SEQUENCING OF PEPTIDES AND PROTEINS

PITC has been used extensively in the sequencing of peptides and proteins and reactions under alkaline conditions with both primary and secondary amino acids. The methods of sample preparation and derivatization follow a stringent procedure which involves many labour-intensive stages. However, the resulting phenylthio-carbamyl-amino acids (PTC-AA s) are very stable, and the timing of the derivatization step is not as critical as when using OPA. 

Nano-electrospray Mass Spectrometry and Edman Sequencing of Peptides and Proteins Collected from Capillary Electrophoresis... 

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. 

Edman degradation was originally developed for determination of the primary structure (i.e., amino acid sequence) of peptides and proteins. Sequence analysis is not regularly performed for quality control in routine peptide synthesis but is more often employed for problem solving. As described earlier in this chapter, efficient characterization of synthetic peptides can be readily obtained by a combination of RP-EDPLC and mass spectrometry. Amino acid analysis is also valuable if MS is not available. If an incorrect mass or a discrepancy in the amino acid composition is found, one obvious alternative is to resynthesize the peptide. But, in order to deduce the cause of a failed synthesis, additional analyses must be performed. Both Edman degradation and tandem MS can be used to obtain sequence information... 

Edman degradation a method for determining the amino acid sequence of peptides and proteins (5.4) electronegativity a measure of the tendency of an atom to attract electrons to it in a chemical bond (2.1) electron transport to oxygen a series of oxidation-reduction reactions by which the electrons derived from oxidation of nutrients are passed to oxygen (19.1) electrophile an electron-poor substance that tends to react with centers of negative charge or polarization (7.5)... 

Wellner, D., et al. (1990). Sequencing of Peptides and Proteins with Blocked N-terminal Amino Acids N-acetylserine or N-acetylthreonine, Proc. Natl. Acad. Sci. USA 87 1947-1949. 

Inglis, A. (1991). Chemical Procedures for C-terminal Sequencing of Peptides and Proteins, Anal Biochem. 195 183-196. 

This reaction has a general application in the preparation of 2-thiohydantoins from pure amino acids but mostly is used for the sequencing of peptide and protein starting from the C-terminal. 

H. Yajima, N. Fujii, Chemical synthesis of ribonuclease A with full enzymatic activity in Chemical Synthesis and Sequencing of Peptides and Proteins (t-Y. Liu, A.N. Schechter, R.L. Heinrikson, P.G. Condliffe eds.) Elseview/North Holland, New York, 1981 p. 21-39... 

---