Polysulfoethyl column

Following the iTRAQ protocol (Applied Biosystems, Foster City, CA), each 100 μg protein was digested with 0.2 mL of a 50 μg/mL trypsin (Promega, Madison, WI, USA) at 37°C for 16 h. Peptides were labeled with isobaric tags 118 (normal control group), 119 (untreated rd12 group), and 121 (treated rd12 group) and incubated at room temperature for 2 h. Then the labeled mixtures were dried by vacuum centrifugation, desalted with Sep-Pak Vac C18 cartridge 1 cm³/50 mg (Waters, USA), and fractionated using a ShimazuLC-20AB HPLC Pump system (Shimazu, Japan) connected to a strong cation exchange (SCX) column (polysulfoethyl column, 2.1 mm × 100 mm, 5 μm, 200 Å, The Nest Group, Inc. USA). SCX separation was performed using a linear binary gradient of 0–45% buffer B (350 mM KCl, 10 mM KH2PO4 in 25% ACN, pH 2.6) in buffer A (10 mM KH2PO4 in 25% ACN, pH2.6) at a flow rate of 200 μL/min for 90 min, and 30 fractions were collected every 3 min. Each fraction was dried down and redissolved in buffer C (5% (v/v) acetonitrile and 0.1% formic acid solution), and the fractions with high KCl concentration were desalted with PepClean C-18 spin Column (Pierce, USA).

The labeled peptides were desalted using a Sep-Pak Vac C18 cartridge(Waters, Milford, USA) and then fractionated by a strong cation-exchange (SCX) chromatography - on a 20AD HPLC system (Shimadzu, Japan) using an SCX column (polysulfoethyl column, 2.1 mm×100 mm, 5 um, The Nest Group, Inc. USA). Mixed peptides were eluted using a linear binary gradient of 0–45% buffer B (350 mM KCl, 10 mM KH2PO4 in 25% ACN, pH 2.6) in buffer A (10 mM KH2PO4 in 25% ACN, pH 2.6) at a flow rate of 200 µL/min for 60 min. A total of 28 fractions were collected. Each SCX fraction was dried, dissolved in buffer C (5% (v/v) acetonitrile and 0.1% formic acid), and analyzed on a QSTAR XL LC-MS/MS system (ABI, USA) with an RPLC column (ZORBAX 300SB-C18 column, 3 um, 75 um×150 mm, Microm, Auburn, CA). The RPLC gradient was 5% to 35% buffer B (95% acetonitrile, 0.1% formic acid) in buffer A (5% ACN, 0.1% formic acid) at a flow rate of 0.3 uL/min for 90 min.

iTRAQ™ Reagents Kit was bought from Applied Biosystems (San Jose, CA, USA). The acetonitrile, formic acid, acetone, trypsilin, and sodium citrate buffer were from Sigma-Aldrich (California, CA, USA). The Zorbax 300SB-C18 reversed-phase column (Microm, Auburn, CA, USA), the Polysulfoethyl column (The Nest Group, Southborough, MA, USA) and QSTAR XL System (Applied Biosystem, California, CA, USA) were for 2D LC-MS/MS. Sep-Pak Vac C18 cartridges was obtained from Millipore Corporation (Minneapolis, Minnesota, USA). The rabbit polyclonal antibodies were purchased from Abcam (London, UK).