Considerations for MS Identification of Phosphorylation Sites

Phosphorylation is a common type of protein post-translational modification (PTM), playing a significant role in biological processes such as cell signal transduction, protein activity and stability regulation. Mass spectrometry is one of the main methods for identifying protein phosphorylation, but the following issues need to be considered in the actual operation:

 

1. Sample Preparation

The level of phosphorylation in the sample is generally low, and special enrichment strategies, such as Metal Oxide Affinity Chromatography (MOAC), are needed to enhance the detection signal. Additionally, phosphorylation is a reversible modification, so phosphatase inhibitors need to be added during sample preparation to prevent the loss of phosphorylation in the sample.

 

2. Data Analysis

Since phosphorylation results in an 80Da increase in the mass of the protein fragment, a special database search algorithm needs to be used to consider this modification.

 

3. Result Interpretation

The identified phosphorylation sites need further study in the lab to reveal their role in biological processes.

 

In actual operation, the above issues still need to be considered to obtain more accurate and comprehensive results.