C18 hypersep plate

Proteins were reduced with adding 1.5 μL of 200 mM dithiothreitol (DTT, Sigma) in 500 mM AmBic and incubated at 37°C for 1 h with shaking at 400 rpm. Alkylation was performed with adding 1.5 μL of 66 mM iodoacetamide (Sigma) in 500 mM AmBic at room temperature for 30 min with shaking at 400 rpm. Thereafter 1 μg of sequencing grade modified Trypsin (Promega) was added to each sample (1:33 trypsin:protein) and incubated for 16 h at 37°C. The digestion was stopped by adding of formic acid at final concentration of 5% and incubating the solution for 20 min at 37°C. Then the samples were cleaned on a C18 Hypersep plate (Thermo Scientific), dried using a Speedvac and re-suspended in 70 μL of 50 mM triethylammonium bicarbonate (TEAB) buffer and 30 μL of TMT-10plex (Thermo Scientific) reagent was added in dry acetonitrile (ACN) following incubation for 2 h at room temperature with shaking at 550 rpm. Labeling reaction was quenched with 11 μL of 5% hydroxylamine. Labeled samples were combined and dried on Speedvac. Following a cleaning on StageTip C18, 20 μL of the combined samples were dissolved in 0.1% formic acid and 2% ACN.

The methods used were the same as reported previously (3 (link)). Frozen samples were powdered by Mikro-dismembrator (B. Braun Biotech International, Germany) on dry ice. Powdered tissue samples were solubilized in 8M urea and 100 mM NaCl with 1% ProteaseMAX (Promega) in 100 mM ammonium bicarbonate (AmBic) and mixed vigorously. Low binding silica beads (400 µm, Ops Diagnostics, Lebanon NJ) were added to each sample and vortexed at high speed. Subsequently, samples were subjected twice to disruption on a Vortex Genie disruptor for 2 min before addition of AmBic, urea and NaCl. Following centrifugation, the 50 mM AmBic was added and vortexed vigorously. Proteins were then reduced with 100 mM dithiothreitol in 50 mM AmBic, incubated at 37°C and alkylated with 100 mM iodoacetamide in 50 mM AmBic. The reaction was stopped with formic acid and the samples were then cleaned on a C18 Hypersep plate (bed volume of 40 µL, Thermo Scientific) and dried in a vacuum concentrator (miVac, Thermo Scientific).