Question

Please explain the process to determine unknown protein in blood stream.

The question should be answered wrt Biomolecular analytical methods on system-level.

Answer 1

Proteins, also known as polypeptides, are organic compounds made up of amino acids. They’re large, complex molecules that play many critical roles in the body.Proteins are made up of hundreds of thousands of smaller units that are arranged in a linear chain and folded into a globular form. There are 20 different types of amino acids that can be combined to make a protein and the sequence of amino acids determines each protein’s unique 3-dimensional structure and its specific function.

The 3 major protein analysis methods are :-
* Protein separation :- is the process of separating or purifying proteins by placing them in a gel matrix and then observing protein mobility in the presence of an electrical field. It’s an important approach to studying protein function and the effect of a particular protein on development or a physical function by introducing it into an organism.
The most commonly used technique for protein separation is sodium dodecyl sulfate polyacrylamide gel electrophoresis Proteins can be separated according to solubility, size, charge and binding affinity. SDS-PAGE separates proteins mainly on the basis of molecular weight as opposed to charge or folding. It’s a technique that’s widely used in biochemistry, forensics, genetics and molecular biology.
* Western blotting :-

The western blot technique uses three elements to identify specific proteins from a complex mixture of proteins extracted from cells: separation by size, transfer to a solid support, and marking target protein using a proper primary and secondary antibody to visualise.The most common version of this method is immuno blotting This technique is used to detect specific proteins in a given sample of tissue homogenate or extract. The sample of proteins is first electrophoresed by SDS-PAGE to separate the proteins based on molecular weight. The proteins are then transferred to a membrane where they are probed using antibodies specific to the target protein.
*protein identification :- There are two methods that are commonly used to identify proteins: Edman Degradation and Mass Spectrometry.
Edman degradation is a method of sequencing amino acids in a peptide. Here, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues.

Protein mass spectrometry :- is an analytical technique that measures the mass-to-charge ratio of charged particles for determining masses of particles and the elemental composition of a sample of molecules as well as for elucidating the chemical structure of molecules such as peptides. Protein mass spectrometry is an important method for the accurate mass determination and characterisation of proteins, and a variety of methods and instrumentations have been developed for its many uses.