Figure 4.

MassSQUIRM can be used to successfully quantify differentially modified peptides. (A) A di-methylated synthetic peptide was analyzed using mass spectrometry and the resulting isotopic envelope was used to determine r₁ and r₂ from equations 3 and 4. This peptide corresponds to that noted in blue in Figure 1A (inset). (B) The same synthetic peptide was incubated with 125 ng GST-LSD1 in demethylase buffer for two hours at 37°C. Samples were then subjected to MassSQUIRM analysis. A mixed population of overlapping peaks represents three different methylation states as seen in Figure 1C (inset). Peptides initially mono-methylated occur at a mass 2 Da heavier than di-methylated peptides due to the addition of one heavy methyl group during reductive methylation. Similarly, peptides initially unmodified will occur 4 Da heavier than di-methylated peptides due to the addition of two heavy methyl groups. Areas under the monoisotopic peaks were noted as A₁, A₂ and A₃. These values were used to determine equations 5–7, which take into account the isotopic overlap noted in (A). Open circles indicate light methylation while closed circles indicate heavy methylation.