Electrospray 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... 

FAB and PD have been replaced by electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) in the analytical mass spectrometry laboratory, because both of these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. 

The major limitation of both UV and MS detectors is that neither can provide quantitative or even semiquantitative information without reference standards. Ultraviolet response depends on the presence of a chromophore in a molecule and evidently might vary from one molecular species to another in a library. Although successful application of electrospray mass spectrometry for quantitative analysis of peptides has been reported [35], one should always keep in mind that signal intensity in a mass spectrum depends on the ability of a molecule to ionize. The ability to produce ions, especially with soft ionization techniques, might be very different for different molecules within one library, and the difference might be even bigger from one library to another. 

Coupling mass spectrometry (MS) to capillary electrophoresis provides detection and identihcation of amino acids, peptides, and proteins based on the accurate determination of their molecular masses [15]. The most critical part of coupling MS to CE is the interface technique employed to transfer the sample components from the CE capillary column into the vacuum of the MS. Electrospray ionization (ESI) is the dominant interface which allows a direct coupling under atmospheric pressure conditions. Another distinguishing features of this soft ionization technique when applied to the analysis of peptides and proteins is the generation of a series of multiple charged, intact ions. 

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. 

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 quantitation of small-sized therapeutic proteins or peptides in plasma can be conducted with EC—MS/MS detection of the intact proteins. Electrospray ionization (ESI) is the most widely used ionization technique for quantitative EC—MS/MS analysis of proteins or peptides. There are some occasions where atmospheric pressure chemical ionization (APCI) was used to circumvent matrix effects (Volosov et al., 2001). ESI typically generates multiply-charged protein or peptide ions, depending upon the number of basic charges in the polypeptide backbone. For small proteins, high abundance peaks of multiply... 

Identification is primarily based on molecular-mass determination, while for an actual structure elucidation LC-MS must be used in combination with tandem mass spectrometry (MS-MS). ESI and APCI are soft-ionization techniques, generating only intact molecule-derived ions, but no fragment ions for most molecules. Therefore, it is frequently applied in combination with MS-MS to achieve more structural information. With respect to qualitative analysis, the use of electrospray LC-MS-MS for peptide sequencing as part of proteomics research is currently an important area. 

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... 

Ionization techniques derived from ESI, such as electrosonic spray ionization (ESSI), are also suitable for studies of fragile molecules. The ESSI-MS approach enables observation of non-covalent complexes of myoglobin, protein kinase A/ATP complex, and other proteins [47]. It can be used to study on-line deprotonation reactions on peptides and proteins [48,49]. Such analyses can be done by introducing volatile bases between the ion source and mass analyzer. This method is fast one reference base can be scanned in a time interval of 1 min. The desorption electrospray ionization (DESI) technique was also implemented in the study of protein conformation in solution [50]. The interaction time between the spray solvent and the protein was estimated to be 1 ms, and it was suggested that this timescale would be too short for the studied proteins to unfold [50]. 

Microcolumn reverse-phase HPLC electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a rapid and sensitive technique for the analysis of complex mixtures of peptides. This technique is used to determine the amino acid sequence of unknown peptides, to verify the structure of proteins, and to determine posttrans-lational modifications (see also the article by Beth L. Gillece-Castro). In particular, the strength of this approach is the analysis of peptides in complicated mixtures, such as amino acid sequence analysis of peptides isolated from class I and II major histocompatibility T-cell receptor complexes (Hunt et al., 1992a). 

In Situ Digestion and Desorption from Membranes. While the electrospray ionization technique enjoys a considerable advantage as an online chromatographic (HPLC) detector, the direct desorption of proteins and peptides from electroblotted membranes using MALDI provides an equally important link to one-dimensional and two-dimensional gel electrophoresis. In such a scheme the laser beam would... 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

Samples containing mixtures of peptides can be analyzed directly by electrospray. Alternatively, the peptides can be separated and analyzed by LC/MS coupling techniques such as electrospray or atmospheric pressure chemical ionization (APCI). 

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