Edman degradation solid-phase

Figure 10.15 Solid-phase peptide sequencing procedure based on Edman degradation technique.

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-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. 

Laursen, R. A (1972). Automatic Solid-Phase Edman Degradation. Enzyme Stmcture, Part B. Methods Enzymol 25 344. 

Pisano, A., Redmond, J.W., Williams, K.L. Gooley, A. A. (1993). Glycosylation sites identified by solid-phase Edman degradation O-linked glycosylation motifs on human glycophorin A. Glycobiology 3,429-35. 

Because C-terminal amides have often been reported in insect neuropeptides (8), a 33 residue C-terminal amide consistent with the sequence data was synthesized by solid-phase methods. The peptide was purified by HPLC and its structure confirmed by automated Edman degradation. Californium-252 time-of-flight plasma desorption mass spectrometry provided additional evidence for the structure via a very broad peak for the singly-charged molecular ion at m/z 3902-3906. Because the calculated MW of Hez-PBAN (3899.6, based on the most abundant ion in the isotope cluster) was seen to differ from that observed in the mass spectrum of the isolated native peptide by ca. 32, it was presumed that the native peptide had undergone oxidation of both its methionines to their respective sulfoxides during the course of its isolation and purification. 

Fig. 3. Alignment of N-terminal sequences of Alt a 1. N-terminal peptide sequences of Alt a 1 [48, 50, 51 ] were compared with the complete open reading frame of Alt a 1 [16, 18]. Identical amino acids are marked in grey. The numbering of the amino acids is according to the complete open reading frame. Curran et al. [48] as well as Aden [50] identified 2 different variants with minor differences in the respective N-terminal sequences. Dashes represent gaps in the sequence. Dots indicate that this part of the protein was not sequenced. The first 5 sequences were determined hy solid-phase Edman degradation of nAlt a 1. The last two sequences are deduced from cDNA sequences.

Laursen, R. A., Automatic solid-phase Edman degradation, Meth. Enzymol., 25, 344-359, 1972. 

Truncated sequences, core sequences, incompletely synthesized peptides. Imperfect conversion during acylation or deprotection of the temporary protecting group in solid-phase synthesis may lead to mismatch sequences that lack some amino acids and truncated sequences (core sequences). They occur, when the accessibility or reactivity of the peptide bound to the solid phase is insufficient difficult sequences). Truncated sequences may be classically identified and quantified by a modified Edman degradation, also called preview analysis. Alternatively, mass spectrometry provides efficient tools for the identification of truncated sequences. 

An example of a reaction on a solid-phase matrix, the Merrifield resin, is shown in Fig. 7.14. The peptide fragments are covalently bonded, either onto a poiymer membrane or onto micrometer sized beads. The solid support is then immersed into a liquid phase and Edman degradation is carried out by sequentially adding the required reagents and removing the products for analysis. 

Carbonyldiimidazole was first used for peptide activation in conjunction with the solid-phase Edman degradation because of its reported high yields and the absence of side reactions (Paul and Anderson, 1960). Activation occurs by way of the imidazolide (Fig. 3). Although this reagent does seem... 

In the future, the technique of solid-phase sequencing will be most aided by improved peptide coupling methods and a reduction in the side reactions of the Edman degradation. 

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