How do I validate the PTM sites identified in PTMScan® experiments?

The validation of PTM sites may be accomplished in several ways. Often, the site of interest may be seen in the context of more than one peptide due to alternative forms caused by incomplete protease digestion. A related approach is to employ an alternative protease in the primary digest that presents the PTM in the context of a different peptide. The identification of these alternative peptides increases confidence in the original identification. The use of site-specific PTM antibodies in conjunction with treatment conditions or knock-out and knock-in models that lead to an increase or decrease in the site can also validate the assignment. Finally, the method considered the gold standard is to synthesize the respective PTM peptide and show that its MS2 chromatogram is identical to the one experimentally determined.

Last updated: February 27, 2024