with the separation because it is a vacuum ionization technique. However, if the pep-tide is large enough (0 1000 Da) MALDI can be useful. For the analysis of peptides in dialysate an appropriate separation is important before mass spectrometric detection. In a comparison with direct sampling of dialysate in MALDI, capillary electrophoresis provides the high efficiency separations necessary to resolve all... 

The past years have seen the development of two important ionization techniques electrospray ionization (ESI) and matrix-assisted laser desorption (MALDI), which have been used especially in conjunction with quadrupole, time of flight (TOF) and ion cyclotron (IC) analyzers. Both ionization techniques have revolutionized the essence of mass spectrometry itself, radically increasing the upper mass limit and thus the applicability of the mass spectrometric technique and indirectly affecting the endeavors for improved resolution and sensitivity. With these techniques, the analysis of peptides, proteins and oligonucleotides has become possible. The aim of this chapter is to demonstrate the capabilities of these techniques, especially electrospray mass spectrometry in conjunction with HPLC techniques, for the analysis of combinatorially generated compounds and libraries, and to exemplify and discuss both the potentials and limits of these analytical techniques. 

MS, especially in combination with advanced separation techniques, is one of the most powerful and versatile techniques for the structural analysis of bacterial glycomes. Modern mass spectral ionization techniques such as electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI) provide detection limits in the high atto- to low femto-mole range for the identification of peptides and complex carbohydrates. Structural characterization of these trace level components can be achieved using tandem MS. This provides a number of specific scanning functions such as product, precursor ion, and constant neutral loss scanning to... 

The techniques of matrix-assisted laser desorption/ ionization-time of flight (MALDI-TOF) and electro-spray/ionization (ESI) have revolutionized biological MS since 1989. All state-of-the-art biochemistry and biology laboratories possess at least one of these ionization techniques and, in general, have access to both of them. Both ionization techniques are applicable to peptides and proteins, DNA and RNA, glycoconjugates, and synthetic polymers. 

MALDI is a soft ionization technique for introducing large and delicate molecules, such as peptides and proteins, into the mass spectrometer for analysis. The methodology as it is known today was pioneered by Hillenkamp and coworkers (1). The essence of the technique is that the analyte molecules are embedded in an excess amount of a suitable matrix. Then, a laser beam (usually a UV laser) is irradiated on the matrix, which subsequently transfers the absorbed energy to the sample, causing the molecules to desorb and ionize into... 

MALDI is a soft ionization technique used for the analysis of biomolecules (biopolymers such as proteins, peptides, and sugars) and other large organic molecules, such as polymers, dendrimers, and other macromolecules, which tend to be fragile and fragment when ionized by more conventional ionization methods. It is most similar in character to ESI both in relative softness and the ions produced. 

Electrospray ionization (ESI) is one of the two newer ionization techniques (the other being MALDI) that has revolutionized the application of mass spectrometry to biochemistry and molecular biology, and may fairly be said to have revolutionized these disciphnes also. This is the result of the ability of ESI-MS to provide molecular mass information of hitherto unthinkable accuracy and precision for fragile biopolymers, particularly proteins, and to provide amino acid sequence information for specific peptides present at trace levels in complex mixtures characteristic of biological extracts. The discipline of proteomics would not exist without the development of ESI and MALDI. However, this book is concerned with quantitation of small molecules present at trace levels in complex matrices, rather than the essentially qualitative data typically acquired in proteomics experiments, although application of the approach to quantitation of proteins and peptides is discussed in Section 11.6. Nonetheless, development of methods to produce and characterize gaseous ions from macromolecules was very important in the development of ESI-MS, and this history will be briefly described here. Excellent reviews of this history as it pertains to macromolecule characterization (Penn 1990 Smith 1991, 1992 Fernandez de la Mora 1992) are available, while... 

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