Edman sequencing

While the first 20-30 residues of a peptide can readily be determined by the Edman method, most polypeptides contain several hundred amino acids. Consequently, most polypeptides must first be cleaved into smaller peptides prior to Edman sequencing. Cleavage also may be necessary to circumvent posttranslational modifications that render a protein s a-amino group blocked , or unreactive with the Edman reagent. 

DNA sequencing reveals the order in which amino acids are added to the nascent polypeptide chain as it is synthesized on the ribosomes. However, it provides no information about posttranslational modifications such as proteolytic processing, methylation, glycosylation, phosphorylation, hydroxylation of prohne and lysine, and disulfide bond formation that accompany mamra-tion. While Edman sequencing can detect the presence of most posttranslational events, technical hmitations often prevent identification of a specific modification. 

Complex peptide mixmres can now be analyzed without prior purification by tandem mass spectrometry, which employs the equivalent of two mass spectrometers linked in series. The first spectrometer separates individual peptides based upon their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses determined. As the sensitivity and versatility of mass spectrometry continue to increase, it is displacing Edman sequencers for the direct analysis of protein primary strucmre. 

In Edman sequencing, the N-terminal amino acids are sequentially cleaved and labeled with a hydrophobic chromophore. Then, the labeled... 

Stony Brook synthesized a photoreactive benzophenone containing inhibitor (39, Fig. 14) which efficiently labeled the active site of the enzyme. The photoinhibition was prevented by adding native Ras to the reaction mixture. That competition indicated that the labeling was specific at the active site. Peptide mapping of the labeled enzyme by HPLC, Edman sequencing and MALDI-MS allowed the identification of key amino acids in the substrate binding, as Asp-110 and Asp-112 in the a-subunit [126]. 

Sequencing peptides with tandem mass spectrometry was carried out in the early 1980s (Biemann, 1986 Hunt et al., 1986 Hall et al., 1993). Usually the sensitivity and the lengths of sequences achievable were not sufficient to compete with Edman sequencing techniques. In 1988 and 1989, two efficient cold ionization techniques for large molecules were discovered MALDI (Karas and Hillenkamp, 1988) and the electrospray... 

The primary goal of peptide mapping is the verification of the amino acid sequence deduced from the genetic code of the recombinant protein. The protein backbone gets cleaved by typically two or three different endoproteinases like Lys-C, trypsin, and Glu-C to achieve maps with sequence-overlapping peptide fragments. These peptide mixtures can then be separated by LC or CE and analyzed on-line by MS to obtain sequence information. Often simple mass analysis matches the predicted primary sequence of the protein. However, sometimes mutations can lead to isobaric masses of peptides that can be overseen, if no further sequence analysis like N-terminal Edman sequencing and MS/MS is carried out. 

Gerhart M.J., Balland A., and Paxton RJ. (1997), Evaluation of CNBr digestion followed by Edman sequencing as a tool for assessing methionine susceptibility to oxidation in native molecules, ABRF 97 Techniques at the Genome-Proteome Interface, Baltimore, MD. 

Analysis of Problem Peptides 7.8.3.1 Edman Sequence Analysis... 

Sequencing by both Edman sequence analysis and mass spectrometric analysis can be complimentary and where one may fail in yielding information concerning the synthesis problem, the other may succeed. This is because every peptide has different properties and each technique has advantages and disadvantages. However, sequence analysis is expensive and time consuming, which must be taken into consideration when solving the problems of peptide synthesis, especially those of routine peptide synthesis. 

The 10 laboratories that correctly identified the error in the reverse peptide used Edman sequencing. 

Of the 20 respondents, 11 use Edman sequencing for problem solving, three use amino acid analysis, three use fragmentation sequendng, two use CID, and five just resynthesize the peptide. 

Isomerization of aspartyl-X residues can occur via a succinimide intermediate (Fig. 132). The isomerized aspartyl residue can be detected by peptide mapping, Edman sequencing, and selective methylation of the iso-Asp peptide using carboxyl methyl transferase enzyme. Succinimide sites... 

Peptides have also been used as tags (Figure 6.12) in a similar manner to DNA oligo nucleotides. The sequence of amino acids in the encoding peptide is determined using the Edman sequencing method. This amino acid sequence is used to determine the history of the formation and hence the structure of the product found on that bead. 

Distilled 4-VP (see Note 1). Keep the solution under nitrogen atmosphere in a deep freezer. Iodoacetamide is also frequently used. An addition of 107 or 57 Da per alkylated cysteine residue is observed with vinylpyridine and iodoacetamide, respectively. Cysteine residues derivatized with V-melhyl iodoacetamide can be also analyzed by the Edman sequencing (no PTH standard commercially available). 

Beyer, K., Bardina, L., Grishina, G., and Sampson, H.A. 2002. Identification of sesame seed allergens by 2-dimensional proteomics and Edman sequencing Seed storage proteins as common food allergens. J Allergy Clin Immunol 110(1) 154-159. 

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