Ultremex scx column

iTRAQ Reagent 4-plex Multiplex Kits were used to label each independent biological replicate (IBR) in the groups. For each group, 15 subjects were recruited, plasma samples of which were randomly mixed to three pools (five individuals for each IBR). Each iTRAQ reagent was diluted in 150 μl of isopropanol before 100 μg of peptide was added. Peptides were labeled as follows: Three IBRs of the nCMS-HPu group were labeled with iTRAQ tags 114; three IBRs of the CMS-HPu group were labeled with iTRAQ tags 115; three IBRs of the nCMS-TPu group were labeled with iTRAQ tags 116, and three IBRs of the nCMS-HPn group were labeled with iTRAQ tags 117. Next, 100 μl ultra-pure HPLC-grade water was added to terminate the labeling after the samples were labeled for 1 h. Samples were combined into one tube for each group, then resolved into 50 fractions using a 5-μm particle Ultremex SCX column (Phenomenex, Torrance, CA, U.S.A.), and finally desalted using a Gemini-NX C18 column (4.6 mm × 250 mm, Phenomenex). After all the fractions were freeze-dried under a vacuum, they were resuspended with 30 μl of mobile phase A (2% acetonitrile [ACN], 0.1% formic acid [FA]), divided into 15 groups based on their peak intensities and then centrifuged at 12,000 rpm for 10 min prior to LC-MS/MS analysis.

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.

For SCX chromatography using a Shimadzu LC-20AB HPLC Pump system, the iTRAQ-labelled peptides were reconstituted with 4ml of buffer A (25mM NaH₂PO₄ in 25% acetonitrile, pH 2.7) and loaded onto a 4.6×250mm Ultremex SCX column that contained 5 μm particles (Phenomenex). The peptides were eluted at a flow rate of 1ml min^–1 with a gradient of buffer A for 10min, 5–35% buffer B (25mM NaH₂PO₄, 1M KCl in 25% acetonitrile, pH 2.7) for 11min and 35–80% buffer B for 1min. The system was then maintained in 80% buffer B for 3min before equilibrating with buffer A for 10min prior to the next injection. Elution was monitored by measuring the absorbance at 214nm, and the fractions were collected every 1min. The eluted peptides were pooled as 12 fractions, desalted using a Strata X C18 column (Phenomenex), and vacuum dried.