Sequence analysis, oligopeptides

The amino-terminal (N-terminal) residue of a protein can be identified by reacting the protein with a compound that forms a stable covalent link with the free a-amino group, prior to hydrolysis with 6 M HC1. The labeled N-terminal amino acid can then be identified by comparison of its chromatographic properties with standard amino acid derivatives. Commonly used reagents for N-terminal analysis are fluorodinitrobenzene and dansyl chloride. If this technique was applied to the oligopeptide above, the N-terminal residue would be identified as Val, but the remainder of the sequence would still be unknown. Further reaction with dansyl chloride would not reveal the next residue in the sequence since the peptide is totally degraded in the acid hydrolysis step. 

The same types of A-acyl-O-alkyl derivatives can also be used for GC analysis of the simplest oligopeptides (at least dipeptides and tripeptides). Other, obsolete, recommendations on the GC analysis of these compounds include various sequences for their reduction by LiAlH4 into polyaminoalcohols, followed by N-acylation or per-methylation and, finally, silylation of OH groups. For example, A-TFA dipeptide Phe-Phe after reduction and silylation gives the compound (III) with a retention index of 2390 on semistandard-phase Dexsil-300. 

This reaction has been used to synthesize dichlorophthalimido derivatives for the analysis of peptides, since the mass spectra of those derivatives are easily recognized due to the characteristic pattern of ions containing two chlorine atoms. The simplicity of this technique is illustrated by the following general example, in which a few milligrams of a peptide is subjected to reaction with hydrazine dissolved in dimethlysulfoxide in a commercially available microwave oven for about 30 min. Samples are then removed from the solution in 5 min intervals, and the FAB MS of corresponding samples are recorded. In the case of oligopeptides, it is often possible to determine the entire sequence of amino acids via this application. 

The experimental approaches employed for these purposes involve the chemical synthesis and conformational studies of model peptide sequences (homo- and co-oligopeptides) and polypeptides, semisynthesis of proteins and the comparison of their function with natural proteins, and structure-activity correlation studies using peptide analogs. The conformational preferences of the model peptides in solution are usually determined by various spectral measurements. The x-ray structure analysis, which is... 

At a first glance, this result seems to generally support the validity of the conformational preference parameters in prediction schemes. However, the statistical analysis of proteins favors the P-structure potential of L-Val over its helix-indudng power. Provided these theoretical prediction methods could be applied not only to proteins but also, in a first approximation, to synthetic oligopeptides, a stable p-strudure for Boc-(L-Ala)s-(L-Val)2-(L-Ala)3-NH-POE-M in TFE should have been expected. The experimental outcome of a partial a-helical conformation for this sequence in TFE points to limitations of the prediction rules which rely on the assumpticm of a dominance of short-range interactions. Consequently, prediction of peptide ctmforma-tion requires more informations than the preference parameters of the constituting amino acids alone. 

The determination of amino acid sequences of individual oligopeptides is achieved by N- and C-terminal analyses for which a number of methods are available. The principle of chemical methods for A -terminal analysis is shown in Figure 4.9. A... 

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