Steps in Proteomic Analysis

The following steps are involved in analysis of proteome of an organism as shown in
Fig. 3:

1.  Purification of proteins: This step involves extraction of protein samples from whole cell, tissue or sub cellular organelles followed by purification using density gradient centrifugation, chromatographic techniques (exclusion, affinity etc.)

2.  Separation of proteins: 2D gel electrophoresis is applied for separation of proteins on the basis of their isoelectric points in one dimension and molecular weight on the other. Spots are detected using fluorescent dyes or radioactive probes.

3.  Identification of proteins: The separated protein spots on gel are excised and digested in gel by a protease (e.g. trypsin). The eluted peptides are identified using mass spectrometry.

Analysis of protein molecules is usually carried out by MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) based peptide mass fingerprinting. Determined amino acid sequence is finally compared with available database to validate the proteins.

Several online tools are available for proteomic analysis such as Mascot, Aldente, Popitam, Quickmod, Peptide cutter etc.



Figure 3. Overview of steps involved in proteomic analysis