There are various methods through which sequence of protein can be determined. When we talk about finding the sequence of protein we salute Bioinformatics. Bioinformatics has provided us various tools which make our work easier. Method for the determination of protein sequence is:
Complete Hydrolysis Process
a) Identification of Protein
First we have to identify a protein for which we want to determine a sequence. Proteins are identified by using different techniques, we can identify them through antibodies (every protein has different antibodies. Specific antibody will bind with the specific protein), Through Affinity Chromatography (a selective technique, specific to study function and interaction of protein in which binding affinity is exploited) and Through Antibody Array (Binding takes place between paratope of antibody and epitope of antigen).
b) Determination of Protein by Complete Hydrolysis
After identifying protein of interest, we have to determine its sequence so that structure and function of protein can be identified.
First step to determine protein sequence is:
• Heat protein at 100-110°C with concentrated HCL.
• Now boil it for 24-72 hours.
Advantage: peptide bonds between amino acids are broken down through this method.
• Degradation chances of tyrosine, tryptophan, glutamine, and cysteine.
• To avoid this degradation Thiol Reagent is used.
• We can also use phenol with concentrated HCL.
c) We will get only amino acids.
d) Now we will place amino acids on a HPLC (High Pressure Liquid Chromatography) column, where we apply high pressure by pump. In HPLC beads are finely divided, pores are small. It has maximum interaction sites. Resolution becomes high in HPLC.
e) One by one amino acids elute out, polar amino acids will elute out first and basic amino acids will elute out after.
f) When amino acids elute out we will put laser beam over it.
g) Graph is generated according to absorption.
h) Graph tells which amino acid is present in what amount. Large peak will show amino acid is in large amount.
i) But still we do not get the sequence.
j) To determine amino acid sequence we will use Bioinformatics tool.
AAcompIdent (Amino Acid Composition Identification) Tool
It is an algorithm which attempts to predict protein sequences on the basis of amino acid composition by searching protein sequence databases for entries that would give a similar composition profile.
k) We can label proteins, if amino acid concentration amount is less than 10 pico mol (pmol) we will use fluorescamine.
l) If amino acid concentration amount is greater than 10 pmol we will use ninhydrin.
Disadvantage of Complete Hydrolysis method
Through complete hydrolysis we cannot determine de-novo sequences, because there is no entry in databases.
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