Protein Phosphorylation


 Protein Phosphorylation Background

Protein phosphorylation is an important post-translational modification on proteins, which is responsible for many biological functions. The neutral hydroxyl groups on the protein (serine, threonine and tyrosine) can be changed to a charged phosphate group by the protein phosphorylation process. As a reverse reaction, the phosphoprotein can also be dephosphorylated by protein phosphatases.

Protein phosphorylation plays a key role in many cellular functions including the nervous and immune system and regulation of transcription. Many human diseases, such as cancer, inflammation and diabetes, occur due to the abnormal activity of protein phosphorylation. For example, aberrant protein phosphorylation in lymphocytes of transgenic mice leads to the development of tumors. As a result, a better understanding of protein phosphorylation is essential to the study of diseases and the development of drugs.

 

Monitor protein phosphorylation

Since protein phosphorylation is a key event in influencing protein activity and regulating cell signaling pathway, it is very important to monitor protein phosphorylation to obtain a better understanding of biological events. However, it is still a challenging task to analyze protein phosphorylation. The common analytical techniques include in vivo 32P labeling, gel electrophoresis, mass spectrometric analysis (MS) and immobilized metal affinity chromatography.

Radioactive labeling with 32P labeled ATP is a classic method to identify protein phosphorylation. First, phosphoproteins are radioactively labeled by incubating cells in a γ-32P-ATP environment. The cells are then lysed and phosphoproteins are separated via gel electrophoresis. The 32P labeled phosphoprotein can be visualized with radioactive decay. It is not efficient since the 32P labeled ATP compete with the unlabeled ATP. After the phosphoproteins are separated using gel electrophoresis and digested to peptide fragments. The resulting peptide fragments are then extracted and identified using mass spectrometry (MALDI or ESI mass spectrometry). MALDI MS was used in this thesis work to identify the phosphopeptide.

Immobilized metal affinity chromatography (IMAC) is a common technique to purify phosphoproteins, because the negative charged phosphoproteins can interact with positive charged metal ions, such as Ga3+ and Fe3+. When the protein sample is loaded to an IMAC column, phosphoproteins will bind to the column, and they will be eluted after the other proteins are washed away. There is a weakness for this purification since the positive charged metal ions can also interact with other negative charged unphosphorylated protein. For example, the proteins containing aspartic acid and glutamic acid cause contamination in the IMAC purification.