Microsequencing Edman degradation

Urea should not be used because its effects on amino groups interfere with the Edman degradation in subsequent microsequencing. For similar reasons, microsequencing can rarely be applied to chemical cleavage fragments. 

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. 

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)...

Collect fractions and identify labeled peptide(s) by scintillation counting of 1/10 of each fraction. Monitor OD220 to assess the concentration of peptides in each fraction. As a rule of thumb, 1 nmol of a 10-mer peptide yields an OD220 of about 0.2 Thus, a peak of 0.002 OD contains 10 pmol of peptide, which should be sufficient for microsequencing However, it is advisable to work with OD peaks of about O.l-.Ol for a better sensitivity of the Edman-degradation reaction and detection of the phenylthioazolinone (PTH) denvatives of amino acids Lyophilize the labeled fraction(s) in glass tubes... 

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