Human colon biopsies (healthy and Ulcerative Colitis) LC-MS/MS

Description
The etiology of the inflammatory bowel diseases, including ulcerative colitis, remains incomplete, but recent findings points to the involvement of complex host-microbial interactions. We hypothesized that an analysis of the proteins on the host-microbial interacting surface, the intestinal mucosa, could reveal novel insights into the diseases. Mucosal colonic biopsies were extracted by standard colonscopy from sigmoideum from 10 ulcerative colitis patients from non-inflammed tissue and 10 controls. The biopsies were immediately following extraction snap-frozen for protein analysis and the protein content of the biopsies was characterized by high-throughput quantative gel-free proteomics.

Sample Processing Protocol
Colon mucosal biopsies for MS were sampled 40 cm from the anus by sigmoidoscopy. Patient biopsies were taken from macroscopically normal tissue, and all controls had normal findings at sigmoidoscopy. The biopsies were immediately transferred to cryotubes and snap-frozen in liquid nitrogen followed by storage at -140°C until proteomics sample preparation. Each biopsy was homogenized with steel beads in 0.5 mL cold lysis buffer (5% sodium deoxycholate, 50 mM triethylammonium bicarbonate, pH 8.5) Protein digestion was performed using a modified filter-aided sample preparation (FASP) protocol, Proteins were reduced for 30 min at room temperature by adding tris(2-carboxyethyl)phosphine (Thermo Scientific, Waltham, MA, USA) to a final concentration of 10 mM. The reduced protein solution was transferred to a 30 kDa molecular weight cutoff spin-filter (Millipore, Billerica, MA, USA), and buffer exchange was facilitated between all steps by centrifugation at 15,000 g for 15 min at 4°C. The proteins were alkylated using 100 µL 50 mM 2-iodoacetamide (Sigma-Aldrich, St. Louis, MO, USA) in lysis buffer. Two µg sequencing grade modified trypsin (Promega, Madison, WI, USA) dissolved in 50 µL digestion buffer (0.5% sodium deoxycholate, 50 mM triethylammonium bicarbonate, pH 8.5) was added to the spin-filter, and the proteins were digested to peptides overnight at 37°C. The peptide material was eluted from the spin-filter and purified by phase inversions. One mL ethyl acetate with 1% formic acid was added to the peptide material, the tube was vortexed, centrifuged at 15,000 g for 5 min to obtain phase separation, and the aqueous phase with peptides was recovered. Five µg peptide material as determined by A280 was analyzed per LC-MS analysis. All biopsies were analyzed in triplicates, and in a random order of biopsy analysis. The peptides were analyzed by LC-MS using an UltiMate™ 3000 UPLC system (Thermo Scientific, Waltham, MA, USA) coupled online to a Q Exactive mass spectrometer (Thermo Scientific, Waltham, MA, USA) using a trapping column setup. The peptide material was loaded onto a 2 cm trap column packed with Acclaim PepMap100 C18, 5 µm 100 Å material (Thermo Scientific, Waltham, MA, USA). Subsequently, the peptides were separated using a 50 cm analytical column packed with Acclaim PepMap100 C18, 2 µm 100 Å material (Thermo Scientific, Waltham, MA, USA). The peptides were eluted from the column with a gradient of 96% solvent A (0.1% formic acid) and 4% solvent B (0.1% formic acid in acetonitrile), which was increased to 8% solvent B on a 5 minutes ramp gradient and subsequently to 30% solvent B on a 225 minutes ramp gradient, with a constant flow rate of 300 nL/min. The column was washed with 90% solvent B for 10 min, and equilibrated for 40 min, resulting in a total analysis-time of 290 min per LC-MS analysis. Both columns were kept at 40°C. The eluting peptides were introduced directly into the mass spectrometer by a picotip emitter for nanoflow ionization (New objective, Woburn, MA, USA). The mass spectrometer was operated in positive mode using a data-dependent acquisition method. A full MS scan in the mass range of m/z 400 to 1200 was acquired at a resolution of 70,000. In each cycle, the mass spectrometer would trigger up to 12 MS/MS acquisitions of eluting ions based on highest signal intensity for fragmentation. The MS/MS scans were acquired with a dynamic mass range at a resolution of 17,500. The precursor ions were isolated using a quadrupole isolation window of 1.6 m/z and fragmented using high-energy collision with a normalized collision energy of 27. Fragmented ions were dynamically added to an exclusion list for 30 s.

Data Processing Protocol
The data-files were searched using MaxQuant 1.4.1.2 against all reviewed Homo sapiens Uniprot proteins (downloaded 08/10/2013, containing 20277 entries).22 All standard settings were employed with carbamidomethyl (C) as a static modification and protein N-terminal acetylation, deamidation (NQR) were included as variable modifications. A target-decoy fragment spectra search strategy was employed and used to adjust the false discovery rate (FDR) of identified peptides and proteins to below 1% FDR.

Contact
Tue Bjerg Bennike, Aalborg University

Submission Date
27/04/2015

Publication Date
26/05/2015

Project PXD001608