Protein or Peptide Sequencing by Edman Degradation
The following topics will be discussed below:
Edman Sequencing: a well-known technology with ever increasing sensitivity Quality of Fluka Reagents
1. Edman Sequencing: a well-known technology with ever increasing sensitivity 1a. Introduction Due to the progress of instrument manufacturing, the traditional Edman chemistry (principle) has gained extremely high sensitivity, allowing sequencing of proteins in the lower pico- or upper femtomol level. The yield of PTH-amino acid is highly dependant on the quality of solvents and reagents used. Fluka reagents for automated sequencing have proofed to fulfil even highest quality requirements (as shown by some typical chromatograms).
1b. The benefits of Fluka reagents are:
- Highest quality of reagents enables sequencing of proteins at the femtomol level
- Composition matches routine procedures for automated sequencing
- Packaging matches the most commonly used instruments, so that bottles can directly be placed into the instrument itself
- Convenient delivery service, no need for individual stock
Table 1. Special solvents and reagents
| Catalog No. | Product Name | Package Size | | 09653 | TFA | 40 ml | | 91709 | 25% TFA solution | 40 ml | | 00712 | 20% Acetonitrile | 200 ml | | 00682 | Acetonitrile | 40 ml | | 09642 | Heptan | 100 ml | | 09652 | Ethylacetate | 450 ml | | 09651 | Butylchloride | 200 ml | | 78787 | 5 % PITC solution | 40 ml | | 53490 | Methylpiperidine solution, new improved formulation | 40 ml | | 68835 | Methylpiperidine solution, classic formulation | 40 ml | | 72827 | Eluent A for separation of PTH amino acids | 500 ml, 1l, 2.5 l | | 80939 | Eluent B for separation of PTH amino acids | 450 ml | | 85278 | Eluent B for separation of PTH amino acids | 1 l, 2.5 l | | 79923 | Premix for separation of PTH amino acids | 100 ml | | 87859 | Acetonitrile reagent 10% | 200 ml |
Table 2. Sequencing grade endoproteinases
| Catalog No. | Product Name | Package Size | | 45173 | Endoproteinase Arg-C | 100 U | | 45168 | Endorpoteinase Asp-N | 2 µg | | 45169 | Endoproteinase recombinant | 10 mg/50 mg | | 45172 | Endoproteinase Glu-C | 1 mg/5 mg | | 45175 | Endorpoteinase Lys-C | 0.1 mg | | 45167 | Endoproteinase Pro C | 1 mg | | 27207 | Chymotrypsin | 0.1 mg |
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1c. Edman Chemistry The figure 1 below shows a simplified scheme of the Edman chemistry. Coupling of N-terminal amino groups of protein (1) with phenylisothiocyanate (PITC, Edman reagent) (2) forming an open chain thiourea derivative (3), the N-terminal amino acid is split off as anilinothiazolinone (ATZ) derivative (5) with trifluoroacetic acid. This unstable ATZ-derivative is transferred into a more stable phenylthiohydantoin (PTH) derivative (6), which is separated by reversed phase HPLC and identified by comparison with a standard. 
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2. Quality of Fluka reagents 2a. Quality of complete range of reagents The yield of the PTH-amino acid (especially lysine and tryptophane) is highly dependant on the quality of solvents and reagents used. Two sequencing cycles representing the sequence –M-K-of a standard peptide performed with: - a series of improved Fluka reagents specially designed to meet current quality requirements of protein sequencing. These chromatograms are characterized by a very low background and especially a strong signal of lysine (figure 4 (b)).
- using ethyl acetate for protein sequencing offered earlier. Although this ethyl acetate contains DTT as a protecting agent [recognizable by the PITC-DTT adduct peak (*)] the yield of PTH-lysine is significantly reduced.
2b. Quality of acetonitrile used for conversion of PTH-derivatives  | Blank using specified Fluka acetonitril (a) and the highest HPLC gradient quality (b) |
2c. Quality of acetonitrile for preparation of PTH amino acid standard  | The PTH amino acid standard
(a) prepared with specified Fluka acetonitrile, 1 month storage
(b) prepared with HPLC acetonitrile and DDT, following 2 days storage
Quantity of PTH amino acid standard: 4 pmol |
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