Sequence grade modified

After protein quantification, 20 µg of each sample were aliquoted to a new tube and brought to an equal volume in 50 mM HEPES, pH 8.0. Samples with less than 20 ug total required use of the full sample for digest and the trypsin ratio was adjusted at the digestion stage. Samples were reduced with 10 mM dithiothreitol at 42°C for 30 minutes on a ThermoMixer with 500 rpm agitation, alkylated with 15 mM iodoacetamide for 20 minutes at room temperature on a ThermoMixer with 500 rpm agitation, and trypsin-digested (Sequence Grade Modified; Promega) with a 1:50 trypsin:protein ratio at 37°C for 20h. Following the digest, all peptide samples were C18-purified using Millipore C18 zip-tips (Millipore, Cat#: ZTC18S096) according to the manufacturer protocol’s and as previously reported (48 (link)). Eluted purified peptides were dried with a vacuum centrifuge and stored at −20°C until tandem mass tag (TMT) labeling was performed.

After protein quantification, 100 µg of each sample were aliquoted and the urea was diluted 3-fold with 50 mM HEPES, pH 8.2 (brought <2M). Samples were reduced with 10 mM dithiothreitol at 42°C for 30 minutes on a ThermoMixer with agitation (500 rpm), alkylated with 15 mM iodoacetamide at room temperature for 20 minutes on a ThermoMixer with agitation (500 rpm), and trypsin-digested (Sequence Grade Modified; Promega) with a 1:100 trypsin:protein ratio at 37°C for 20 hours. Following the digest, all peptide samples were C18-purified using C18 MacroSpin Columns (Harvard Apparatus, Cat. #74–4101) according to the manufacturer’s protocol. Eluted purified peptides were dried with a vacuum centrifuge and stored at −20°C until TMT labeling was performed.