<P> Posttranslational modification (PTM) isoforms are easily detected on 2D gels . Indeed, phosphorylation replaces neutral hydroxyl groups on serines, threonines, or tyrosines with negatively charged phosphates with pKs near 1.2 and 6.5 . Thus, below pH 5.5, phosphates add a single negative charge; near pH 6.5, they add 1.5 negative charges; above pH 7.5, they add 2 negative charges . The relative amount of each isoform can also easily and rapidly be determined from staining intensity on 2D gels . </P> <P> In some very specific cases, the detection of the phosphorylation as a shift in the protein's electrophoretic mobility is possible on simple 1 - dimensional SDS - PAGE gels, as it's described for instance for a transcriptional coactivator by Kovacs et al. Strong phosphorylation - related conformational changes (that persist in detergent - containing solutions) are thought to underlie this phenomenon . Most of the phosphorylation sites for which such a mobility shift has been described fall in the category of SP and TP sites (i.e. a proline residue follows the phosphorylated serine or threonine residue). </P> <P> More recently large - scale mass spectrometry analyses have been used to determine sites of protein phosphorylation . Over the last 4 years, dozens of studies have been published, each identifying thousands of sites, many of which were previously undescribed . Mass spectrometry is ideally suited for such analyses using HCD or ETD fragmentation, as the addition of phosphorylation results in an increase in the mass of the protein and the phosphorylated residue . Advanced, highly accurate mass spectrometers are needed for these studies, limiting the technology to labs with high - end mass spectrometers . However, the analysis of phosphorylated peptides by mass spectrometry is still not as straightforward as for "regular", unmodified peptides . Recently EThcD has been developed combining electron - transfer and higher - energy collision dissociation . Compared to the usual fragmentation methods, EThcD scheme provides more informative MS / MS spectra for unambiguous phosphosite localization . </P> <P> A detailed characterization of the sites of phosphorylation is very difficult, and the quantitation of protein phosphorylation by mass spectrometry requires isotopic internal standard approaches . A relative quantitation can be obtained with a variety of differential isotope labeling technologies . There are also several quantitative protein phosphorylation methods, including fluorescence immunoassays, Microscale thermophoresis, FRET, TRF, fluorescence polarization, fluorescence - quenching, mobility shift, bead - based detection, and cell - based formats . </P>

Phosphorylation is a common form of covalent modification