Analysis of Protein Spots Derived from 2D Gels.

After the staining procedure is completed, the individual proteins are visible as discrete spots in the polyacrylamide gel. The number of spots depends on the diversity of proteins that are found in that particular cell or tissue.  In whole cell preparations, it is common to observe thousands of protein spots, and a major technical challenge is the separation of such a rich mixture of proteins using the 2D gel electrophoresis.  Other types of sample may have fewer protein spots, which will simplify the separation and characterization.  A major problem associated with a high density of protein spots is that of cross contamination.  A protein spot partially superimposed on another will result in the  cross contamination of the polypeptides between one region and another on the gel. 

Well separated protein spots may be physically cut out from the gels, and prepared for further analysis. The first step is to dry the gel slice using lyophilization, after which the sample is rehydrated using a solution containing the proteolytic enzyme trypsin. Trypsin hydrolyses polypeptide chains specifically at lysine or argentine residues, and will cleave the protein in the spot into a number of smaller, defined peptide fragments. These fragments are eluted from the gel, and concentrated by micro scale reverse-phase cromatograhpy. Although the resulting sample will contain a mixture of defined peptides derived from a single protein spot, the individual masses of each peptide in this mixture may be found using mass spectrometry.