Proteins affinity-based enrichment

A number of affinity-based or chromatography methods have been used to prefractionate protein samples for 2D electrophoresis. For example, proteins of low abundance can be enriched from crude lysates by affinity-... 

Affinity-based proteomics. In affinity-based proteomics, a compound of interest is immobilized to the solid surface by means of functional groups (e.g., NHj) or using the biotin-streptavidin interaction. Cell lysate that contains the target proteins is then incubated with the immobilized compound and this leads to enrichment of proteins on the matrix (1). Stringent washing removes proteins that unspecifically bind to the solid surface. Bound proteins are then released from the matrix by elution (e.g., with an excess of unmodified compound) or by heating... 

Many of the techniques used to enrich PTMs at the protein level are applicable at the peptide level. A popular method for enrichment of phosphopeptides is to use an immobilized metal affinity column. The molecular basis for the enrichment is the phosphate affinity to transition metal ions, such as copper, nickel, cobalt, iron, aluminum, gallium, and zinc (142). A comprehensive source for phosphopeptide enrichment strategies can be found in a recent review (143). Lectin-based chromatography is used to enrich for glycopep-tides (144). Lectins are proteins that have an affinity for carbohydrates. With multiple available enrichment options, choosing the right one for the experiment is important. For determining which proteins have a certain PTM, protein-level enrichment can be performed. However, if a type of PTM is of... 

The equalization approach is based on the opposite strategy, in that only low-abundance proteins are collected and subjected to LC-MS analysis. The method relies on bead-bound combinatorial peptide ligand libraries (10) that enrich low-abundance proteins. This strategy is much cheaper than antibody precipitation, but it has the clear drawback that only proteins that have affinity to one of the millions of beads present in the library used can be equalized for further analysis. 

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