Mass spectrometry building blocks

The first part of this book is dedicated to a discussion of mass spectrometry (MS) instrumentation. We start with a list of basic definitions and explanations (Chapter 1). Chapter 2 is devoted to the mass spectrometer and its building blocks. In this chapter we describe in relative detail the most common ion sources, mass analyzers, and detectors. Some of the techniques are not extensively used today, but they are often cited in the MS literature, and are important contributions to the history of MS instrumentation. In Chapter 3 we describe both different fragmentation methods and several typical tandem MS analyzer configurations. Chapter 4 is somewhat of an outsider. Separation methods is certainly too vast a topic to do full justice in less than twenty pages. However, some separation methods are used in such close alliance with MS that the two techniques are always referred to as one combined analytical tool, for example, GC-MS and LC-MS. In effect, it is almost impossible to study the MS literature without coming across at least one separation method. Our main goal with Chapter 4 is, therefore, to facilitate an introduction to the MS literature for the reader by providing a short summary of the basic principles of some of the most common separation methods that have been used in conjunction with mass spectrometry. 

Molecular weight. This is a two-part criterion, as both the absolute molecular weight and its uniqueness are important for each building block. The first of these is particularly important in the context of libraries focused on drug-like molecules, since one generally wants to keep the total molecular weight low. Uniqueness is critical if one plans to employ mass spectrometry for analysis of library results. 

The assembly of the p- and y-amino-acid building blocks to peptidic chains was achieved by simply using the established methods of peptide synthesis - in solution [6], on solid phase [11], or in a synthesizer machine [39] also, the so-called native ligation can be applied with p-peptides [54]. Furthermore, the methods of analyzing and studying the structures of a-peptides and natural proteins can mostiy be applied to P-peptides as well (the same is true for y-peptides [51,55-60]). These methods are CD [35,37] and NMR [6, 49] spectroscopy, mass spectrometry [27,35], X-ray analysis [6,21,24,25,36], molecular dynamics (MD) calculations [9,13,18,31,38] and biological investigations [6, 15,20,26,30,41-43,45,46,48]. All of this sounds like routine, but the results are rather spectacular. 

The quality of solvents and inorganic salts is an important consideration. Soluble impurities can give noisy baselines and spurious peaks or can build up on the surface of the packing material, eventually changing chromatographic retentions. Furthermore, the eluate may need to be collected for further analysis (e.g. mass spectrometry) and all contamination must be avoided. In addition, particulate matter should be removed, otherwise pump filters, meshes, and tubing can become blocked. 

In spite of the incomplete coverage in classical reviews and the absence of references in chemical and biochemical texts on mass spectral studies applied to nucleic acids and their derivatives, mass spectrometry is a very promising technique for studying these compounds. The analysis of the relevant building blocks (nucleosides and nucleotides) is satisfactorily achieved, whereas the analysis of the polymers (oligonucleotides and nucleic acids) still needs refinement despite the sophistication level of the techniques used. The future looks very promising for the sequence analysis of nucleic acids and this, along with structural elucidation studies of modified bases, for example, could establish mass spectrometry as a routine technique in this area. 

FIA is the simplest form of sample introduction into the mass spectrometer, and this injection format has been widely used in the analysis of combinatorial library samples. This technique offers the highest throughput combined with ease of use and facile automation. Richmond et al. [67-69] reported methods to minimize sample carryover for the FIA-MS analysis of combinatorial libraries. Samples were sorted before the analysis to maximize the molecular-weight difference between samples in the analysis queue and to minimize the conditions where consecutively measured wells contain samples similar to building blocks. Cycle times of less than a minute were reported with a carryover of 0.01%. A software appUcation was developed to automatically report the sample purity and calculate sample carryover by an automatic spectrum comparison method [70,71]. A quasi-molecular ion discovery feature was also implemented [72] in the automated data-processing program. Automated FIA-MS analysis and reporting were also used in the analysis of fractions from the purification of combinatorial libraries [73]. Whalen et al. developed software to allow automated FIA-MS analysis from 96-well plates [74]. The system optimizes the interface for mass spectrometry and MS/MS conditions, and reports the results in an unattended fashion. 

Heparan sulfate-like oligomers 81 were assembled on a soluble PEG resin using repeating disaccharide building blocks (Fig. 14) [97]. The coupling efficiency was about 95% and, after deprotection and cleavage from the resin, the products were sulfated. The structure of the products was confirmed by H NMR and MALDI-mass spectrometry. 

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