Fig. 1

Analysis of histone H3 N-terminal tails from HeLa cells labeled with heavy lysine/arginine residues (heavy KR). a Workflow for metabolic labeling of histone sequences. Histones were extracted from HeLa S3 cells grown in equal amounts in light and heavy medium. After extraction, histone H3 was purified using C₁₈ chromatography and digested using GluC. MS/MS-based quantification was performed using isoScale labels. b Nano-liquid chromatography (nLC)-MS ion map of the histone H3 N-terminal tail in its unlabeled and isotopically heavy labeled form. The y axis represents m/z, while the x axis represents retention time. The pattern above is the sequential elution of modified histone tails with heavy KR label, while the pattern below is the unlabeled tails. Histone tails elute from the most modified to the least modified, which is why the m/z value decreases as function of time. c Full MS spectrum representing differently methylated histone tails co-eluting from chromatography. Unlabeled (light) forms are underlined in blue, while heavy KR-labeled peptides are underlined in red. d Number of MS/MS spectra performed for light and heavy histone tails. Their ratio indicates that both species are equally subjected to MS/MS selection; error bar represents standard deviation of 4 replicates. e MS/MS events selecting either unlabeled (black) or heavy KR-labeled (red) peptides for fragmentation. No clustering of colors indicates that there is no bias in MS/MS selection for the two forms. f Correlation between quantified polypeptides for each of the two labels. Axes represent the average relative abundance of given modified polypeptides quantified in the two forms. Axes are Log₁₀ scaled to facilitate visualization. g MS/MS ion spectrum of a selected histone tail in its unlabeled form and h the same histone tail in its heavy KR-labeled form. The MS scan of their precursor mass is displayed on the square at the top right of the MS/MS spectrum