Proteomic grade trypsin

Water, acetone, acetonitrile, trifluoroacetic acid (TFA), 2,2′:5′,2″-terthiophene (TER), 1,5-diaminonapthalene (DAN), trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 4-hydroxy-α-cyano cinnamic acid (CHCA), 4-chloro-α-cyano cinnamic acid (CClCA), Nickel(II) phthalocyanine (NiPc), Nickel(II) 2,9,16,23-tetraphenoxy-29H-31H-phthalocyanine (NiPcPhe), Cu(II) phthalocyanine (CuPc), Zinc(II) Protoporphyrin IX (ZnPP), Cobalt chloride (II) Protoporphyrin IX (CoPP), cytochrome c (horse heart), myoglobin, human hemoglobin, and proteomic-grade trypsin, were obtained from Sigma-Aldrich (Milan, Italy). Livers of cod and bovine as well as mussel were purchased from a local supermarket. Plasma was drawn from a healthy voluntary subject. All solvents used were LC–MS grade. A mass standards kit containing bradykinin (2–9 clip), angiotensin I, glu1-fibrinopeptide, ACTH (1–17, 18–39, 7–38 clips) for calibration was purchased from AB Sciex (Concord, ON, Canada).

On day 8 post-crypt-isolation, Paneth-cell-enriched 3D organoids were extracted from Matrigel using Cell Recovery Solution (BD Bioscience), washed in PBS and centrifuged at 300 g for 5 min. Organoid pellets were lysed by sonication in 1% (w/v) sodium deoxycholate (SDC) in 50 mM ammonium bicarbonate. Samples were heated at 80°C for 15 min before centrifugation at 12,000 g to pellet debris. The supernatant was retained, and proteins reduced with 3 mM DTT (Sigma-Aldrich) at 60°C for 10 min, cooled, then alkylated with 9 mM iodoacetamide (Sigma-Aldrich) at room temperature for 30 min in the dark; all steps were performed with intermittent vortex-mixing. Proteomic-grade trypsin (Sigma-Aldrich) was added at a protein:trypsin ratio of 50:1 and incubated at 37°C overnight. SDC was removed by adding trifluoroacetic acid (TFA) to a final concentration of 0.5% (v/v). Peptide samples were centrifuged at 12,000 g for 30 min to remove precipitated SDC.

Protein from cell lysates was dispensed into low protein-binding microcentrifuge tubes (Sarstedt, Leicester, UK) and made up to 160 μl by addition of 25 mM ammonium bicarbonate. The proteins were denatured using 10 μl of 1% (w/v) RapiGest (Waters MS Technologies, Manchester, UK) in 25 mM ammonium bicarbonate followed by three cycles of freeze-thaw, and two cycles of 10 min sonication in a water bath. The sample was then incubated at 80°C for 10 min and reduced with 3 mM dithiothreitol (Sigma-Aldrich, Dorset, UK) at 60°C for 10 min then alkylated with 9 mM iodoacetamide (Sigma-Aldrich, Dorset, UK) at room temperature for 30 min in the dark. Proteomic grade trypsin (Sigma-Aldrich, Dorset, UK) was added at a protein:trypsin ratio of 50:1 and samples incubated at 37°C overnight. Three biological replicates were prepared for each cell type.

The LESB65, pmrB SNP and ∆pmrB strains were incubated on LB agar plates for 18 h at 37 °C. A sweep of colonies were inoculated into 5 ml of LB broth and incubated for a further 18 h at 37 °C and 180 r.p.m. A fresh dilution in LB broth was made by incubating 1.2 ml of the overnight culture into 28.8 ml of fresh LB media. The bacteria were incubated at 37 °C and 180 r.p.m. to an OD₆₀₀ between 0.5 and 0.6 and pelleted down at 4500 r.p.m. for 12 min. The pellet was washed once in phosphate buffered saline and stored at −80 °C until processing for proteomic analysis. Five replicates per condition were used. Bacterial cell pellets were lysed by sonication 1% (w/v) SDC in 50 mM ammonium bicarbonate. Samples were heated at 80 °C for 15 min before centrifugation at 12,000×g to pellet debris. The supernatant was retained, and proteins reduced with 3 mM DTT (Sigma) at 60 °C for 10 min, cooled, then alkylated with 9 mM iodoacetamide (Sigma) at RT for 30 min in the dark; all steps were performed with intermittent vortex-mixing. Proteomic-grade trypsin (Sigma) was added at a protein:trypsin ratio of 50:1 and incubated at 37 °C overnight. SDC was removed by adding TFA to a final concentration of 0.5% (v/v). Peptide samples were centrifuged at 12,000×g for 30 min to remove precipitated SDC.

Apical wash secretions from six batches of ALI day 14 mMEC cultures were pooled. TCA precipitation was performed (30% TCA in acetone) at −20°C for 2 h to precipitate soluble proteins (50 µg). Proteins were pelleted at 12,000 g for 10 min at 4°C and pellets washed three times with ice-cold acetone, air dried and resuspended in 50 mM ammonium bicarbonate, 0.1% RapiGest SF (Waters). Samples were heated at 80°C for 10 min, reduced with 3 mM DTT at 60°C for 10 min, cooled and then alkylated with 9 mM iodoacetamide (Sigma) for 30 min. All steps were performed with intermittent vortexing. Proteomic-grade trypsin (Sigma) was added at a protein:trypsin ratio of 50:1 and incubated at 37°C overnight. The samples were precipitated using 1% TFA at 37°C for 2 h and centrifuged at 12,000 g for 1 h (4°C) to remove RapiGest SF. The peptide supernatant was desalted using C₁₈ reverse-phase stage tips (Thermo Scientific Pierce) according to the manufacturer's instructions, dried and re-suspended in 3% (v/v) acetonitrile, 0.1% (v/v) TFA for analysis by mass spectrometry (MS).