Protein sequencing

Introduction:Protein: polymer of amino acid protein’s st. and function depends

upon the amino acid sequence.

Protein Sequencing:technique to find out the amino

acid sequence in protein Imp for understanding cellular

process Imp in targetting drugs to

specific metabolic pathways.

SEQUENCING METHODS

1-N-terminal sequencing

2-C-terminal sequencing

3-Prediction from DNA sequence

The N-terminal sequencing is done through

1. Sanger’s method

2. Dansyl chloride method

3. Edman’s degradation method

N-TERMINAL SEQUENCING:

Sanger’s method

• Treat with DNFB to form a derivative of the amino-terminal amino acid

• Acid hydrolysis

• Extraction of DNP-derivative with organic solvent

• Identification of DNP-derivative by chromatography and comparison with standards

• Forms a highly fluorescent derivative of the amino-terminal amino acid

• Identified by chromatography & fluorescence detection after acid hydrolysis.

• Highly sensitive.

• Best for small amounts.

Dansyl chloridemethod

Edman’s degradation method

Used to sequence the peptides

It removes one amino acid from the N-terminal end of the peptide under ideal conditions the sequence of 30-60 amino acids can be determined

• Protein purification

• Protein denaturation

• Protein digestion

• N-terminal labeling

• Separation by chromatography

• Detection by mass spectrometry

• Data analysis

Steps

Protein purification

Source Material

Homogenization &Extraction

Precipitation

Chromatography

Concentration

Purity

C-TERMINAL SEQUENCING• Add carboxypeptidases to a solution of the protein

• Take samples at regular intervals

• Determine the terminal amino acid by analyzing a plot of amino acid concentrations against time.

From DNA sequencing

Protein sequence can also be determined indirectly from the mRNA

Design primers from the amino acid sequence and amplify the gene

Sequence the gene and determine the amino acid sequence of protein

Peptide Mass Fingerprinting (PMF)• Proteolytic enzyme digest protein

• 2-D gel electrophoresis is used forprotein separation.

• Separated spots are then identified by PMF.

• Trypsin cleaves lysine and arginine sites.

• After digestion a set of diff masses of peptides. Mass of each peptide = amino acids present Search for peptide seq.

Mass spectrometry

Recombinant protein synthesis

Drugs production

Functional genomics

Determine the protein folding patterns

Applications of Protein Sequencing

THAT’s

ALL.