Protein Sequencing

Research

Reagents for Edman degradation method

Edman Degradation Method

Pehr Edman developed the Edman degradation method for sequencing
amino acids in 1950. During the reaction phenylisothiocyanate (PITC)
reacts with the amino acid residue at the amino terminus under basic
conditions, to form a phenythiocarbamyl derivative (PTC-protein).
Trifluoracetic acid (TFA) is then used to cleave the first amino acid as its
anilinothialinone derivative (ATZ-amino aid) and leaves the new amino
terminus available for the next degradation cycle.

The ATZ-amino acid is then removed by extraction with ethyl acetate

and converted to a phenythiohydantoin derivative (PTH-amino acid) with
25% TFA. PTH-amino acids are transferred to a reversed-phase C-18
column for detection at 270nm.

A standard mixture of amino acids is separated on the column during

the first run to give standard retention times for different amino
acids. The resulting chromatogram is then used for comparison with
subsequent chromatograms from the sample of interest.

An HPLC chromatogram is produced for each Edman degradation

cycle and are collected using a computer data analysis system. To
determine the amino acid present at a particular residue number, the
chromatogram from the residues of interest is compared with the
chromatogram from the previous residue by overlaying one on top
of the other. From this, the amino acid for the particular residue can
be determined. This process is repeated sequentially to provide the
N-terminal sequence of the protein/peptide.

Typically up to 30 amino acid residues can be determined during one

sequencing run.

Reagents for Protein Sequencing

Wako offer a range of quantity chemicals for protein sequencing - from
enzymes for pre-digestion of samples such as lysyl endopeptidase,
solvents and reagents for Edman degradation including phenyl
isothiocyanate, n-heptane, and PTH columns and standards for
subsequent HPLC analysis.

Shimadzu, an established manufacturer of Automated Protein

Sequencing Analysers, recommends using the Wako reagents with their
Edman sequencers, PSQ-1, PSQ-2 and PPSQ-10.

Also available for these analysers are TFA treated glass fibre filters for
sample immobilisation and a dedicated PTH column (S-PSQ).
 Ordering Information
Cat. No. Description Pack Size

Research
Enzymes
125-02543 Lysyl Endopeptidase 2 AU
129-02541 Lysyl Endopeptidase 10 AU
056-05921 Endoproteinase Asp N 2µg
050-05941 Endoproteinase Glu C 50µg
164-13982 V8 Protease 2mg
Reagents
082-03363 Heptafluorobutyric Acid 5 x 1ml
080-03364 Heptafluorobutyric Acid 50ml
162-08473 Phenyl Isothiocyanate 5 x 1ml
166-08476 Phenyl Isothiocyanate 5 x 1ml
161-08605 1-Propanol 500ml
201-06393 Trifluoroacetic Acid 5 x 1ml
209-06394 Trifluoroacetic Acid 50ml
Shimadzu Reagents
161-27341 5% Phenyl Isothiocyanate n-Heptane 40ml
200-20021 12% Trimethylamine Solution 200ml
207-20031 Trifluoroacetic Acid 40ml
201-10781 25% Trifluoroacetic Acid (in glass) 40ml
204-20041 25% Trifluoroacetic Acid 40ml
084-05501 Heptane 200ml
054-14981 Ethyl Acetate 200ml
033-24371 1-Chlorobutane 200ml
014-12471 Acetonitrile 200ml
168-27351 PTH-amino Acids Mobile Phase (Eluent) 1L
018-26041 37% Acetonitrile Solution 200ml
071-03131 Glass Fibre Disc TFA Treated 12mm 50 sheets
072-06461 Glass Fibre Disc TFA Treated 8mm 50 sheets
Standards
165-27361 PTH-Amino Acids Mixture Standard 2ml x 4
013-08391 Amino Acids Mixture Standard Solution, Type H 5ml
019-08393 Amino Acids Mixture Standard Solution, Type H 5 x 1ml
Columns
235-63951 Wakopak Wakosil-PTH 4.6 x 250mm (S-PSQ) Each

For more information, visit our website at www.alphalabs.co.uk/proteomics

JBN163A 11.21

Tel: +44 (0)23 8048 3000

Email: sales@alphalabs.co.uk
Web: www.alphalabs.co.uk