Edman degradation instrumentation

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. 

Automated Edman degradation was performed on. 200 pmol of purified Hez-PBAN using the pulsed-liquid sequencer. Data were obtained from 33 cycles (Table I, run 3). However, the residues at positions 23 and 32 could not be ambiguously assigned. A second attempt on the same instrument using sample purified from the PBAN II zone confirmed the earlier sequence and established the residues at positions 23 and 32 (Table I, run 4). 

Figure 7 Edman degradation sequence analysis, (a) Chromatograms for each sequence analysis cycle (b) [see p. 786] relative yield per cycle. The analysis was performed on a PE Biosystems cLC Precise 490 sequencer. Two microliters of an approximately 2 nM solution of the peptide in 0.2% TEA was applied to a PVDF membrane (0.4 X 0.4 mm) and allowed to air dry. Biobrene-T (2.5 /xL of a solution containing Biobrene-T/methanol/0.2% TEA, 1 7 2, v/v/v Biobrene-T from PE Biosystems) was then added on top of the dried peptide. Sequence anMysis of the dry sample on the membrane was accomplished by means of instrument protocols provided by the manufacturer. A standard mixture containing 19 PTH-amino acids (all of the common amino acids except Cys) was utilized to determine the HPLC retention times for each derivatized residue.

In Steps 3 and 4, the protein is cleaved into smaller fragments, and the amino acid sequence is determined. Automated instruments can perform a stepwise modification starting from the N-terminal end, followed by cleavage of each amino acid in the sequence and the subsequent identification of each modified amino acid as it is removed. This process is called the Edman degradation. 

Preview analysis, an on-resin monitoring of SPPS based on modified Edman degradation. For the analysis of peptide resins only very little sample is required as modern instruments are capable of analyzing 10 to 100 pmol quantities of amino acids [R. A. Laursen, Eur.J. Biochem. 1971, 20, 89 G. W. Tregear et al., Biochemistry 1977, 16, 2817]. 

Further advancement in sequence determination by mass spectra was brought about through the introduction of new modes of ionization, such as field desorption and fast electron bombardment. Double focusing high resolution instruments and sophisticated computation greatly increased the number of peptides that could be sequenced by mass spectroscopy. For compounds which are unsuited for Edman degradation, such as cyclic peptides or peptides with an acyl group at their N-termini mass spectra can turn out to be the most practical avenue to sequence determination. 

Figure 24.4 PTH amino acid standards run on a Procise instrument see Table 24.1 for amino acid abbreviations. Peaks marked dmptu (dimethylphenylthiourea) and dptu (diphenylthiourea) represent side-reaction products of the Edman degradation. Courtesy of Applied Biosystems.)...

An automated instrument known as a sequencer allows about 50 successive Edman degradations of a polypeptide to be performed (100 with more advanced instruments). The entire primary structure cannot be determined in this way, however, because side products accumulate that interfere with the results. 

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