Tmt 6 plex reagent

The dried peptides were dissolved with super pure water (18.2 MΩ) and labeled by using the tandem mass tags (TMT) 6-plex reagent (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. The labeling reaction was quenched by using hydroxylamine (Alfa Aesar, Tianjin, China). Each TMT labeled sample group contained an internal control for reference. Then the TMT labeled peptides were pooled together and desalted by using Sep-Pak columns (Waters, MA, USA) and dried again. The pooled TMT-labeled peptides were separated on a UPLC 3000 system (Thermo Fisher Scientific, Waltham, MA, USA) with a XBridgeTM BEH300 C18 column (Waters, Milford, MA, USA). The mobile phase A was H₂O and the mobile phase B was 98% acetonitrile and 2% H₂O. The flow rate used was 1 mL/min. TMT labeled peptides were eluted with a gradient solution of which the percent of phase B increased from 8% to 18% for 30 min, from 18% to 32% for 22 min. Forty-seven fractions were collected and dried by using a SpeedVac Vacuum concentrator and combined into 12 fractions. The TMT-labelled fractions were dissolved with 25 μL 0.1% formic acid (FA, Dikma Technologies Inc., CA, USA) and prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

C18 cartridges were primed using 70% acetonitrile (ACN) with 0.1% formic acid (FA) (100 μl, 300 μl/min), equilibrated in 2% ACN with 0.1% FA (50 μl, 5 μl/min), and eluates described above were loaded (280 μl, 5 μl/min). For performing reduction and alkylation, peptides were reduced in HEPES buffer containing 5 mM Tris (2-carboxyethyl) phosphine (TCEP; 100 μl, 5 μl/min), alkylated with 40 mM IAA (100 μl, 2 μl/min), and washed with HEPES buffer/5 mM TCEP (100 μl, 10 μl/min) followed by HEPES buffer alone (100 μl, 10 μl/min). For performing oxidation, the oxidant solution (5% formic acid, 1X H₂O₂) was prepared by mixing (1:1) 10% formic acid with 2X H₂O₂ (concentration varies). H₂O₂ was diluted with water from fresh 30% H₂O₂ (Sigma) to the appropriate concentration. Peptides were then washed with this oxidant solution (150 μl, 5 μl/min) followed by HEPES buffer (100 μl, 10 μl/min). For TMT tagging, 85 μg of TMT6plex reagent (Thermo Fisher) in HEPES containing 8% acetonitrile (ACN) was loaded over 25 min (50 μl at 2 μl/min). Finally, peptides were washed with 2% ACN with 0.1% FA (100 μl, 10 μl/min) and eluted in 30% ACN with 0.1% FA (50 μl, 5 μl/min). Fractions were transferred to glass LCMS vials (Agilent), dried for 10 min by speedvac, and stored at −80 °C.