Edman degradation position

In 1950 an alternative to the Sanger procedure for identifying N-terminal amino acids was reported by Edman—reaction with phenyl-isothiocyanate to give a phenylthiocarbamide labeled peptide. When this was heated in anhydrous HC1 in nitromethane, phenylthiohy-dantoin was split off, releasing the free a-NH2 group of the amino acid in position 2 in the sequence. While initially the FDNB method was probably the more popular, the quantitative precision which could be obtained by the Edman degradation has been successfully adapted to the automatic analysis of peptides in sequenators. 

A number of ribosomal proteins contain modihed amino acids at the N terminus or at other positions of the protein chain (Table IV). The N termini of three proteins (S5, S18, and L7) are acetylated, thus they cannot be subjected successfully to manual or automatic Edman degradation because of their blocked N termini. Mutants have been isolated in... 

This modification of the DMAA program permitted identification of the first 14 residues. Identification of the polar amino acids in positions 15 and 16 could not be achieved whereas glycine in position 17 could again be recognized. The remaining unknown section of the sequence was elucidated by the manual Edman degradation procedure after tryptic cleavage of C3... 

The fluorescent beads are manually isolated, washed with 8 M guanidine-HCl and water, and then submitted for sequence analysis. We expect that some of the peptides on the positive beads will be cleaved at the proteolytic site. As a result, the PTH-amino acids detected during each cycle of Edman degradation will have been generated from both of these peptides. This complicates the sequence analysis somewhat. However, it also allows us to determine the proteolytic cleavage site of the peptide, in addition to its uncleaved sequence. 

In the selected example by Lam et al. [101] many peptide libraries were prepared using the mix and split technique and tested in different on-bead screens. Incomplete libraries were tested (the population of most of them was more than a million compounds), and the positive structures were exploited through focused libraries. Some libraries were screened against an anti-insulin monoclonal antibody tagged with alkaline phosphatase, which allowed an enzyme-linked colorimetric detection. Only the beads bound to the murine MAb showed a tourquoise color, while the vast majority remained colorless (details of the technical realization of the assay can be found elsewhere [101, 102]). The chemical structure linked to the positive beads was then easily determined via Edman degradation of the peptide sequences. 

The critical feature of the Edman degradation is that it allows the N-terminal amino acid to be removed without cleaving any of the other peptide bonds. Let s see how this occurs. The mechanism of the reaction is shown in Figure 26.3. First the nucleophilic nitrogen of the N-terminal amino acid attacks the electrophilic carbon of phenyl isothiocyanate. When anhydrous HF is added in the next step, the sulfur of the thiourea acts as an intramolecular nucleophile and attacks the carbonyl carbon of the closest peptide bond. II is the intramolecular nature of this step and the formation of a five-membered ring that result in the selective cleavage of only the N-terminal amino acid. The mechanism for this part of the reaction is very similar to that for acid-catalyzed hydrolysis of an amide (see Section 19.5). However, because no water is present, only the sulfur is available to act as a nucleophile. The sulfur is ideally positioned for intramolecular attack at the carbonyl carbon of the N-terminal amino acid, so only this amide bond is broken. 

Automated Edman degradation was performed on. 200 pmol of purified Hez-PBAN using the pulsed-liquid sequencer. Data were obtained from 33 cycles (Table I, run 3). However, the residues at positions 23 and 32 could not be ambiguously assigned. A second attempt on the same instrument using sample purified from the PBAN II zone confirmed the earlier sequence and established the residues at positions 23 and 32 (Table I, run 4). 

Over the years, several techniques have been developed to elucidate the structure of a new lantibiotic. The extensive post-translational modifications limit Edman degradation to a stretch of amino acids from the N-terminus to the first modification. Various chemical derivatization techniques have been used to allow continuation of the sequence and to reveal the position of the posttranslationaUy modified residues. Originally, these techniques relied on treatment with ethanethiol under highly basic conditions that result in elimination reactions of the thioethers... 

For the synthetic library method that uses the affinity chromatography selection approach, the bound peptides can be eluted and microsequenced by Edman degradation. Concurrent microsequencing of the retrieved peptide mixture can be performed rather than sequencing individual peptides. Sequeuce motifs then can be defined in a fast and efficient way. However, the amino acid sequence obtained wiU be the result of the summation of the peptide mixture. Uuless a predomiuaut, distinct motif and an alignment of one or more of the critical residues exists within the peptide sequence of the library (e.g., with a fixed residue at a specific position), the result could be very difficult if not impossible to interpret. 

The term pool sequencing refers to the multiple sequence analysis through Edman degradation of peptide libraries composed of highly complex nnixtures. This analytical technique is valuable for the characterization of peptide hbraries in that it enables the qualitative and quantitative determination of each annino acid at each position of the sequence, thus, not only verifying equimolar representation of annino acids at each position, but also reconfirming completeness of couphng at each step. 

The presence of proline in positions 7, 10, and 13 of a preparation of AFGP-8 was confirmed by Edman degradation (Morris et al., 1978), but no sequences with prolines in all of the 7, 10, and 13 positions were found by mass spectrometry. Preparations of AFGP-8 from T. borchgrevinki consist almost entirely of a mixture with the following three sequences in the approximate proportions indicated (Morris et al, 1978) ... 

The presence of an /V-alkylatcd terminal amino-acid residue (/V,TV-dimethyl-valine), deduced from the mass spectrum, means that chemical sequencing (Edman degradation) is ruled out. The sequence followed from the interpretation of the mass spectrum. The usual site of ionisation, the C-terminal carbonyl group, was the starting point for initial speculation on interpretation of the mass spectrum to solve the sequence. The positions of valine, /V-methylvaline, proline, hydroxyvaline (i.e. 2-... 

Fig. 1. Amino acid sequence of GMF-beta. The sequence was established for bovine brain GMF-beta by automated Edman degradation (microsequencing) and tandem mass spectrometry. Identical sequence was obtained for recombinant hGMF-beta, which was deduced from nucleotide sequence of the cDNA and verified by microsequencing of the first ten NH2-terminal residues and by carboxylpeptidase de adation of the first four COOH-terminal residues. The one-letter abbreviations for the amino acids are A, Ala C, Cys D, Asp E, Glu F, Phe G, Gly H, His I, He K, Lys L, Leu M, Met N, Asn P, Pro Q, Gin R, Arg S, oer T, Thr V, Val W, Trp and Y, Tyr. The three cysteine residues (at positions 7, 86 and 95) are underlined. (Adapted from ref. 13)...

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