Protein Phosphorylation Mass Spectrometry Analysis

Protein phosphorylation is a type of post-translational modification catalyzed by protein kinases and phosphatases (PP), playing crucial roles in many cellular processes including the cell cycle, growth, apoptosis, and signal transduction pathways. To understand these mechanisms, it is essential to analyze protein phosphorylation.

 

There are many methods for phosphorylation analysis, including kinase activity assays, development of phosphorylation-specific antibodies, protein blotting, enzyme-linked immunosorbent assay (ELISA), as well as intracellular flow cytometry and immunocytochemistry/immunohistochemistry (ICC/IHC). In addition to these methods, mass spectrometry (MS) analysis also plays an important role in phosphorylation analysis. There are two common strategies for identifying protein phosphorylation sites by bottom-up proteomics, including the identification of phosphorylation sites from individual proteins and small protein complexes, and the identification of global phosphorylation sites from whole cell and tissue extracts.

 

Identifying the phosphorylation sites of specific target protein is very important for fully studying the functional role of protein phosphorylation. 

 

Analysis Workflow

1. Sample Preparation

Because phosphorylated peptides have a lower stoichiometric level compared to unmodified peptides, it is necessary to purify as much protein as possible to successfully perform liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This is usually done by using epitope tags such as FLAG, HA, or Myc, or by immunoprecipitation (IP) using an appropriate antibody. In addition, proteins can be purified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or precipitation. There are several methods available for the enrichment of phosphorylated peptides, including immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO2) beads.

 

2. Analysis via LC-MS/MS

Peptides can be introduced into the mass spectrometer via liquid chromatography combined with electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). Peptides are typically sequenced by MS/MS using collision-induced dissociation (CID), where collision with an inert gas causes protonated peptides to break at the amide bonds of the backbone. Electron capture dissociation (ECD) and electron transfer dissociation (ETD) can be applied for phosphorylation analysis.

 

3. Data Analysis

Informatics software is needed to identify the peptide sequences and their phosphorylated counterparts. Several database search engines are available, including Mascot, Sequest, MaxQuant, and X!Tandem.

 

In order to truly compare global phosphorylation, a quantitative proteomics strategy is necessary. Quantitative phosphoproteomics enables researchers to study signaling pathways in different model systems, identifying phosphorylation events that change in abundance and duration due to a given stimulus.