Phosphorylation and Phosphoproteomics

This posttranscriptional modification occurs in more than 30% of proteins in mammals. This involves the addition of one or more PO4 groups to particular amino acids in a protein. In mammalian cells, PO4 groups are added to the amino acids threonine, serine, or tyrosine of the protein. In contrast, amino acids such as aspartic, glutamic, and histidine are phosphorylated in prokaryotes, instead of tyrosine, serine, and threonine in eukaryotes. Occasionally in both prokaryotes and eukaryotes, phosphorylation occurs in arginine, lysine, and cystein residues of the protein. The ratio of phosphorylation of the three amino acid residues in mammalian cells is 1000 100 1 for threonine, serine, and tyrosine. Proteins are phosphorylated at more than one site, and usually a mixture of phosphorylated isomers with different levels of phosphorylation exists in the cell. Phosphorylation adds a negative charge to the proteins with the addition of the PC 4 group. 

Phosphorylation is carried by enzyme protein kinases. The phosphorylated proteins may undergo the process of dephosphorylation by removal of phosphate group(s). The dephosphorylation is catalyzed by another enzyme 

Phophoproteomics involves several steps requiring the preparation of sample proteins, their enrichment, and tryptic digestion before analysis by the mass spectrometer. 

The sample proteins are usually prepared by a several methods. Among these methods the following are commonly used phosphoprotein isotope-coded affinity tag (PhlAT), isotope-coded affinity tag, (ICAT) and stable isotope labeling with amino acids in cell culture (SILAC). PhlAT introduces isotopes directly into phosphoserine and phosphothreonine residues of the protein. 

The other two methods introduce isotopes in the protein at sites other than the phosphorylation sites. PhlAT and ICAT are used to label the proteins in vitro, whereas SILAC is used to label proteins in vivo. SILAC is useful for in vivo labeling of the proteins in cell cultures grown under different conditions that may influence the extent of phosphorylatiom. The ICAT and SILAC methods are described in Chapter 3. 

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