Edman degradation, sequencing peptides with

As everybody knows, a sequencing machine digests the amino acid chain starting at the N-terminus and it identifies the amino acid derivatives via a connected HPLC. A requirement is a free N-terminus. The Pierce Catalog, for example, describes the chemistry of the Edman degradation of peptides with phenylisothiocyanate. Baumann (1990) compares the effectiveness of different sequencing techniques. 

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). 

The most efficient method for sequencing peptides is the Edman degradation. A peptide is treated with phenyl isothiocyanate, followed by acid hydrolysis. The products are the shortened peptide chain and a heterocyclic derivative of the N-terminal amino acid called a phenylthiohydantoin. 

Hundreds of proteins have been sequenced by Edman degradation of peptides derived from specific cleavages. Nevertheless, heroic effort is required to elucidate the sequence of large proteins, those with more than 1000 residues. 

Determining the structure of a peptide or protein is carried out in several steps. The identity and amount of each amino acid present in a peptide is determined by amino acid analysis. The peptide is hydrolyzed to its constituent a-amino acids, which are then separated and identified. Next, the peptide is sequenced. Edman degradation by treatment with phenyl isothiocyanate (PITC) cleaves one residue from the N terminus of the peptide and forma an easily identifiable phenylthvobydantoin (PTH) derivative of the N-terminal amino acid. A series of sequential Edman degradations allows the sequencing of a peptide chain up to 50 residues in length. 

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 remaining peptide has another amino unit, and it is treated with more phenyl isothiocyanate and then cleaved to examine and identify the next amino acid. If a pentapeptide (ala-val-ser-leu-ile) is subjected to the sequential Edman degradations by treatment with Edman s reagent and loss of the phenylthiohydantoin, each amino acid can be identified, in order. Because the process begins at the N-terminus and progresses toward the C-terminus, both the identity of the amino acids and their exact sequence in the peptide are known. This method can be used to identify from 30 to 60 amino acid residues in a long peptide under the right conditions. 

DNS-Edman refers to the danysl Edman technique and sub-Edman to the subtractive Edman approach to peptide sequencing. Non-automated Edman degradation of peptides coupled with PTH identification is referred to as manual Edman. L/P and S/P refer to liquid pha.se and solid phase automatic sequencing, respectively. 

The Edman degradation involves removing one amino acid residue at a time and identifying each residue as it is removed. The process is repeated until the entire peptide has been sequenced. To perform an Edman degradation, a peptide is first treated with phenyl isothiocyanate followed by trifluoroacetic acid. These two reagents remove the amino acid residue at the N terminus and convert it into a phenylthiohydantoin (PTH) derivative. 

The classical method is the Edman degradation reaction. It involves stepwise degradation of peptides with phenylisothiocyanate (cf. 1.2.4.2.3) or suitable derivatives, e. g. dimethylaminoa-zobenzene isothiocyanate (DABITC). The resultant phenylthiohydantoin is either identified directly or the amino acid is recovered. The stepwise reactions are performed in solution or on peptide bound to a carrier, i.e. to a sohd phase. Both approaches have been automated ( sequencer ). Carriers used include resins containing amino groups (e. g. amino polystyrene) or glass beads treated with amino alkylsiloxane ... 

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... 

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. 

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