Edman chemistry sequencing

In the case of E. coli leader peptidase, 45 brightly fluorescent beads were isolated and analyzed by Edman chemistry sequencing (Fig. 3). 

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. 

Enzymatic cleavage is often requested when sequence information is too limited for cDNA cloning experiments (if this strategy is appropriate to the animal model), database search or when no N-terminal sequence is obtained by Edman chemistry, and for cysteine pairing elucidation. 

Increase in sensitivity and efficiency of analysis in structural studies of enzymes with a gas phase sequencer have made it possible to determine the primary structure in a shorter period of time with a small amount of enzyme at the picomole or even femtomole level. In addition, thanks to the DNA sequencing technique the number of enzymes (or proteins) whose amino acid sequences are registered in a data base file has expanded explosively. The introduction of mass spectrometry on the primary structure determination of protein has stimulated the search for a new methodology other than Edman chemistry. 

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). 

For more than 25 years, automated Edman chemistry [1,2] has remained the favoured method for routine protein sequence analysis. Several limitations, however, have never been overcome. The procedure is inherently slow and does not allow direct identification of many post-translational modifications. In addition, current detection limits are only at the level of hundreds of femtomoles [3]. Large-format 2D-electrophoresis systems now make it possible to resolve several thousand proteins from whole-cell lysates in the low- to upper-femtomole concentration range [4,5] and more versatile and sensitive methods of protein sequencing are needed to meet analytical problems of this scale. 

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. 

For staining the blotted membrane, a variety of stains including Amido Black and Ponceau S are compatible with Edman chemistry. However, without a special reason for using another stain, Coomassie R-250 is recommended, because advice on whether sufhcient material is likely to be present is best based on a common stain. Because most blots submitted to sequence laboratories are stained with Coomassie, sequencing personnel are quite accustomed to looking at Coomassie-stained membranes and they have a feel for the amount of material present based on stain intensity. It does not help to use a dye with a sensitivity higher than that of the laboratory sequencing facility to which you submit your sample. 

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. 

The protein transfer from the gel to the blot should be as efficient as possible in order for the protein on the blot to be easily identifiable. The blot membrane should be stable against the reagents used for sequencing. Nitrocellulose and nylon membranes do not withstand the solvents of the Edman chemistry. The remaining choice for the experimenter is between coated glass fiber and PVDF membranes. 

This article has summarized the most significant developments in the field of automated protein sequencing since the introduction of the Edman chemistry. It is not unreasonable to expect that the next major advance in protein sequencing by the end of the 1990s and into the twenty-first century will be based on the routine use of mass spectrometry, giving... 

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