Fig. 4

Histone PTM quantification from breast normal and tumor samples. a H&E staining of a Luminal A-like breast cancer section. Normal epithelial cells and tumor cells (areas containing 1800–4500 cells from four consecutive sections) in the indicated areas were collected by LMD and analyzed by MS. Scale bar: 2 mm. Of note, histone H1 variants were analyzed from the same tissue areas in a previous publication [36]. b Unsupervised tissue segmentation (k-means clustering, 10 clusters) based on the MALDI MSI data. Scale bar 2 mm. In the magnification, the detail of the clusters used to define tumor 3 and tumor 4 regions, with the corresponding histological image. Scale bar 330 µm. c PCA analysis based on histone PTM data obtained from the normal and tumor areas highlighted in a, b. d Graphs showing significant differences among the tissue areas collected by LMD. Error bars represent the standard error of the mean (SEM) from 2 to 3 LMD areas. Samples were compared by one-way ANOVA, followed by multiple comparison test. The lines indicate a p value < 0.05, with the exception of the comparison of the H4 4–17 4ac peptide between tumor 3 and 4, where p = 0.07. e Heatmap display of the log2 of L/H ratios obtained with the super-SILAC strategy (light channel: laser microdissected sample, heavy channel: spike-in standard) obtained for the indicated histone PTMs in normal and tumor samples. The heatmap on the left shows the results for the tissue areas selected by the pathologist, the one on the right the areas selected by MALDI-MSI. The samples were normalized over the average value across all the samples shown in each heatmap. The grey color indicates peptides that were not quantified. f Graphs showing the levels of the H4 4–17 4ac peptide in normal and tumor samples for Luminal A-Like breast cancer (BC) samples (n = 5, matched), triple-negative BC samples (n = 3, matched), ovarian cancer (OC) and head and neck cancer (HNC). Error bars represent the standard error of the mean (SEM). Samples were compared by multiple t test (**FDR < 0.01)