Edman chemistry peptide sequencing

Recently, Laursen realized the concept of a novel Peptide-Sequencer based on the chemistry of the Edman degradation, adapted to solid phase chemistry. The peptide under investigation is covalently linked to a resin packed into a column. The reactions are carried out by pumping the reagents and solvents through the column as required. This method appears to be particularly suitable for shorter peptides and may be regarded as an excellent supplement to the Sequencer based on Edman s design. 

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). 

In 1991, we first introduced the one-bead one-compound (OBOC ) combinatorial library method.1 Since then, it has been successfully applied to the identification of ligands for a large number of biological targets.2,3 Using well-established on-bead binding or functional assays, the OBOC method is highly efficient and practical. A random library of millions of beads can be rapidly screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The amount of acceptor needed is minute compared to solution phase assay in microtiter plates. The positive beads with active compounds are easily isolated and subjected to structural determination. For peptides that contain natural amino acids and have a free N-terminus, we routinely use an automatic protein sequencer with Edman chemistry, which converts each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivatives, to determine the structure of peptide on the positive beads. 

The structure of peptides containing 20 eukaryotic natural amino acids is now routinely determined by the use of automatic protein microsequencer, which uses Edman chemistry to convert each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivative. The formed PTH-amino acids can be identified by their retention times on HPLC systems by comparison with reference standards derived from the 20 natural amino acids. For an OBOC peptide library composed of natural amino acids, the sequencing protocols of the automatic sequencer are well developed and standardized. However, structure determination of peptides composed of unnatural a-amino acids requires modification of the standard sequencing program.32 For peptides composed of non-a-amino acids, one can use an encoding strategy or mass spectrometry if a cleavable linker is employed. In this chapter, we shall focus on the new sequencing method we have developed for unnatural a-amino acids. 

The appropriate fraction was collected and rechromatographed on the same column with the same gradient but a different ion-pairing reagent (10 mM triethylamine adjusted to pH 6.5 with trifluoroacetic acid). SchistoFLRFamide was isolated on the basis of cross-reactivity to antibodies raised against the molluscan peptide FMRFamide. Sequence analysis was performed with a pulsed-liquid phase sequencer using Edman chemistry (25). 

Sequence Analysis Low pH elution with 3 N HCl / 50% MeOH / DIW as desorbant maintains compatibility with Edman chemistry. The recovered sample is spotted onto glass fiber filter for sequencing. Higher concentrations of HCl in desorbant and 50% IPA may be required for the recovery of strongly hydrophobic peptides. 

The most popular method is automated Edman chemistry (mn on a sequencer), a method which removes one amino acid at a time from the N-terminus, resulting in the sequential liberation of phenylthiohydantoin (PTH) amino acids, which are identified by on-line HPLC analysis. Edman sequanators are completely automated high-sensitivity instrument systems that can routinely detect and identify as little as 0.2 pmol to 1 pmol of amino acid in a given cycle and carry out more than 20 cycles with 1 to 5 pmol of protein. Most peptides of length 3-30 amino acids can be sequenced completely. Although amino... 

Since the sensitivity of MS methods is similar to that of Edman chemistry, both methods will continue to be used. Because MS methods cannot determine the N- or C-terminal sequences in intact proteins, there will be a continued need for the Edman sequencer. However, MALDI- and ESI-MS methods can give accurate molecular masses for intact proteins, which when compared to their predicted sequences and databases (Eng et al., 1994), can verify a given structure, giving confidence to the N- and C-terminal sequence predicted from the peptide maps. MS-MS provides the fast and sensitive approach to sequence determination of peptides/proteins. 

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