DOI: 10.25345/c5qr4p406
title: MassIVE MSV000099466 - Nasal polyp biopsies from patients with chronic rhinosinusitis
title: Nasal polyp tissue samples that were technically suitable for proteomic study were processed as follows. The dewaxed tissues were solubilized in RapigestTM SF reagent (Waters Co, Milford, MA, USA) at the final concentration of 0.5% (w/v) and incubated under stirring, first at 100C for 20 minutes and sequently at 80C for 2 hours. Protein concentration was assayed using SPNTM Protein Assay kit (G-Biosciences, Maryland Heights, MO, USA). From each sample, 50 microg +- 0.5 microg of proteins were digested by adding Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA) at an enzyme/substrate ratio of 1:50 (w/w) and incubating them overnight at 37C; an additional aliquot of Trypsin was added (enzyme/substrate ratio of 1:100 (w/w)) in the morning and the digestion continued for 4 hours. The enzymatic reaction was chemically stopped with the addition of 0.5% TFA (Sigma-Aldrich Inc., St. Louis, MO, USA), and a subsequent incubation at 37C for 45 minutes completed the acidic hydrolysis of RapiGest. The water immiscible degradation products were removed by centrifuging at 13,000 x g for 10 minutes. Finally, the tryptic digested mixtures were desalted using PepCleanTMC18 spin column (Thermo Fisher Scientific, Waltham, MA, USA), concentrated in SpeedVac (Savant Instruments, Farmingdale, NY, USA) at 60 C and resuspended in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) at a concentration of 0.1 microg/microL. Trypsin digested mixtures were analyzed by means of a platform consisting of a nano-liquid chromatographic system, Eksigent nanoLC-Ultra 2D System (Eksigent, part of AB SCIEX Dublin, Dublin, CA, USA) configured in trap-elute mode, coupled with a high-resolution mass spectrometer. Briefly, samples (0.8 microg injected) were first loaded on a peptide trap (200 microm x 500 microm ChromXP C18-CL, 3 microm, 120 A) and washed with the loading pump running in isocratic mode with 0.1% formic acid in water for 10 min at a flow of 3 microL/min. The automatic switching of a ten-port valve then eluted the trapped mixture on a nano-reversed phase column (75 microm x 15 cm ChromXP C18-CL, 3 microm, 120 A) through a 150 min gradient of eluent B (eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile) at a flow rate of 300 nL/min. In depth, the gradient was: from 5-10% B in 3min, 10-40% B in 130 min, 40-95% B in 10 min and holding at 95% B for 10 min. Mass spectra were acquired using a LTQ-Orbitrap XL-ETD mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA), equipped with a nanospray ionization source (Thermo Fisher). The spray capillary voltage was set at 1.7 kV and the ion transfer capillary temperature was held at 220 C. Full mass spectra were recorded in positive ion mode over a 400-1600 m/z range and with a resolution setting of 30000 FWHM and scan rate of 2 spectra per second, followed by five low-resolution MS/MS events, sequentially generated in a data-dependent manner on the top five most intense ions selected from the full MS spectrum (at 35% collision energy), using dynamic exclusion of 0.5 min for MS/MS analysis. Mass spectrometer scan functions and high-performance liquid chromatography solvent gradients were controlled by the Xcalibur data system version 1.4 (Thermo Fisher Scientific). Sample legend: CS--&gt; Controls R-Pre--&gt; Responders pre treatment R-Post--&gt; Responders post treatment NR-Pre--&gt; Non-Responders pre treatment NR-Post--&gt; Non-Responders post treatment P-Pre--&gt; Placebo pre treatment P-Post--&gt; Placebo post treatment