How to Detect Protein Phosphorylation Without Site Information or Available Antibodies
Detecting protein phosphorylation without prior knowledge of the modification sites or access to site-specific antibodies can be challenging. However, several alternative strategies are available:
Mass Spectrometry (MS)
1. Protein Digestion
Initially, proteins are enzymatically cleaved into peptides using proteases such as trypsin.
2. Enrichment of Phosphorylated Peptides
Phosphorylated peptides are selectively enriched using techniques like Immobilized Metal Affinity Chromatography (IMAC) or titanium dioxide (TiO₂)-based chromatographic media.
3. LC-MS/MS Analysis
The enriched peptides are analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), enabling precise identification of phosphorylation sites.
2D-Gel Electrophoresis
Protein phosphorylation often leads to a shift in the isoelectric point (pI) of the protein. Two-dimensional gel electrophoresis can be used to detect such shifts, offering insights into phosphorylation-dependent changes in protein charge states.
Radioactive Labeling
Radiolabeling assays using [³²P]-orthophosphate allow for direct detection of phosphorylated proteins by tracking radioactive phosphate incorporation.
Other Phosphorylation Antibodies
Even in the absence of antibodies targeting specific phosphorylation sites, commercially available antibodies that recognize phosphorylated serine (phospho-Ser), threonine (phospho-Thr), or tyrosine (phospho-Tyr) residues can provide initial evidence of phosphorylation.
Among these approaches, mass spectrometry is the most widely used and reliable method, offering high sensitivity and site-specific resolution for phosphorylation analysis. However, it requires advanced instrumentation and skilled technical personnel.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
