Figure 1

Design of the combinatorial PTM histone peptide microarray. (A) Construction of the combinatorial PTM peptide library. Combinatorial PTM histone peptide microarray featuring a tiled design and a high content of linked PTMs. Peptide sequence (underlined) covers core histones and histone variants; colored amino acids were modified according to the key. Colored arcs represent new PTM combinations and black arcs display PTM combinations used in previous arrays. (B) Histone peptide microarray binding assay. Peptides from Figure 1A were synthesized as cellulose conjugates and spatially spotted onto microarray, on which reader binding was detected and quantified by dual-channel fluorescence. Red star represents the binding signal at 635 nm, which was used for reference binding intensities. (C) Layout of the combinatorial PTM histone peptide microarray. On the left is a representative image of an entire microarray before protein binding (detected at 532 nm, green channel). On each microarray, two full libraries were included and each peptide was printed as triplicate spots. On the right is a zoom-in of a subarray from the big library detected at dual-channel (532 nm/635 nm, green/red channel) after protein binding. On each subarray, biotin peptides were included as positive controls (boxed in red dashed line). Misprinting (boxed in white dashed line) can be easily identified from the green channel. Protein bindings (boxed in yellow dashed line) were quantified from the signal intensities at 635 nm.