C18 ziptip micropipette tips

A gel containing 100 μg of proteins was mixed with dithiothreitol (final concentration of 10 mM) and kept at 60 °C for 70 min [15 (link),16 (link)]. After the sample was cooled, iodine acetamide with a final concentration of 66 mM was added and reacted away from light at room temperature for 40 min [16 (link)].
After that, 200 μL NH₄HCO₃ solution (50 mM) was added to the filter and centrifuged at 14,000× g for 25 min. Finally, 1.25 μg of trypsin was added and the mixture incubated at 40 °C overnight [16 (link),17 (link)]. The digestion procedure was stopped by adding formic acid (10%), and digested peptides were collected by centrifuging at 14,000× g for 15 min. After desalting with the C18 tips, the collected peptides were vacuum-dried [17 (link),18 (link)]. After trypsin digestion, Ziptip C18 micropipette tips (Millipore) were used to purify the peptides prior to adding 0.1% formic acid for LC-MS/MS analysis.

Mass spectrometry analysis was performed with a 4800 Plus (Sciex) or Microflex (Bruker Daltonics) MALDI-TOF mass spectrometer using 0.25 M aqueous 3-hydroxypicolinic acid as the matrix and 10 mM diammonium citrate as an additive. Spectra were recorded for positive ions. Before analysis, the samples were desalted and concentrated using ZipTip C18 micropipette tips (Millipore).

TmT duplex isobaric tags reagent set (TMT²) and trypsin for MS were purchased from Thermo Scientific (San Jose, CA, USA). EDTA-free protease inhibitor cocktail was purchased from Roche Diagnostics (Indianapolis, IN, USA). Phosphatase inhibitors were purchased from Pierce (Idaho, ID, USA). RIPA Lysis Buffer was purchased from Thermo Scientific (San Jose, CA, USA). The Bradford assay was purchased from Applygen (Beijing, China). C18 ZipTip micropipette tips were purchased from Millipore (Bedford, MA, USA). Generic chemicals were purchased from Sigma-Aldrich (St. Louis, USA). All of the chemicals and reagents used in this study were of analytical chromatography grade.