Protein sequencing is a technique to determine the amino acid sequence of a protein as well as which conformation the protein adopts and the extent to which it is complexed with any non-peptide molecules. Discovering the structures and functions of proteins in living organisms is an important tool for understanding cellular processes. It is also possible to generate an amino acid sequence from the DNA or mRNA sequence encoding the protein, if this is known. A protein sequencer is a machine that is used to determine the sequence of amino acids in a protein. They work by tagging and removing one amino acid at a time, which is analysed and identified. This is done repetitively for the whole polypeptide, until the whole sequence is established. 

Determination of amino acid sequences : 

Prior to amino acid sequence determination, it is essential to know the protein’s purity, molecular weight and its amino acid composition. A pure protein, if multimeric must be dissociated into its individual polypeptide chains and the molecular weight of the chains is determined. The molecular weight of a protein roughly tells about the number of amino acid residues contained in it. Each amino acid residue has an average molecular weight of 110 D. So if the molecular weight of a protein is 11000 D, we can predict that 100 amino acid residues are present. The amino acid composition tells the protein chemist about the strategies which could be employed in protein sequencing. 

The following protein sequencing techniques are employed : 

The first protein to be sequenced was the hormone ‘insulin’ whose deficiency leads to the disease diabetes. The Nobel Laureate, Fred Sanger invented a method of protein sequencing using a stepwise release and identification of amino acids starting from the N-terminal amino acid. The reagent employed by Sanger, known as fluoro-dinitro-benzene (Sanger’s reagent) reacted specifically with the free NH₂ group of the N-terminal amino acid and on acid hydrolysis, it yielded a yellow colored dinitro phenyl (DNP) derivative of the original N-terminal amino acid. 

The polypeptide was subsequently, shortened by one amino acid. The DNP-amino acid was identified by comparison with other standard DNP amino acids using chromatography techniques. This procedure was repeated on the shortened polypeptide. In order to complete the sequence of insulin, Sanger used more than a gram of the hormone purified from a large number of pancreatic glands. However
Sanger’s method has become obsolete and historical. The following steps summarise the Sanger’s method for determining the amino acid sequence. 

• Hydrolysis by heating a sample of protein in 6M hydrochloric acid. • Separation of polypeptide chains by ion – exchange chromatography. 

• Fragmentation of the polypeptide chain by 

• Enzymatic methods using proteolytic enzyme, highly selective in hydrolyzing peptide bond, 

• Chemical methods using cyanogen bromide (CNBr) cleaves selectively the peptide bond following a methionine residue. 

• Identification of N-terminal amino acid using Sanger’s reagent (fluoro-dinitrogen-benzene). 

• Identification of DNP amino acid by comparison using chromatography by interpreting chromatogram. 

• Repeating the procedure on the shortened polypeptide to identify rest of amino acids one by one. 

Limitation : The method cannot be used in case the sample material is less in amount.