Ammonium bicarbonate

The proteins were centrifuged and precipitated in ice-cold acetone (1:5 v/v). After precipitation, the protein pellet was reconstituted in 0.25% RapidGest SF (Waters™, USA) in 15 mM ammonium bicarbonate (Sigma-Aldrich, USA.). 60 µg of protein in each group was subjected to gel-free digestion. Then, sulfhydryl bond reduction and sulfhydryl alkylation were performed using 5 mM DTT (Sigma-Aldrich, USA) in 15 mM ammonium bicarbonate at 72 °C for 1 h and adding IAA (Sigma-Aldrich, USA) in 15 mM ammonium bicarbonate at room temperature for 30 min in the dark, respectively. The solution was desalted by a Zeba Spin Desalting Column (Thermo Scientific, USA), digested with trypsin (Promega Co., Madison, WI, USA), and incubated at 37 °C for 3 h. The digested solution was dried and reconstituted in 0.1% formic acid before being subjected to LC–MS/MS. The experiment was conducted in 3-biological replications.
The LC–MS/MS spectrum data were collected in the positive mode with an HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer combined with an EASY-nLC1000 nano-LC system equipped with a nano-C18 column. Mobile phase A comprises 0.1% formic acid, and mobile phase B comprises 90% acetonitrile with 0.1% formic acid. The samples were loaded into an analytical C₁₈ column.

Gel pieces were de-stained with 50% acetonitrile in 50 mM ammonium bicarbonate (Merck). The disulfide bonds in the proteins were reduced by treatment with 4 mM DTT in 50 mM ammonium bicarbonate at 60 °C for 15 min. Subsequently, the proteins were alkylated by incubation with 250 mM iodoacetamide at room temperature in the dark for 30 min. The reaction was quenched by 4 mM DTT in 50 mM ammonium bicarbonate for 5 min at room temperature. All solutions were removed, and the gel pieces were dehydrated by acetonitrile. The gel pieces were then air dried and resuspended in 0.1 μg/μl of Trypsin Proteomics Grade (Sigma Aldrich, Missouri, USA) in 50 mM ammonium bicarbonate. The digestion was performed overnight at 37 °C. The digested peptides were extracted by acetonitrile and dried using a centrifugal evaporator.

The cell pellets were resuspended in 100 µL 8 M urea (Merck KGaA, Darmstadt, Germany) and 100 mM ammonium bicarbonate (Merck KGaA, Darmstadt, Germany), and sonicated for 10 min at 50% power at three 10 s intervals. The samples were then heated to 95 °C on a heat block for 10 min, then centrifuged for 1 min at 5000× g. The solution was then reduced and alkylated by adding a final concentration of 10 mM tributyl-phosphate (TBP, Merck KGaA, Darmstadt, Germany) and 20 mM acrylamide (Merck KGaA, Darmstadt, Germany), then vortexed and spun down on a mini-centrifuge (Qik Spin QS7000 Edwards Instruments, Elkhorn, WI, USA) at 2000× g for 2 s. The samples were incubated for 90 min at room temperature then quenched with a final concentration of 50 mM dithiothreitol (DTT, Merck KGaA, Darmstadt, Germany) and again vortexed and spun down on a mini-centrifuge at 2000× g for 2 s. The samples were then diluted 1:8 in 100 mM ammonium bicarbonate. We then added 0.5 µg of trypsin to digest at 37 °C for a minimum of 12 h. The samples were then desalted using Stop and Go Extraction (STAGE) tips solid phase extraction columns. The peptide concentration was determined using the Pierce quantitative colorimetric peptide assay (Thermofisher Scientific, Sydney, NSW, Australia) and prepared for LC-MS/MS analysis.