The Proteomic Workflow, continued

Once proteins are separated according to their isolectric point we apply the IPG strips to the second dimension, which separates proteins according to their molecular mass.  The strip is placed on top of a large-format so-called SDS-PAGE (sodium dodecyl sulfate – poly acrylamide gel electrophoresis).  SDS is a negatively charged detergent that binds to proteins proportional to their mass and thus makes it possible to separate proteins when they migrate through an electric field (and an acrylamide gel with a porous matrix).  The result is a two-dimensional map of protein spots (Fig. 2).

Figure 2: First dimension separation is followed by second dimension (SDS-PAGE), which separates proteins according to their molecular mass.

The next task is to identify the proteins on the gel, especially those that changed their levels of expression.  In order to accomplish this we employ mass spectrometry.  By measuring the mass of protein fragments (so-called peptides) that we obtained through digestion with other proteins (proteases), we can create a unique fingerprint of peptide masses that identifies a protein.  In order to do this we require a state-of-the-art mass spectrometer (matrix assisted laser desorption/ionization or MALDI – tandem time-of-flight mass spectrometer; Fig. 3).

Figure 3: MALDI- TOF-TOF mass spectrometer by Bruker Daltonics (Ultraflex II). Lab Projects