Edman degradation sequences

Peptide-resin assembly was performed by Fmoc solid-phase methodology. All peptide-amphiphiles were synthesized as C-terminal amides to prevent piperazine-2,5-dione formation. 62 Peptide-resins were characterized by Edman degradation sequence analysis as described previously for embedded (non-covalent) sequencing. 67 Peptide-resins were then lipidated with the appropriate (Cn)2-Glu-C2-OH tail to give the dialkyl peptide-amphiphile-resin. 

Peptide 35 was synthesized using Fmoc chemistry with Rink amide resin. Deprotection and cleavage were performed by treatment with TFA/EDT/anisole (95 1.25 3.75) for 1.5 h, and the peptide was precipitated with the addition of ice-cold EtjO. The precipitate was dissolved in aq AcOH and purified by preparative RP-HPLC using 0.1% TFA in a H20/MeCN gradient. The purified peptide sequence was confirmed by Edman degradation sequence analysis. FAB-MS analysis gave [M + H]+ 2331.2 Da (calcd 2331.1 Da). 

Edman degradation sequence analysis of selected samples (dissolved in 0.1% TFA-20% acetonitrile) was performed on an Applied Biosystems 477A Protein Sequencer/120A Analyzer using BioBrene Plus as described (7). In order to identify deprotection products and deletions, 800-900 pmol of sample was sequenced. 

Modified or unusual amino acids such as hydroxyproline and phos-phoserine and protected amino acids can also be identified by Edman degradation sequence analysis [38], However, it is important to remember that in this method, identification of a residue is made solely on the basis of HPLC retention time. It is, therefore, essential to support the identification of a modified residue by comparison with authentic standards. Furthermore,... 

Figure 7 Edman degradation sequence analysis, (a) Chromatograms for each sequence analysis cycle (b) [see p. 786] relative yield per cycle. The analysis was performed on a PE Biosystems cLC Precise 490 sequencer. Two microliters of an approximately 2 nM solution of the peptide in 0.2% TEA was applied to a PVDF membrane (0.4 X 0.4 mm) and allowed to air dry. Biobrene-T (2.5 /xL of a solution containing Biobrene-T/methanol/0.2% TEA, 1 7 2, v/v/v Biobrene-T from PE Biosystems) was then added on top of the dried peptide. Sequence anMysis of the dry sample on the membrane was accomplished by means of instrument protocols provided by the manufacturer. A standard mixture containing 19 PTH-amino acids (all of the common amino acids except Cys) was utilized to determine the HPLC retention times for each derivatized residue.

In summary, Edman degradation sequence analysis of synthetic peptides can be very useful for identifying problems that occur during synthesis. However, because of time requirements in addition to the expense of the analysis, this method is not practical for routine use. As described earlier, the utilization of HPLC combined with mass spectrometry is more suitable for routine assessment of peptide purity and quality. 

Thermooptical detection has been combined with CE and used for protein and peptide Edman degradation sequencing detection, native protein detection, as well as the analysis of derivatized amino acid mixtures. " Frequency-doubled argon-ion lasers have been employed to supply an UV (257 nm) pump beam for the analysis of dansylated amino acids as well as the analysis of etopside and etopside phosphate in human blood plasma. In addition, two-color thermooptical absorbance detectors have been constructed, where each laser serves a dual role of pump and probe this system can be used to detect analytes that absorb in differing regions of the electromagnetic spectrum. 

The N-terminal sequence containing 12 amino acids of the purified CNBE from lipase were determined by Edman degradation sequencing.and N-terminal sequence of CNBE was tested as Ala - Leu - Arg - Leu - Asn - Ser - Pro - Asn - Asn - lie - Ala - Val.which was... 

Only the N terminal amide bond is broken m the Edman degradation the rest of the peptide chain remains intact It can be isolated and subjected to a second Edman procedure to determine its new N terminus We can proceed along a peptide chain by beginning with the N terminus and determining each ammo acid m order The sequence is given directly by the structure of the PTH derivative formed m each successive degradation... 

Modem methods of peptide sequencing follow a strategy similar to that used to sequence insulin but are automated and can be carried out on a small scale A key feature is repetitive N terminal identification using the Edman degradation... 

Somatostatin is a tetradecapeptide of the hypothalamus that inhibits the release of pituitary growth hormone Its ammo acid sequence has been determined by a combination of Edman degradations and enzymic hydrolysis expenments On the basis of the following data deduce the pnmary structure of somatostatin... 

With the identities and amounts of amino acids known, the peptide is sequenced to find out in what order the amino acids are linked together. Much peptide sequencing is now done by mass spectrometry, using either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) linked to a time-of-flight (TOF) mass analyzer, as described in Section 12.4. Also in common use is a chemical method of peptide sequencing called the Edman degradation. 

The general idea of peptide sequencing by Edman degradation is to cleave one amino acid at a time from an end of the peptide chain. That terminal amino acid is then separated and identified, and the cleavage reactions are repeated on the chain-shortened peptide until the entire peptide sequence is known. Automated protein sequencers are available that allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by products interferes with the results. So efficient are these instruments that sequence information can be obtained from as little as 1 to 5 picomoles of sample—less than 0.1 /xg. 

Edman degradation (Section 26.6) A method for N-terminal sequencing of peptide chains by treatment with Af-phenylisothiocyanate. 

Amino acid sequencing may be carried out in a number of ways. The most widely used is the Edman degradation procedure in which phenylisothiocyanate is used to react with the amino acid residue at the amine end of the protein chain. This derivatized residue is removed from the remainder of the protein and converted to a phenylhydantoin derivative which is identified by using, for example, HPLC. 

Enzymes are now being used in conjunction with LC-MS to provide sequence information as an alternative to the Edman degradation procedure. 

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