Bottom-up proteomics

Samples were lysed with lysis buffer (2% SDS, 50mM Tris-HCl pH 8.5, 10mM TCEP, 40mM chloroacetamide and protease inhibitor cocktail tablet [EDTA-free, Roche]). Samples were incubated for 5 minutes at 95°C before sonication with Sonic Vibra Cell at 1s ON/ 1s OFF pulse for 30s at a maximal amplitude of 30% to shear genomic DNA. After sonication, samples were incubated for 10min at 95°C. Proteins were precipitated using 3 volumes of ice-cold methanol, 1 volume Chloroform and 2.5 volumes ddH2O. After centrifugation at 14,000g for 45min at 4°C, the upper aqueous phase was aspirated and 3 volumes of ice-cold methanol were added. Samples were mixed and proteins pelleted by centrifugation at 14,000g for 5 min at 4°C. Supernatant was discarded and pellets washed one additional time with ice-cold methanol. Protein pellets were dried at RT for further use. Proteins were resuspended in 8M Urea, 50mM Tris pH 8.2 and protein concentration determined using a BCA assay (ThermoFisher Scientific, 23225). Samples were then diluted to 4M urea using digestion buffer (50mM Tris pH 8.2) and incubated with LysC (Wako Chemicals) at 1:50 (w/w) ratio for 4 hours at 37°C and were further diluted to 1M Urea using digestion buffer with 1mM CaCl2 final concentration and incubated at a 1:100 (w/w) ratio of Trypsin (Promega, V5113) overnight at 37°C. Digests were acidified using trifluoroaceticacid (TFA) to a pH of 2-3 and peptides purified using using Empore C18 (Octadecyl) resin material (3M Empore). Material was activated with Methanol, followed by one wash each with 70% acetonitrile / 0.1% TFA and 0.1% TFA. Samples were resuspended in 0.1% TFA and loaded to resin material. Peptides were washed with 0.1% TFA and eluted with 70% acetonitrile (ACN). Eluates were dried and stored for further processing. Peptides were resuspended in TMT labelling buffer (0.2M EPPS pH 8.2, 10% Acetonitrile) and peptide concentration determined by µBCA (ThermoFisher Scientific, 23235). Peptides were mixed with TMT reagents (ThermoFisher Scientific, 90111, A37724, 90061) in 1:2 (w/w) ratio (2µg TMT reagent per 1µg peptide). Reactions were incubated for one hour at RT and subsequently quenched by addition of hydroxylamine to a final concentration of 0.5% at RT for 15min. Samples were pooled in equimolar ratio (unless stated otherwise), acidified, and dried for further processing. Before MS-analysis, peptide samples were purified using Empore C18 (Octadecyl) resin material (3M Empore) as described before, except peptides were resuspended in 3% Acetonitrile/ 0.1%TFA and washed with 3% Acetonitrile/ 0.1% TFA. After elution, samples were dried and resuspended in 2% Acetonitrile/1% formic acid (FA) for LC-MS2/3 . Peptides were fractionated using a Dionex Ultimate 3000 analytical HPLC. For high pH reversed phase fractionation on the Dionex HPLC, 500 μg of pooled and purified TMT-labeled samples were resuspended in 10 mM ammonium-bicarbonate (ABC), 5% ACN, and separated on a 250 mm long C18 column (Aeris Peptide XB-C18, 4.6 mm ID, 2.6 μm particle size; Phenomenex) using a multistep gradient from 100% Solvent A (5% ACN, 10 mM ABC in water) to 60% Solvent B (90% ACN, 10 mM ABC in water) over 70 min. Eluting peptides were collected every 45 s into a total of 96 fractions, which were cross-concatenated into 12 fractions and dried for further processing.