Chapter 2 4 protein analysis

Why do we analyse proteins?• Explore how amino acid sequences specify the structure of proteins • How proteins bind to substrates and other molecules to perform their functions.. Prote

Protein analysis

Progress in science depends on new techniques, new discoveries and new ideas, probably in that order.

Sydney Brenner

Tools for protein analysis

Why do we analyse proteins?

• Explore how amino acid sequences specify the

structure of proteins

• How proteins bind to substrates and other

molecules to perform their functions.

• Understand the function of the protein based on its structure.

Main Steps in Protein Analysis

1 Extraction of proteins.

2 Purification of the protein.

3 Structural Characterization of the protein

Protein Characterization

• Characterization of proteins and peptides: 3

different processes:

1 Determining the Amino Acid Composition

• finding out the amino acids that make up the protein and their

number.

2 Determining the Amino Acid Sequence

• finding out the sequence of amino acids of the proteins in their

order.

3 Determining the Molecular mass of the Protein

Determination of Amino Acid Composition

• peptide  hydrolyzed  amino acids by heating in 6M HCl at 110ºC

for 24 hrs

• The amino acids  separated by HPLC

– measured by reacting them with ninhydrin Alpha Amino acids 

intense blue color , imino acids (proline, hydroproline)  yellow color

– The concentration of a single amino acid is proportional to the light absorbance of the solution after adding ninhydrin

– The elution profile of the amino acids is obtained

– The identity of the amino acid is revealed by its elution volume

Determination of protein Molecular Mass by Mass Spectrometry

• Mass Spectrometry: include an ionization source, a mass

analyzer and a detector The ion hits the mass analyzer The time the ions take in flight is recorded by triggering a laser

pulse

• It depends on one principle

– The smaller ions will fly faster and hit the detector first

then the larger ions reach The ions hit the detector in order by mass

Chromatography for protein purification

Chromatography-3 matrices

Protein purification by chromatography

Genetically-engineered tags provide an easy way to purify proteins

- antigenic determinant, or epitope, recognized

by a specific antibody

- short string of histidines (His-tag) binds to an affinity column containing immobilized nickel ions

- Glutathione S-transferase (GST) fusion protein binds to an affinity column containing

glutathione

Purification of protein complexes by using a GST-tagged fusion protein

Protein analysis

 SDS PAGE ( sodium dodecyl sulfate polyacrylamide-gel electrophoresis

 Protein separation

SDS PAGE

Separation of proteins by isoelectric point

Specific proteins can be detected by Western blotting

The diffraction of X-ray by protein crystals can reveal a protein’s exact structure

Determing Primary Structure of an Entire

Protein