Identifying Protein Phosphorylation Sites via Mass Spectrometry

In mass spectrometry analysis, proteins or peptides are ionised into charged particles and then separated by a mass spectrometer. The mass spectrum displays the relationship between the mass-to-charge ratio (m/z) of these particles and their intensity (signal size). The interpretation of the mass spectrum requires an understanding of the fragmentation patterns of peptides and the mass changes caused by phosphorylation. Below are the basic steps to interpret the mass spectrum to identify protein phosphorylation sites:

 

Identify Phosphorylation Signature Signals

Phosphorylated peptides have specific signals in the mass spectrum. The addition of the phosphate group increases the mass of the peptide (~79.966 Da). Therefore, on the mass spectrum, the phosphorylated peptide will show a corresponding mass increase based on the unmodified peptide. In MS/MS analysis, the fragmentation of phosphorylated peptides often leads to the loss of the phosphate group (-98 Da), which can be observed in the mass spectrum.

 

Analyse B-Ion and Y-Ion Sequences

In tandem mass spectrometry (MS/MS), peptide fragmentation produces b-series and y-series ions. By analysing the sequence of these ions, the amino acid sequence of the peptide can be determined. Ion peaks near the phosphorylation site will show a change in mass. The phosphorylation site can be located by comparing the peptide mass before and after modification.

 

Use Mass Spectrometry Data Analysis Software

Various software tools (such as MaxQuant, Mascot, etc.) can help identify and validate phosphorylation sites. These software can automatically match peptide sequences and annotate potential modification sites.