File:Phosphorylation.jpeg [Phosphorylation] is the most common post-translational modification. Phosphorylation changes a peptides molecular weight by about 80 mass units. This change is clearly discernible using mass spectrometry, but the negative charge on the phosphate group decreases the peptides charge state and makes peptide ionization difficult. Peptides with lower charge states can appear to be machine noise, and when a peptide becomes neutral or negative it cannot be read by the mass spectrometer at all. Also, the percent of phosphorylated peptides in a cell is very low compared to the percent of dephosphorylated peptides, and the mass spectrometer only selects the most abundant peptides for MS2 identification. Therefore, phosphopeptides are rarely identified with high confidence in complex samples. Fortunately techniques have been developed to isolate phosphopeptides from complex samples overcoming many of these challenges including ionization competition, and relative abundance.
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