Sep pak vac tc18 100 mg cartridge

Approximately 100 µg of total soluble protein extract samples were adjusted in volume to 100 µL with 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 8.0). Reduction with 10 mM dithiothreitol in HEPES (pH 8.0) was applied and incubation took place for one hour at 37 °C, followed by alkylation with 25 mM iodoacetamide in HEPES (pH 8.0), incubation for two hours in dark and precipitation with 10 volumes of cold acetone overnight −20 °C. The samples were then centrifuged at 16,000 g, supernatant was decanted, and pallets were re-suspended in 100 mM HEPES (pH 8.5) and sonicated. After that, samples were subjected to digestion with trypsin in HEPES (pH 8.5) 1:40 ratio over-night at 37 °C, label with isobaric tandem mass tag (TMT™) ten-plex (Thermo Fisher Scientific, Waltham, MA, USA) for two hours at room temperature, quench with 8 µL of 5% hydroxylamine in 100 mM HEPES (pH 8.5) for one hour, and then further quenched with 100 µL water overnight at 4 °C. Each biological replicate was labelled separately then pooled together for further analysis. In a final step, the pooled TMT labelled peptides were desalted using Sep-Pak Vac tC18 100 mg cartridge (Waters, Milford, MA, USA). The desalted sample was then dried with a Speed Vac concentrator.

Approximately 1 mg of total soluble protein extract was reduced with 5 mM dithiothreitol for 2 h at 37°C, cooled and then proteins were alkylated with 14 mM iodoacetamide for 30 min at room temperature in the dark. Unreacted iodoacetamide was quenched by increasing dithiothreitol concentration to 10 mM with a further incubation for 15 min at room temperature in the dark. Proteins were diluted to 1.5 M urea with 50 mM triethylammonium bicarbonate (TEAB) buffer (Sigma-Aldrich) and incubated at 50:1 ratio with sequencing-grade modified trypsin (Promega, Madison, WI, USA) overnight at 37°C with gentle agitation. Protein digestion was stopped by acidification of the mixture to pH 2.0 with trifluoroacetic acid. Acidification serves to precipitate lipids that would interfere with downstream purification and importantly to prepare the samples for desalting which requires peptide mixture to be acidic (Hsu et al., 2009 (link)). Peptides were then purified using Sep-Pak Vac tC18 100 mg cartridge (Waters, Milford, MA, USA), as described previously (Groen et al., 2013 (link)), and completely dried in a Speed Vac concentrator (Thermo Scientific, Bremen, Germany).

At 10 days post-subculturing, three biological replicate flasks were treated with 100 nM of 8-bromo-cAMP and cells of each mock (equal volume of water) or cAMP treated were collected at 0, 5 and 10 min post-treatment. Media were drained off using Stericup^® filter unit (Millipore, Billerica, MA, USA), and the cells were immediately snap frozen in liquid nitrogen and stored at −80 °C until use. Approximately 1 g of cells was ground to a fine powder with mortar and pestle in liquid nitrogen and proteins were precipitated in trichloroacetic acid in acetone, vortexed and incubated overnight. Precipitated proteins were pelleted, washed and re-suspended in urea lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate). Approximately 100 µg of total soluble protein extract was reduced, alkylated, digested with trypsin and purified using Sep-Pak Vac tC18 100 mg cartridge (Waters, Milford, MA, USA), as described previously [68 (link)], prior to drying in a Speed Vac concentrator (Thermo Scientific, Bremen, Germany).