Experiment 5

Protein Sequencing
I. Objectives:
At the end of the lab simulation video, the students will be able to:
1. determine the protein sequence by Edman degradation method

II. Materials
PITC (Edman’s reagent), Peptide sample, 6M HCl, ninhydrin or fluorescamine

III. Apparatus
Chromatography, mass spectroscopy

Introduction
Proteins are the most abundant and functionally diverse molecules in living organisms.
Proteins are macromolecules consisting of one or more polypeptides. Each polypeptide consists of a
sequence of amino acids linked by peptide bonds. Polypeptide folds up to different structures with unique
three-dimensional conformation.
Proteins can be in different forms and possess different functions.
For examples:
1. Enzymes - catalyze biochemical reactions. e.g. trypsin, subtilisin
2. Transport proteins - e.g Hemoglobin
3. Contractile or motile proteins - e.g. actin and myosin in skeletal muscle cells
4. Structural proteins - to give strength and protection to biological structures. e.g. tendon, cartilage,
leather, ligament, hair, fingernail, spider web.
5. Defense proteins - e.g antibodies
6. Regulatory proteins - e.g. hormones, transcription factors
Amino acid sequence of a protein is rich in information. Mutation will result in malfunctioning of the protein
or disease. Knowledge of a sequence provides valuable clues to its evolutionary relationship with other
proteins, conformation and function

Principle:

In 1950 Pehr Edman developed a method of protein sequencing. It involves sequential identification of
amino acids from N to C terminal. Phenyl isothyocynate (PITC) reagent is used for the Edman degradation.
The amino terminal of a protein can be identified by reacting the protein with PITC that forms a stable
covalent linkage with the free amino group prior to hydrolysis with 6M HCl
Mechanism of protein sequencing

A. Proteins Are Built from a Repertoire of 20 Amino Acids

Amino acids are the building block of proteins. An amino acid consists of a central carbon atom, called α-
carbon, linked to an amino group, a carboxylic acid group, a hydrogen atom, and a distinctive R group. The
R group is often referred as the side chain.
                                                                       

Figure 1. Amino Acid Structure

Figure 2 Polypeptide Chain

B. Amino Acid Sequence Can Be Determined by Automated Edman Degradation

The amino acid composition of a protein can be ascertained by hydrolyzing it into its constituent amino
acids in 6 M HCl at 110℃. The amino acids can be separated by ion-exchange chromatography and
quantitated by reacting them with ninhydrin or fluorescamine. Amino acid sequences can be determined by
Edman degradation, which removes one amino acid at a time from the amino end of a peptide. Phenyl
isothiocyanate reacts with the terminal amino group to form a phenylthiocarbamoyl derivative, which
cyclizes under mildly acidic conditions to give a phenylthiohydantoin-amino acid and a peptide shortened
by one residue. Automated repeated Edman degradation by a sequencer can analyze a peptide sequence
of about 50 residues.
Longer polypeptide chains are broken into shorter ones for analysis by specifically cleaving them with a
reagent such as cyanogen bromide, which splits peptide bonds on the carboxyl side of methionine
residues.
Trypsin, which cleaves on the carboxyl side of lysine and arginine residues (if the next residue is not
proline), is also useful in splitting proteins.

IV. Procedure
Kindly visit this site to start your journey http://www.bch.cuhk.edu.hk/vlab2/animation/protein/index.html
Table 1. PTH amino acid produced
Peptide Sample PTH amino acid produced
A Phe
B Arg
C Gly

Guide Questions:
1. How do you sequence Edman Degradation?
In sequencing the amino acid sing Edman Degradation method is by removing one amino acid at a
time from the amino end of a peptide.
2. What are the three amino acids produced in the degradation of the peptide sample?
The three amino acids that produced in degradation of the peptide sample was Phe
(phenylalanine), Arg (arginine), Gly (glycine).
3. Describe the “Release” process in protein sequencing.
The release process of protein sequencing is where by cleaving the first peptide bond without
breaking any other peptide bond also known as a mildly acidic condition. The cyclic compound is
phenylthiohydantoin (PTH) amino acid.
4. What happen during the “Labelling” round?
In labelling round, phenyl isothiocyanate was being used in order to label the amino acid. If this
reagent will react and label the amino acid at the N-terminus of the peptide to form a
phenylthiocarbamoyl derivative. We should label the amino acid because in the second step were
going to cleave the peptide bond that is holding the amino acid, by cleaving it were going to
separate from the rest of the polypeptide.
5. What is the protein sequence produced in the experiment?
The sequence produced in this experiment was
Phe -Arg-Gly
 V. Conclusion
Proteins are polypeptide structures consisting of one or more long chains of amino acid
residues. Protein sequencing was created to determine the amino acid sequence of a protein.
In Edman's Degradation, there are first and second steps. So, the first step is labelling. The
point of this step is to label the first amino acid in the protein with the used
phenylisothiocyanate to form a phenylthiocarbamoyl derivative. In the second step, the first
peptide bond cleaves without breaking any other peptide bonds. The separated amino acid will
be determined by the used chromatography technique. The cyclic compound is
phenylthiohydantoin (PTH) amino acid.  In this experiment, it was concluded that Edman
Degradation is very useful because it gives us a systematic approach to basically cleaving that
first peptide and no other peptide bonds are cleaved within that polypeptide chain and also
determines exactly the correct sequencing. The process of this experiment is specifically
repeated until we get everyone the amino acid. The first amino was phenylalanine, the second
was arginine, and the last was glycine.
VI. References:
 Protein Sequencing. (n.d.). School of Life Sciences. The Chinese University of
Hongkong Retrieved May 15, 2022, from
http://www.bch.cuhk.edu.hk/vlab2/animation/protein/index.html

 26.7: The Edman Degradation. (2015, August 26). Chemistry LibreTexts. Retrieved

May 20, 2022 from

https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_(McM

urry)/26%3A_Biomolecules_Amino_Acids_Peptides_and_Proteins/

26.07%3A_The_Edman_Degradation
 Protein Sequencing of Edman Degradation – Creative Proteomics Blog. (2018,

January 25). Creative-Proteomics.com. Retrieved May 20,2022 from

https://www.creative-proteomics.com/blog/index.php/protein-sequencing-of-edman-

degradation/