Pierce fluorometric peptide assay

Desalted peptides were reconstituted in 40 μl of 50 mM TEAB and quantified using Pierce Fluorometric Peptide Assay (Thermo Scientific). Each sample was diluted with 50 mM TEAB to 0.5 μg/μl for a total of 50 μg of peptide per replicate and labeled with TMT 6 Plex Mass Tag Labeling Kit (Thermo Scientific). Briefly, 41 μl of each TMT label (126–131) was added to each digested peptide sample and incubated for 1 h. The reaction was quenched with 8 μl of 5% hydroxylamine and incubated for 15 min. All labeled samples were then mixed together and lyophilized to almost dryness. TMT labeled samples were reconstituted in 0.1% trifluoroacetic acid (TFA) and the pH was adjusted to 2 with 10% TFA. The combined sample (20 μg) was separated into 8 fractions by Pierce High pH Reverse-Phase Peptide Fractionation Kit (Thermo Scientific) with an extra wash before separation to remove excess labels. The 8 fractions were dried almost to completion.

Proteins were prepared for mass spectrometry using a modified version of the S-trap protocol (Protifi). Proteins in lysates were reduced with 5 mM TCEP for 10 min at 55°C and alkylated with 15 mM MMTS for 10 min at room temperature. The lysates were acidified to a final concentration of 1.2% v/v phosphoric acid. A 6× volume of S-trap binding buffer (90% methanol, 100 mM TEAB, pH 7.55) was mixed to each sample to precipitate proteins. The solution was loaded onto S-trap micro columns (Protifi) and spun at 4000 × g for 1 min until all the solution had passed through the column. The columns were washed four times with 150 µL of S-trap binding buffer and centrifuged at 4000 × g for 1 min between each wash. Proteins were digested on-column with 0.75 µg of trypsin (Promega) in 50 mM TEAB pH 8.5 overnight at 37°C in a humidified incubator. Digested peptides were eluted in three steps at 4000 × g for 1 min: 40 µL of 50 mM TEAB, 40 µL of 0.2% formic acid, and 35 µL of 50% acetonitrile/0.2% formic acid. The peptide concentrations of eluted peptides were quantified using the Pierce Fluorometric Peptide Assay (Thermo Fisher Scientific) according to the manufacturer’s instructions. The remaining samples were frozen in liquid nitrogen and lyophilized.