Bond breaker tcep solution

The following stock solutions were prepared: 2 mg·mL⁻¹ brefeldin A (BFA; Nacalai Tesque, Kyoto, Japan, 05325‐81) in methanol; 5 mm thapsigargin (Wako, Osaka, Japan, 209‐17281) in DMSO; 10 mg·mL⁻¹ tunicamycin (Wako, 202‐08241) in DMSO; 1 m DTT (Wako) in water; 400 mm diamide (Sigma, St Louis, MO, USA, D3648) in PBS; 100 mm 4‐acetamido‐4ʹ‐maleimidylstilbene‐2,2ʹ‐disulfonic acid (AMS; Thermo Fischer Scientific, A485) in water; 10 mm menadione (Nacalai Tesque, 36405‐71) in DMSO; 100 mm l‐buthionine sulfoximine (BSO; Wako, 021‐14121) in water; and 500 mm N‐acetyl‐l‐cysteine (NAC; Sigma, A9165) in 1 m HEPES. N‐Ethylmaleimide (NEM) was obtained from Wako (058‐02061), and Bond‐Breaker TCEP solution from Thermo Fischer Scientific (77720).

Deionized (DI) water (18.2 MΩ·cm) was produced by a Milli-Q system (Millipore, Bedford, MA, United States) and used in all experiments. HPLC grade acetonitrile (ACN) and methanol as well as analytical grade acetone and hydrochloric acid (HCl, 37%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Analytical grade formic acid was purchased from J&K Scientific Ltd. (Beijing, China). Iodoacetamide (IAA), trizma base, urea, sodium dodecyl sulfate (SDS), and ammonium bicarbonate (NH₄HCO₃) were purchased from Sigma-Aldrich (St. Louis, MI, United States). Bond-breaker TCEP solution (0.5 mol/L) and protease inhibitor cocktail (100×, EDTA-free) were purchased from Thermo Fisher Scientific (Rockford, United States). Trypsin used for protein digestion was bought from Hualishi Technology Co., Ltd. (Beijing, China). Trypsin–EDTA solution (0.25%, with phenol red) used for cell digestion and phosphate buffered saline (PBS, 1×) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China).

The biphenylsulfonyl
(BPS) and phosphorothioate (PS) ligators used for CL-qPCR were synthesized
on a MerMade 12 Oligonucleotide Synthesizer (BioAutomation) as previously
described.^{36 (link),40 (link)} Briefly, the synthesis of the
PS ligator was performed on a 3′-Phosphate-ON solid support
(ChemGenes N9977-05) by sulfurization of the coupling between the
support and first nucleotide, following standard DNA synthesis. The
BPS ligator was synthesized under “UltraMild” conditions
using dT-Q-CPG support (Glen Research 21-2030). The dA and dG amidites
were exchanged for Pac-dA-CE (Glen Research 10-1601) and iPr-Pac-dG-CE
(Glen Research 10-1621), respectively. Capping of failed sequences
was performed using 5% (w/v) phenoxyacetic anhydride (TCI Chemicals
P0111) in THF/pyridine 90:10 (v/v) in place of acetic anhydride. Cleavage
and deprotection were performed using 30% ammonium hydroxide at ambient
temperature for 4 h. The PS ligator bears a 3′ terminal PS
group that enables dimerization. To prevent spontaneous sulfur–oxygen
exchange, the PS ligator was stored as a dimer. Immediately prior
to use, an aliquot of the PS ligator was reduced by adding tris(carboxyethyl)phosphine
hydrochloride (TCEP HCl; Fluorochem M02624) or Bond-Breaker TCEP Solution,
Neutral pH (Thermo Scientific 77720) to a final concentration of 50
μM.

Site-specific citrulline antibodies against HP1γ R38/9-citrulline were raised by Orygen Antibodies Ltd. Sheep were immunised with HP1γ peptides (aa 34–44) citrullinated at residues 38 and 39 (KVLD(Cit)(Cit)VVNGKC) that was C-terminally coupled to KLH. After the initial immunisation, 3 boost injections every 4 weeks were performed. Crude serum of the final bleed was purified against the HP1γ R38/9-citrulline peptide (same as immunogen) columns. In brief, peptides were first reduced using Bond-Breaker™ TCEP Solution (Thermofisher Scientific) and then coupled to SulfoLink™ Coupling Resin (Thermofisher Scientific) according to the manufacturer’s instructions. Non-specific binding of the column was blocked using quenching reagent (50 mM l-cysteine in coupling buffer), followed by 3 washes in wash buffer (1 M NaCl in H₂O) and twice in 1xTBS. Crude serum was centrifuged at maximum speed for 10 min; supernatant was added to the resin and incubated at RT for 1 h on a rotating wheel. Resin was then washed 4x in 1xTBS. To elute the antibodies, elution buffer (0.2 M glycine HCl pH 2.5) was added and tube was quickly flicked and directly centrifuged at 800 rpm for 1 min. Supernatant was moved to a fresh tube and placed on ice. Immediately ice-cold neutralisation buffer (1 M Tris HCl pH 8.8) was added and mixed thoroughly to neutralise the pH to 7.

HV1 was reduced by an incubation in Bond-Breaker TCEP solution (neutral pH) (Thermo Scientific) with TECP at a final concentration of 50 mM at 70°C for 10 min, and then desalted by using a SPE C-TIP(C18) column (Nikkyo Technos, Co., Ltd., Japan), followed by elution in 80% acetonitrile and 0.5% TFA. The obtained samples were analyzed by MALDI-TOF MS in a TOF/TOF 5800 system (AB SCIEX) in the positive mode with sinapinic acid (Tokyo chemical industry Co. Ltd., Japan) or 2′, 6′-dihydroxyacetophenone (Sigma-Aldrich) as the matrix.