The main methods for identifying phosphorylation are as follows: a. In vitro phosphorylation analysis: Radiometric kinase reactions using 32P-gamma-ATP can be used to detect phosphorylation status in vitro; this method is semi-quantitative, but can determine the molecular weight of phosphorylated proteins. b. Radioactive pulse labeling: To detect phosphorylation in vivo, radioactive pulse labeling can be used; cells are grown in the presence of 32P-orthophosphate, and then a certain protein is immunoprecipitated with a specific antibody. The radioisotope obtained by precipitation is analyzed by SDS-PAGE electrophoresis and quantified by X-ray film exposure. This method can detect phosphorylation under various physiological conditions. c. Phosphorylation-specific antibodies: There are two categories of phosphorylation-specific antibodies. The first category is a universal phosphorylation tyrosine/serine/threonine antibody that will bind to any phosphorylated tyrosine, phosphorylated serine, and phosphorylated threonine molecule, regardless of adjacent amino acid residues; the second category of antibodies is a phosphorylation-specific amino acid epitope antibody. Of course, the above methods can be combined with data acquisition mode or after mass spectrometry to identify (putative) phosphorylation sites, use universal antibodies for phosphorylation sites to determine whether the target protein is phosphorylated at a specific type of site through immunoprecipitation (IP).
Glycosylation analysis can be performed by enzymatic digestion (such as trypsin digestion) of the protein to be analyzed, followed by analysis of glycopeptides using liquid chromatography-mass spectrometry (LC-MS) or hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) to identify all proteins with specific glycan molecules, or the glycan binding sites in the target protein. In addition, for some glycopeptides, mass spectrometry (MS) analysis of intact proteins can provide data on protein species identification and their glycosylation status, but at a lower resolution.
In summary, although the methods for identifying protein phosphorylation and glycosylation are different, both can be analyzed using mass spectrometry, including the identification of phosphorylated/glycosylated proteins, phosphorylation/glycosylation modification sites, and modification levels. Mass spectrometry has also become one of the most popular post-translational modification research technologies due to its superior resolution, accuracy, and sensitivity.