Mass spectrometry (MS) is an analytical technique that produces a spectrum containing the atomic or molecular masses of a material sample. Spectra are used to determine the elements, isotopic signatures, masses of particles and molecules of a sample, and to elucidate the chemical structure of molecules such as peptides and other compounds. In a typical MS identification of phosphoproteomics, a protein sample is ionized, and the sample ions are separated according to their different masses and relative abundances. The detection of MS phosphorylation is based on the actual detection mass spectrum produced by trypsin digestion of the peptide matching its theoretical peptide mass, and a mass shift between mass and charge (m/z) of 79.9 or a neutral loss (m/z) of 80 or 98 is generally considered to have occurred. It is not only suitable for phosphorylation detection, but also for the identification of phosphorylation sites.
Mass spectrometry is one of the core technologies for protein identification and has become the mainstream method for proteomics research. At the same time, there are some defects in the identification of phosphorylation sites by mass spectrometry. First, phosphorylated peptides are negatively charged, and electrospray mass spectrometry is ionized in positive charge mode. The phosphorylated peptides detected by mass spectrometry are usually weak and the ionization effect is poor. Secondly, it is difficult for the mass spectrometer to detect the signal of low-abundance proteins under the high background of a large number of non-phosphorylated proteins. The above defects can be avoided by purifying and enriching the phosphorylated peptides in the sample.
Mass spectrometry has been used to identify phosphorylation sites and quantify them. However, mass spectrometry is expensive, requires specific equipment, and often requires more quantitative data, so mass spectrometry is often combined with other biochemical methods to analyze post-translational modifications of proteins.