Metabolic Tracing of Methyl Donor Utilization in Histone Methylation via Relative Quantification of Isotopomer Distribution Mass Spectrometry

Histone methylation depends on one-carbon metabolism, with methyl groups donated by methionine-, serine-, and glucose-derived intermediates. To dissect the metabolic origins of histone methylation, we developed Relative Quantitative Methyl Isotopomer Distribution Mass Spectrometry (RQMID-MS), a high-resolution mass spectrometry-based method that uses diagnostic low-mass fragment ions to quantify methyl group transfer from isotope-labeled precursors. Using this method, we mapped methylation sources to histone lysines in glioblastoma cells under nutrient and oxygen stress. Methionine was the dominant methyl donor under replete condition. Under combined serine and methionine depletion or prolonged methionine depletion alone, glucose emerged as a key compensatory source, particularly in U87 cells with elevated 3-phosphoglycerate dehydrogenase (PHGDH) expression. In contrast, U251 cells favored exogenous serine and glycine, correlating with higher levels of serine hydroxymethyltransferase 2 (SHMT2) expression. Hypoxia initially enhanced glucose-derived methylation but later suppressed it, likely due to impaired vitamin B₁₂-dependent remethylation of homocysteine. RQMID-MS enables precise tracking of methyl donor routing to histones and offers a robust platform for studying metabolic and epigenetic crosstalk in cancer and beyond.