Introduction to Peptide Structure Determination

First the general structure and chemistry of the amino acids is presented. Then several methods that can be used to prepare them in the laboratory are discussed. After an introduction to the structure of peptides and proteins, chemical methods that can be used to determine the amino acid sequence in proteins are presented. Next, the synthesis of peptides in the laboratory is introduced. Finally, the three-dimensional structure of proteins and the mechanism of action of enzymes are briefly addressed. 

Mass spectrometry has been applied to the structure elucidation of peptides and proteins for some time. Depending on the problem, it is used as both a primary and a complementary technique and although many difficulties still remain it is now an established means of determining amino acid sequences. As instrumental methods and new derivatives have developed, the different approaches employed, with particular combinations of chemical pretreatment and sample introduction to the MS, have been dictated by the size and type of the peptide and the information required. 

Equally important as NMR spectroscopy is the development of mass spectrometry where advances have been even more spectacular, partly due to its far greater sensitivity. A broad introduction is provided by Hocart (Chapter 9.10), who describes the range of available spectrometers and techniques. In the next chapter (Chapter 9.11), Dorrestein and his coauthors describe applications to the elucidation of biosynthetic pathways, focusing on nonribosomal peptides and polyketides, while Das provides a detailed account of the structure determination of proteins and peptides in Chapter 9.12. In yet another illustration of the power of mass spectrometry to analyze biomacromolecules, Li and Altman extend the technique to a study of bacterial polysaccharides (Chapter 9.13). 

The particle beam LC/FT-IR spectrometry interface can also be used for peptide and protein HPLC experiments to provide another degree of structural characterization that is not possible with other detection techniques. Infrared absorption is sensitive to both specific amino acid functionalities and secondary structure. (5, 6) Secondary structure information is contained in the amide I, II, and III absorption bands which arise from delocalized vibrations of the peptide backbone. (7) The amide I band is recognized as the most structurally sensitive of the amide bands. The amide I band in proteins is intrinsically broad as it is composed of multiple underlying absorption bands due to the presence of multiple secondary structure elements. Infrared analysis provides secondary structure details for proteins, while for peptides, residual secondary structure details and amino acid functionalities can be observed. The particle beam (PB) LC/FT-IR spectrometry interface is a low temperature and pressure solvent elimination apparatus which serves to restrict the conformational motions of a protein while in flight. (8,12) The desolvated protein is deposited on an infrared transparent substrate and analyzed with the use of an FT-IR microscope. The PB LC/FT-IR spectrometric technique is an off-line method in that the spectral analysis is conducted after chromatographic analysis. It has been demonstrated that desolvated proteins retain the conformation that they possessed prior to introduction into the PB interface. (8) The ability of the particle beam to determine the conformational state of chromatographically analyzed proteins has recently been demonstrated. (9, 10) As with the ESI interface, the low flow rates required with the use of narrow- or microbore HPLC columns are compatible with the PB interface. 

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