Strata x c18 column

All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) in the best available grade, unless otherwise noted. Rat brains, stripped of the meninges, were obtained from Pel-Freez Biologicals (Rogers, AR). Sodium peroxynitrite (100–200 mM in 4.7% sodium hydroxide) was obtained from EMD Millipore (Billerica, MA) and was aliquoted and stored at −80°C for up to 6 months and then thawed immediately before use. Sulfo-NHS-SS-Biotin and Aminolink aldehyde-agarose beads were purchased from Thermo Fisher Scientific (Waltham, MA), and Strata-X C18 columns were purchased from Phenomenex (Torrance, CA). The histone H1.2-derived peptide, ALAAAG(Y-NO₂)DVEK, was custom-synthesized by Genscript (Piscataway, NJ).

After trypsin digestion, the peptides were dissolved by adding 30 µl of 0.5 M TEAB, and the iTRAQ labeling reagents were transferred and combined with samples at room temperature. Peptide labeling was performed using the iTRAQ reagent 8-Plex kit, according to the manufacturer’s operating procedures. Labeled peptides of different reagents were desalted with a combination of Strata X C18 columns (Phenomenex) and vacuum-dried according to manufacturer specifications. The peptides were separated by the Shimadzu LC-20AB HPLC Pump system coupled with a high pH RP column. The peptides were reconstituted with buffer A [ACN:H₂O (1:19), pH = 9.8 adjusted with ammonia] to a total volume of 2 ml and loaded onto a column containing 5 μm particles (Phenomenex). The peptides were separated at a flow rate of 1 ml/min in the following sequence: 5% buffer B [H₂O:ACN (1:19), pH = 9.8 adjusted with ammonia] for 10 min, 5%–35% buffer B for 40 min, and 35%–95% buffer B for 1 min. The system was maintained in 95% buffer B for 3 min and then in 5% buffer B for 1 min before equilibration with 5% buffer B for 10 min. Elution was monitored by measuring the absorbance at 214 nm, and its fractions were collected every minute. The eluted peptides were pooled as 20 fractions and dried by vacuum.

Protein extraction and digestion procedures were performed essentially as described previously (He et al., 2019 (link)). Briefly, the cells were suspended in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris-HCl, pH 8.5, 1 mM PMSF, 2 mM EDTA) and sonicated on ice. The protein mixtures were precipitated by adding 4× volume of chilled acetone and leaving the mixtures overnight at -20°C. After centrifugation at 30,000 g and 4°C, each pellet was dissolved in 0.5 M triethylamine borane (TEAB; Applied Biosystems, Milan, Italy) and sonicated on ice. After centrifuging again at 30,000 g and 4°C, an aliquot of the supernatant was taken for determination of protein concentration by the Bradford method using BSA as a standard. For each example, 100 μg of the extracted proteins were digested at 37°C for 4 h with Trypsin Gold (Promega, Madison, WI, USA) with a protein-to-trypsin ratio of 40:1. Trypsin Gold was then added to each sample again with the same ratio, and the protein was digested for another 8 h. The digested peptides were desalted using a Strata X C18 column (Phenomenex, Torrance, CA, USA) and vacuum-dried.

Modified sequence grade trypsin (Promega Corporation, Madison, WI) was added to each sample at a 1:30 ratio (3.3 μg trypsin : 100 μg target) and digested overnight at 37°C.
Each isobaric tag was solubilized in 70 μL isopropanol. Tags (113, 114, 115, 116, 117 and 121) were added to respective pooled samples (3 pooled replicates in each group) individually and incubated at room temperature for 2 h. Additional isopropanol was added to samples to ensure an organic composition > 60% prior to incubation.
The strong cation exchange fractionation protocol followed a previous report [28 (link)] with slight modification. Briefly, the samples were loaded onto a strong cation exchange column (Phenomenex Luna SCX 100A) equilibrated with buffer A (10 mM KH₂PO₄ in 25% acetonitrile, pH 3.0) using an Agilent 1100 (Santa Clara, CA) system. The peptides were separated using a linear gradient of buffer B (10 mM KH₂PO₄ and 2 M KCl in 25% acetonitrile, pH 3.0) increasing to 5% after 36 min, 50% after 66 min and 100% after 71 min, at a flow rate of 1 ml/min. Elution was monitored by setting the absorbance at 214 nm. The eluted peptides were pooled into 10 fractions, desalted with a Strata X C18 column (Phenomenex) and vacuum-dried.

Total protein (100 μg) was taken out of each sample solution and then the protein was digested with Trypsin Gold (Promega, Madison, WI, USA) with the ratio of protein: trypsin = 30:1 at 37 °C for 16 hours. After trypsin digestion, peptides were dried by vacuum centrifugation and then reconstituted in 0.5 M TEAB and labeled with 8-plex iTRAQ reagent according to the manufacture’s protocol (Applied Biosystems). Samples from Han BB, Han ++ and Dorset groups were labeled with the iTRAQ isobaric tags 116, 121 and 114, respectively. The labeled peptide mixtures were incubated at room temperature for 2 h and then pooled and dried by vacuum centrifugation. The iTRAQ labeled peptide mixtures were resuspended in a 4 ml buffer A (25 mM NaH₂PO₄ in 25% ACN, pH 2.7) and loaded onto a 4.6 × 250 mm Ultremex SCX column containing 5-μm particles (Phenomenex). The peptides were eluted at a flow rate of 1 ml/min with a gradient of buffer A for 10 min, 5–60% buffer B (25 mM NaH₂PO₄, 1 M KCl in 25% ACN, pH 2.7) for 27 min, 60–100% buffer B for 1 min. The system was then incubated in 100% buffer B for 1 min before equilibrating with buffer A for 10 min prior to the next injection. Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every 1 min. The eluted peptides were pooled into 20 fractions, desalted with a Strata X C18 column (Phenomenex) and vacuum-dried.

The proteins were extracted from EA.hy926 cells using the Lysis buffer (7 mol/L Urea, 2 mol/L Thiourea, 4% CHAPS, 40 mmol/L Tris-HCl, pH 8.5) containing protease inhibitors PMSF (1 mmol/L) and EDTA (2 mmol/L). The protein concentrations were determined using BCA kits (Beyotime Institute of Biotechnology). After trypsin digestion and peptide measurement, iTRAQ labeling was performed according to the manufacturer’s protocol for 8-plex iTRAQ reagent (Applied Biosystems). The labeled samples were mixed and divided into 20 fractions, via strong cation exchange chromatography, using an LC-20AB HPLC system (Shimadzu, Kyoto, Japan). After desalting in a Strata X C18 column (Phenomenex, Torrance, CA, USA), each fraction’s supernatant was loaded on an LC-20AD nanoHPLC system (Shimadzu). Mass spectrometry (MS) using a TripleTOF 5600 System (AB SCIEX, Concord, ON, Canada) was performed after HPLC.
The Mascot search engine (Matrix Science, London, UK; version 2.3.02) was applied to analyze the MS data for protein identification. The proteins that possessed at least two unique spectra were considered for further analysis. p < 0.05 and fold change >1.2 denoted significance.