The identification methods of phosphorylated proteins can be divided into two types: direct detection and indirect detection. Here are several common methods:
Western Blotting (WB): Detect the phosphorylation status of target proteins by using specific antibodies. After electrophoretic separation of proteins, anti-phosphorylation-specific antibodies can be used to identify phosphorylation sites and the phosphorylation level can be quantified by the intensity of the signal.
Mass spectrometry (MS): Mass spectrometry is an efficient method to directly identify phosphorylated proteins and phosphorylation sites. It can be used to identify the composition of overall phosphorylated proteins, as well as to identify specific phosphorylation sites.
a. Phosphorylation-induced digestion: perform protease digestion before mass spectrometry, and use a mass spectrometer to identify the phosphorylation sites in the protein enzymatic hydrolyzate.
b. Direct neutron transfer mass spectrometry (DTMS): Use a mass spectrometer to directly detect the neutron mass of phosphorylated proteins to identify phosphorylation sites.
Phospho-specific antibody array: This method uses multiple specific phospho-antibodies and immobilizes them on the chip. After reacting the sample with the chip, multiple phosphorylation sites can be identified and quantified by detecting signal intensity.
Affinity chromatography: Use a column containing affinity materials to purify phosphorylated proteins from a mixture. Then, its phosphorylation status is determined by methods such as Western blotting or mass spectrometry analysis.
These methods each have their own advantages and disadvantages. Choosing an appropriate method should consider factors such as sample properties, experimental purposes, and equipment availability. Often, combining multiple methods can yield more comprehensive and accurate information on the identification and quantification of phosphorylated proteins.