Peptide ionization techniques

Until 1981, mass spectrometry was limited, generally, to the analysis of volatile, relatively low-molecular-mass samples and was difficult to apply to nonvolatile peptides and proteins without first cutting them chemically into smaller volatile segments. During the past decade, the situation has changed radically with the advent of new ionization techniques and the development of tandem mass spectrometry. Now, the mass spectrometer has a well-deserved place in any laboratory interested in the analysis of peptides and proteins. 

Fast-atom bombardment (FAB) is an ionization technique that produces a protonated or deprotonated molecular ion, hence a molecular mass for the sample. It can be used for analysis of peptides up to m/z about 5000. 

The use of mass spectrometry for the analysis of peptides, proteins, and enzymes has been summarized. This chapter should be read in conjunction with others, including Chapter 45, An Introduction to Biotechnology, and Chapters 1 through 5, which describe specific ionization techniques in detail. 

The use of the dynamic-FAB probe (see Section 4.4 above) has allowed the successful coupling of HPLC to this ionization technique but there is an upper limit, of around 5000 Da, to the mass of molecules which may be successfully ionized. Problem solving, therefore, often involves the use of chemical methods, such as enzymatic hydrolysis, to produce molecules of a size more appropriate for ionization, before applying techniques such as peptide mapping (see Section 5.3 below). 

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... 

Kbnig, W.A. Aydin, M. Schulze, U. Rapp, U. Hbhn, M. Pesch, R. Kalik-hevitch, V.N. Fast-Atom-Bombardment for Peptide Sequencing - a Comparison With Conventional Ionization Techniques. Int. J. Mass Spectrom. Ion Phys. 1983, 46, 403-406. 

ESI has become the most commonly used interface for LC/MS. It was recognized by John Fenn and co-workers as an important interface for LC/MS immediately after they developed it as an ionization technique for MS. ESI transforms ions in solution to ions in the gas phase and may be used to analyze any polar molecule that makes a preformed ion in solution. The technique has facilitated the ionization of heat-labile compounds and high-molecular-weight molecules such as proteins and peptides. ESI is a continuous ionization method that is particularly suitable for use as an interface with FiPLC. It is the most widely accepted soft-ionization technique for the determination of molecular weights of a wide variety of analytes and, has made a significant impact on drug discovery and development since the late 1980s. 

Great care has to be taken in the analytical characterization of synthetic cyclic peptides.[73] The major side reactions during cyclization are epimerization of the C-terminal amino acid residue and cyclodimerization. Cyclodimers can be detected by mass spectrometry, although the analysis is not trivial, because artifacts do occur in some ionization techniques such as ES-MS as a result of aggregation.1 1 Ll 121 Real dimers can be detected as double-charged particles with mlz values identical to the cyclic monomers, but with a mass difference of 0.5 amu in the resolved isotope signals. The mass difference of the corresponding monomer is 1 amu. The cyclodimerization has received some attention as a direct method for the synthesis of C2-symmetrical cyclic peptides.[62 67 94113 115]... 

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. 

Fast atom bombardment mass spectrometry (FABMS) has become an important addition to the ionization techniques available to the analytical chemist in recent years. It has been particularly useful in a number of diverse applications which include molecular weight determinations at high mass, peptide and oligosaccharide sequencing, structural analysis of organic compounds, determination of salts and metal complexes, and the analysis of ionic species in aqueous solutions. This paper will focus on some aspects of the quantitative measurement of ionic species in solution. The reader is referred to a more comprehensive review for more details of some of the examples given here as well as other applications (1). 

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. 

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. 

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