Panoramically Identify Crotonylation Sites and Rapidly Discover New Types of Acylation within H3 through FPFIP

Grasping the panoramic PTM (post-translational modification) map of a functionally important protein could be the base to discover the actual functional mechanism and develop precise therapies. However, there are no panoramic PTM maps due to the limitation of the current identification strategy (protease digestion followed by LC-MS). Recently, we have developed a simple, generic, and high-efficiency strategy named FPFIP (Fragmenting-based protease-free identification of PTM sites) to identify PTM sites panoramically. In short, we divided H3 CDS into fragments about 15-25 AA long with lysine inside and mixed all DNA fragments followed by one-time ligation and prep of fragment plasmid mixture; next, one-time transformation, expression, and purification of all GST-labelled H3 fragments was performed. Next, the mixture was re-bound to glutathione resin and incubated with subject lysate to achieve PTM. Finally, the GST tag was cleaved off and the H3 fragment mixture was sent for direct LC-MS identification without protease digestion. Through this method, we have identified 6 new H3 crotonylation sites compared with the published report with the highest coverage. Among this six, H3-K115 crotonylation was important for DSB repair and embryo development. Moreover, using this H3 fragment mixture, we have identified three novel types of acylation, temporarily named as tropatylatyion, urocanylation, nervonylation induced by tropate, urocanic acid, and nervonic acid respectively. In all, FPFIP could be potentially employed for building panoramic PTM map of any protein and rapid, high-efficient screening novel types of PTM. Supplementary datasets Supplementary dataset 1. All second-generation DNA sequencing results matching H3L1-H3L5 coding sequences in 51 randomly-picked DH5α colonies Corresponds to Fig 1A (left). This file includes 6 sheets. From left to right, “H3L1”, “H3L2”, “H3L3”, “H3L4”, and “H3L5” include second-generation DNA sequencing results matching H3L1-H3L5 coding sequences in 51 randomly-picked DH5α colonies transformed with the one-time H3L1-H3L5 fragment mixture ligation (into pGEX-6P1) product. “Percentage of each plasmid” shows the percentage of each group of colonies matching H3L1-H3L5. Supplementary dataset 2. All second-generation DNA sequencing results matching H3S1-H3S9 coding sequences in 90 randomly-picked DH5α colonies Corresponds to Fig 1A (right). This file includes 10 sheets. From left to right, “H3S1”, “H3S2”, “H3S3”, “H3S4”, “H3S5”, “H3S6”, “H3S7”, “H3S8”, and “H3S9” include second-generation DNA sequencing results matching H3S1-H3S9 coding sequences in 90 randomly-picked DH5α colonies transformed with the one-time H3S1-H3S9 fragment mixture ligation (into pGEX-6P1) product. “Percentage of each plasmid” shows the percentage of each group of colonies matching H3S1-H3S9. Supplementary dataset 3. All LC-MS-identified non-crotonylated and crotonylated peptides matching H3L1-H3L5 AA sequences in one-time purified H3 fragment mixture incubated with ovary lysate, testis lysate, and NIH3T3 cell lysate Corresponds to Fig 2C. This file includes 7 sheets. From left to right, “H3L1-5 ref seq” includes AA sequence for H3L1 to H3L5; “H3L1”, “H3L2”, “H3L3”, “H3L4”, and “H3S5” include LC-MS-identified non-crotonylated and crotonylated peptides matching H3L1-H3L5 AA sequences in one-time purified H3 fragment mixture incubated with ovary lysate, testis lysate, and NIH3T3 cell lysate. “Percentage of each fragment” shows the percentage of each group of peptides matching H3L1-H3L5 AA sequences in one-time purified H3 fragment mixture incubated with ovary lysate, testis lysate, and NIH3T3 cell lysate. Supplementary dataset 4. All LC-MS-identified non-crotonylated and crotonylated peptides matching H3S1-H3S9 AA sequences in one-time purified H3 fragment mixture incubated with ovary lysate, testis lysate, and NIH3T3 cell lysate Corresponds to Fig 2D. This file includes 11 sheets. From left to right, “H3S1-9 ref seq” includes AA sequence for H3S1 to H3S9; “H3S1”, “H3S2”, “H3S3”, “H3S4”, “H3S5”, “H3S6”, “H3S7”, “H3S8”, and “H3S9” include LC-MS-identified non-crotonylated and crotonylated peptides matching H3S1-H3S9 AA sequences in one-time purified H3 fragment mixture incubated with ovary lysate, testis lysate, and NIH3T3 cell lysate. “Percentage of each fragment” showed the percentage of each group of peptides matching H3S1-H3S9 AA sequences in one-time purified H3 fragment mixture incubated with ovary lysate, testis lysate, and NIH3T3 cell lysate. Supplementary dataset 5. All mRNA FPKM values and Log2 values for the heat map from RNA seq. Corresponds to Fig. 3E. This file includes 2 sheets. “All samples-fpkm” includes all FPKM values and other info. for all mRNAs identified in MCA205 cells transfected with Ctr plasmid (pcDNA3.1-flag empty plasmid), pcDNA3.1-H3-WT-flag plasmid, and pcDNA3.1-H3-K115A-flag plasmid. “Heat-H3-WT vs Ctr>1.5 & <0.667” includes all info. for Ctr & WT & K115A groups at the threshold of WT/Ctr ≥ 1.5 or ≤ 0.667. Log2(FPKM) values were used for the heat map in Fig. 3E. Supplementary dataset 6. All peak values and Log10 values for the heat map from Chip seq. Corresponds to Fig. 3F. This file includes 4 sheets that include all peak values, Log10(peak value) and other info. identified in Chip seq with Flag antibody or control IgG in MCA205 cells transfected with Ctr plasmid (pcDNA3.1-flag empty plasmid), pcDNA3.1-H3-WT-flag plasmid, and pcDNA3.1-H3-K115A-flag plasmid. Log10(peak) values were used for the heat map in Fig. 3F. Supplementary dataset 7. All peak values and FPKM values for the joint analysis of Chip seq and RNA seq. Corresponds to Fig. 3G. This file includes 4 sheets. From left to right, “Chip-H3-WT vs Ctr > 1.5” include all peak values and other info. for all peaks identified in Chip seq at the threshold of H3-K115A/Ctr ≥ 1.5; “Chip-H3-K115A vs Ctr > 1.5” include all peak values and other info. for all peaks identified in Chip seq at the threshold of H3-K115A/Ctr ≥ 1.5; “RNA-H3-WT vs Ctr >1.5&<0.667” include all FPKM values and other info. for all genes identified in RNA seq at the threshold of H3-WT/Ctr ≥ 1.5 or ≤ 0.667; “RNA-H3-K115A vs Ctr >1.5&<0.667” include all FPKM values and other info. for all genes identified in RNA seq at the threshold of H3- K115A/Ctr ≥ 1.5 or ≤ 0.667. Plasmid transfection, RNA seq, or Chip seq were the same as above. Supplementary dataset 8. All intensity values of H3-WT or H3-K115A-interacting proteins. Corresponds to Fig. 3G. MCA205 cells transfected with Ctr plasmid (pcDNA3.1-flag empty plasmid), pcDNA3.1-H3-WT-flag plasmid, and pcDNA3.1-H3-K115A-flag plasmid. Anti-Flag antibody or control IgG IP-mass spec were performed. The file includes three sheets. From left to right, “All proteins” include intensity values and other info. for all identified proteins in control IgG IP-mass spec in control group, Flag IP-mass spec in H3-WT group, and Flag IP-mass spec in H3-K115A group; “H3-WT vs Ctr IgG > 1.5” includes intensity values and other info. for all identified proteins at the threshold of H3-WT/Ctr IgG ≥ 1.5; “H3-K115A vs Ctr IgG > 1.5” includes intensity values and other info. for all identified proteins at the threshold of H3-K115A/Ctr ≥ 1.5.