Peptides sequential analysis

Sequential analysis of amino acids in purified peptides and proteins is best initiated by analysis of the terminal amino acids. A peptide has one amino acid with a free a-amino group (NH2-terminus) and one amino acid with a free a-carboxyl group (COOH-terminus). Many chemical methods have been developed to selectively tag and identify these terminal amino acids. 

Even more versatile than the dansyl method is the Edman method (Figure E2.4). The NH2-terminal amino acid is removed as its phenylthiohydan-toin (PTH) derivative under anhydrous acid conditions, while all other amide bonds in the peptide remain intact. The derivatized amino acid is then extracted from the reaction mixture and identified by paper, thin-layer, gas, or high-performance liquid chromatography. The intact peptide (minus the original NH2-terminal amino acid) may be isolated and recycled by reaction with phenylisothiocyanate. Since this method is nondestructive to the remaining peptide (aqueous acid hydrolysis is not required) and results in good yield, it can be used for stepwise sequential analysis of peptides. The method is now automated. 

Thiohydantoins (see Scheme 4.25, p. 78) play an important role as derivatives of amino acids, particularly in the sequential analysis of peptides. When the sequence is being determined from the carboxyl end, ammonium thiocyanate is dissolved in acetic acid and acetic anhydride and this mixture is allowed to react with the carboxyl end of the peptide with the formation of l-acyl-2-thiohydantoin. 2-Thiohydantoin is released from the peptide by the action of a base, and a new carboxyl end of the amino acid is exposed. Prior to GC analysis, thiohydantoins must be further modified, e.g., by silylation [271], as follows. A 25-/A volume of ethyl acetate and 25 /d of BSA are added to 500 nmol of 2-thiohydantoin and the mixture is shaken until it is homogeneous. The mixture is then heated in a stoppered test-tube at 80°C for 5 min, cooled and centrifuged before injection. 

The identification of N-terminal amino acids in peptides and proteins is of considerable practical importance because it constitutes an essential step in the process of sequential analysis of peptide structures. 

Dithiocarboxylates are particularly useful reagents in the thioacylation of the terminal amino group in peptides. Combination with a subsequent cleavage by anhydrous TEA results in a modified Edman degradation and a protocol has been worked out with conditions that are compatible with the ones required for an automatic sequential analysis (Scheme 4). ... 

For the most part the derivatives described in this section have found their greatest utility for N-terminal end group analysis, for the fingerprinting of peptides and for the sequential analysis of protein amino acids (B7, Zl). 

Sequential analysis can be accomplished by using the Edman technique. Treatment of an intact polypeptide with phenylisothiocyanate derivatizes the N- amino acid leaving the rest of the peptide intact for further Edman degradation. Large chains must be fragmented into shorter peptides, more easy to work with chemically. Cleavage of peptide bonds at specific amino acid residues is accomplished using enzymes such as trypsin (Lys, Arg), chymotrypsin (aromatics), and carboxypeptidase (C-terminus amino acids). 

The molecular weight of tyrocidine A was found to be about 1,270 . Total hydrolysis and quantitative amino acid analysis revealed the exact composition . Partial hydrolysis, fractionation of the resulting peptide mixture by countercurrent distribution, ion-exchange chromatography and paper-chromatography, followed by sequential analysis, led Paladini and Craig to the structure of tyrocidine A see Table 1.6). 

Owing to the success of cobalt(III)-mediated hydrolysis of amino acid esters, the next step was to examine how these complexes reacted with peptides. If similar hydrolytic results could be obtained with peptides, then one of the potential uses of cobalt(III) complexes would be in the N-terminal determination and sequential analysis of polypeptides. This area has been investigated by several groups. Peptides... 

Figure 6.29 (a) Sequential analysis of tyrosinase activity and thrombin activity by the tyrosinase-induced oxidation of the tyrosine-containing peptide 35 associated with the CdSe/ ZnS QDs results in electron transfer quenching of the QDs, followed by the thrombin-induced cleavage of the dopaquinone-modified peptide to restore the QDs fluorescence (b) (spectrum... 

Sequential Analysis of the Peptide Chain by FAB Mass Spectrometry... 

Greenstein, J. P. and Winitz, M., Sequential Analysis of Peptides in Chemistry of the Amino Acids, p. 1512. New York, J. Wiley and Sons 1961 Gray, W. R., Dansyl Chloride Proc ure in Methods of Enzymology, Vol. 11 (Hirs, C. H. E., ed) pp. 139-151, New York, Acad. Press 1967 Ambler, R. A. Enzymic Hydrolysis with Carboxypeptidase, ibid. pp. 155-156... 

The identification of N-terminal amino acids in peptides and proteins is of considerable practical importance because it constitutes an essential step in the process of sequential analysis of peptide structures. Many N-amino acid derivatives have been proposed for this purpose and the ones most commonly studied by TLC are 2,4-dinitrophenyl (DNP)-and 5-dimethylaminonaphthalene-l-sulfonyl (dansyl, Dns)-amino acids, and 3-phenyl-2-thiohydantoins (PTH-amino acids). Recently, 4-(dimethylamino)azobenzaie-4/-isothio-cyanate (DABITC) and phenyl-isothiocyanate (PITC) have also been investigated as derivatizing agents of amino acids. 

The order in which the tryptic peptides occur must then be determined by another technique, e.g. according to a scheme of overlapping cleavages with other proteases (pepsin, ch3onotrypsin, papain, and others). The first extensive sequence of amino acids was determined on insulin (Sanger 1954), which is a polypeptide consisting of 51 amino acids. The sequential Analysis of true proteins was still beset with serious obstacles. Finally in 1959, ribonuclease vith 124 amino acid... 

A further application of direct injection APl-electrospray to the structural elucidation of peptides is to partially fragment the peptide. In this way, one can often determine the partial or complete amino acid sequence of the peptide of interest (Ramstrom et al., 2003). This is particularly useful in rapidly estabhshing amino acid sequences of synthetic peptides in which the confirmation of a sequence can be done in a few moments rather than going through a complex amino acid analysis derived from a sequential hydrolysis process. 

The sections presented above provide an account of the separate topics into which translation can be divided. These act as an introduction to the current section, in which a description of the individual reactions in peptide synthesis is presented in a single diagram, i.e. a diagram that encapsulates the whole process (Figure 20.22). An analysis of each separate reaction provides a simple explanation of the interactions that are required in a sequential manner to form the various complexes in the pathway, the activities of which result in the synthesis of, initially, a dipeptide but then a growing peptide. The repetition of the formation of the complexes for each amino acid results in the synthesis of the final peptide, as dictated by the base sequence in the mRNA. 

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