L 3000 system

The digest samples were separated by additional high-pH reversed-phase chromatography. The RIGOL L-3000 system was utilized for the separation of mixed peptides in a 30 μg digest specimen using a reverse chromatography column (RIGOL, Beijing, China). After dissolution of peptides in mobile phase A (100 μL; 2% (v/v) acetonitrile (Thermo A955-4, USA), 98% (v/v) ddH₂O, pH 10), the mixture was spun down (14,000 g) for 20 minutes.
Then the mobile phase B (98% (v/v) acetonitrile, 2% (v/v) ddH₂O, pH 10) was injected into the supernatants at 1 mL/min in the column in a stepwise elution mode. Mobile phase B step gradients were used to acquire individual 15 minutes eluant fractions.

The pooled peptides were dissolved in 60 μL of buffer A (98% H₂O and 2% ACN). The sample was then centrifuged at 13,000× g for 15 min and the supernatant was harvested. This step was repeated two times. Finally, 50 μL of the supernatant was used for reverse-phase chromatography on a RIGOL L-3000 system (Rigol, Beijing, China) equipped with an Agela Durashell C18(L) column (4.6 mm × 250 mm, 5 μm, 150A). The peptides were eluted at a flow rate of 0.7 mL/min with buffer B (98% ACN and 2% H₂O). The absorbance at 214 nm was monitored. The fractions were collected every 90 s and then dried in a speedvac. Before mass spectrometry, all the fractions were redissolved in 0.1% FA for the next LC-MS/MS analysis.

The colon tissue samples were lysed by addition of 600 μl of 8 M urea (lysate: protease inhibitor, 50:1), sonicated for 1 s, stopped for 2 s; this procedure was repeated for a total of 120 s. Samples were centrifuged at 14,000 × g for 20 min at 4°C. Protein was quantified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The protein solution rapid prototype high performance liquid chromatograph (RP-HPLC) was separated using an RIGOL L-3000 system (Rigol Technologies, inc.; China) according to the manufacturer’s protocol.

Peptides from 100 μg of each depleted serum were labelled with 8‐plex iTRAQ reagents (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's protocol. In short, the proteins were denatured, alkylated and trypsin‐digested at 37°C overnight. Then the peptides were labelled with one unit of iTRAQ reagent that was reconstituted in 150 μl isopropanol. The labelling arrangement was shown in Table 1. After the labelling, the samples were combined into one tube and dried in vacuo. Dried peptides were resuspended in 100 μl of mobile phase A and were centrifuged at 14,000 g for 20 min. The supernatants were loaded on the column (Durashell‐C18,4.6 × 250 mm, 5 μm, 100 Å, Agela, DC952505‐0) and eluted stepwise by injecting mobile B in the RIGOL L‐3000 system (RIGOL, Beijing, China). Mobile phase A consisted of 2% (v/v) acetonitrile, 98% (v/v) ddH₂O and pH 10, and phase B consisted of 98% (v/v) acetonitrile, 2% (v/v) ddH₂O and pH 10. The 60‐min. gradients comprised 5% mobile B for 5 min., 5–30% mobile B for 35 min., 30–95% mobile B for 10 min. and equilibrated with 5% mobile B for 10 min. at a 300 nl/min. flow rate. The fractions were eluted at 1.5‐min. intervals and collected using step gradients of mobile B.

Frozen tissue (500 mg) was ground in 1 mL of 20 mM cold HCl. Norleucine was used as an internal standard for measuring recovery. After derivatization, the mixture was transferred into a 100 μL glass insert in an amber vial and analyzed by HPLC (Rigol L3000-system, Rigol, Beijing, China) as described by Zhang et al. (2010) [35 (link)].