Peptide synthesis

All the histone H3 peptides 1–34 containing site-specific lysine acetylation (Kac) were synthesized by Fmoc-SPPS by Fmoc-Gly-Wang resin. For 0.2 mmol resin, 0.8 mmol (four eq) Fmoc-amino acid, 3.2 mmol (3.75 eq) HATU and 1.6 mmol (eight eq) N-methylmorpholine (NMM) in 8 mL DMF was added to do the peptide coupling for 90 min, followed by the deprotection of Fmoc with 20% piperidine in dimethylformamide (DMF) for 15 min. The resin was then completely washed with DMF before entering the next coupling cycle. The first depsipeptide TOG was coupled following a similar protocol. After all the coupling steps and deprotection steps, the resin was washed by DMF and dichloromethane (DCM) to dry. Reagent B (5% water, 5% phenol, 2.5% triisopropyl silane (TIPS), 87.5% trifluoroacetic acid (TFA)) for 2.5 hr at room temperature. The crude peptide was concentrated before the addition of cold diethyl ether to precipitate and dried over nitrogen gas flow. After being dissolved in 10% acetonitrile (CH₃CN)-water, the crude peptides were further purified by reversed phase HPLC with the C18 column (Varian Dynamax Microsorb 100), with a gradient of 5% CH₃CN/0.05% TFA in H₂O/0.05% TFA for 2 min and a linear gradient from 5% CH₃CN/0.05% TFA to 32% CH₃CN/0.05% TFA over 25 min, and the flow rate is 10 mL/min. All the synthetic peptides were lyophilized to powders, followed by the characterization by MALDI-TOF at the Molecular Biology Core Facilities of Dana Farber Cancer Institute (Figure 2—figure supplement 1, Figure 5—figure supplement 1).

H3K9ac(1-34) TOG [M + H]+ calculated as m/z 3494.9, observe at m/z 3493.0;

Sequence: ARTKQTARKS-TGGKAPRKQL-ATKAARKSAP-A-TOG-G

H3K14ac(1-34) TOG [M + H]+ calculated as m/z 3494.9, observed at m/z 3493.5;

H3K18ac(1-34) TOG [M + H]+ calculated as m/z 3494.9, observed at m/z 3492.9;

H3K23ac(1-34) TOG [M + H]+ calculated as m/z 3494.9, observed at m/z 3493.7;

H3K27ac(1-34) TOG [M + H]+ calculated as m/z 3494.9, observed at m/z 3493.1;

H3K9acR8G(1-34) TOG [M + H]+ calculated as m/z 3394.8, observed at m/z 3393.8;

H3K14acG13R(1-34) TOG [M + H]+ calculated as m/z 3594.1, observed at m/z 3591.7;