INF380 – Proteomics Chapter 2 – Protein separation

1. INF380 – Proteomics Chapter 2 – Protein separation • Common to most of the separation method is that the proteins or peptides mixture is migrated through a substance. • This substance is chosen to influence the speed of a molecule's migration depending on some property of the molecule (for example size) and the molecules are therefore separated over time based onthis property. • Most of the different separation methods can thus briefly be described by • the substance through which the molecules migrate • the external force that causes the molecules to migrate • the preprocessing of the molecules, making them able to migrate through the substance • The most commonly used technique for protein separation is gel electrophoresis INF380 - Proteomics-2

2. Separation on molecular mass - SDS-PAGE • SDS-PAGE is a technique for separation on molecular size,since the speed of migration is inversely proportional to the size of the molecule. • However, size is usually considered to be sufficiently proportional to mass. • The substance used in this separation technique is polyacrylamide. • When this polymer is formed under the right circumstances, it turns into a porous gel with a lot of small holes or tunnels. • It is this network of tunnels that impedes the transit of large molecules while small molecules move much more readily through them. • The force applied to move the molecules through the gel is supplied by an electric field. • PAGE thus stands for PolyAcrylamide Gel Electrophoresis INF380 - Proteomics-2

3. Separation on molecular mass - SDS-PAGE • SDS-PAGE: Sodium Dodecyl Sulfate INF380 - Proteomics-2

4. Separation on molecular mass - SDS-PAGE INF380 - Proteomics-2

5. Separation on isoelectric point - IEF • Separation on isoelectric point is performed by IsoElectric Focusing • A pH gradient is used for the separation. • The pH gradient is built from certain polymerized chemicals (acrylamide) that are arranged in (continuously) increasing pH values. • The proteins are entered into the gradient by absorption from a buffer with the sample, often strengthened by applying an electric current. • After sample transfer, an electric field is applied across the pH gradient. • The proteins will move in this electric field, initially towards the electrode with the opposite charge. • When the protein reaches the point in the pH gradient that is equal to its pI, the net charge will become zero, and the migration will end. INF380 - Proteomics-2

6. Separation on isoelectric point - IEF INF380 - Proteomics-2

7. Separation on mass and isoelectric point, 2D SDS-PAGE • The SDS-PAGE- or IEF-separation methods alone do not have sufficient separating power for protein samples containing large number of proteins. • However, because these two methods are orthogonal, they can be combined in a method called Two-Dimensional SDS-PAGE (2D SDS-PAGE). • The proteins are separated on pI in the first dimension, and on mass in the other. • This method, first published in 1975, remains the most widely used protein separation technique today. INF380 - Proteomics-2

8. Separation on mass and isoelectric point, 2D SDS-PAGE INF380 - Proteomics-2

9. Problems with 2D SDS-PAGE • Not all proteins will appear on the gel. These are typically: • hydrophobic proteins, with GRAVY values above 0.4 • proteins with an isoelectric point below 3 or above 10 • proteins with a mass below 8 kDa, or above 150-200 kDa • proteins of low abundance • The gels may contain contaminant components. • Protein complexes may stay intact and will therefore behave as a single, very large molecule. • Difficult to observe membrane proteins, which are usually quite hydrophobic • Mediocre reproducibility. It is difficult to obtain the same patterns for repeated experiments. • The relative positions of the spots may also vary inside one gel (local variations). This means that an equal mass difference between proteins may be observed as slightly different spot distances on different areas of the gel. INF380 - Proteomics-2