8 plex itraq reagent

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.

The proteins (100 μg) were digested with 3 μL trypsin gold for 16 h (protein:trypsin, 30:1). The peptides were reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for 8-plex iTRAQ reagent (Applied Biosystems). The labelled mixtures were then reconstituted with buffer A (25 mM NaH₂PO₄ in 25% acetonitrile (ACN), pH 2.7) and loaded onto an Ultremex SCX column containing 5 μm particles (Phenomenex, CA, USA). The peptides were eluted with a gradient of buffer A for 10 min, 5–60% buffer B (25 mM NaH₂PO4 and 1 M KCl in 25% ACN, pH 2.7) for 27 min, and 60–100% buffer B for 1 min. Elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every 1 min^{75 (link)}.