Agilent 6520 q tof tandem mass spectrometer

HPLC was performed using an UltiMate 3000 liquid chromatographic system (Thermo-Dionex Corporation, USA) equipped with a DAD, an auto sampler, and a column compartment. Separation was carried out in a C₁₈ column (250 × 4.6 mm, 5.0 mm particle size, Thermo Scientific Syncronis, USA). A centrifuge (Star Scientific Instrument Co., Ltd., China) and ultrasonic instrument (China) were used during sample preparation.
Mass spectrometry was conducted on an Agilent 6520 Q-TOF tandem mass spectrometer equipped with an electrospray ionization (ESI) source (Agilent Corp., USA).

In-gel digestion with trypsin was carried out using the In-Gel Tryptic Digestion Kit (Thermo-Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, bands were destained in 200 mM ammonium bicarbonate, washed with 50:50 acetonitrile:water, reduced with 10 mM TCEP in 50 mM ammonium bicarbonate (pH 8.6) buffer for 10 minutes at 60 °C, and alkylated in 55 mM iodocetamide in 100 mM ammonium bicarbonate for one hour. Trypsin in ammonium bicarbonate at 20 ng/µL was added to cover gel pieces and incubated overnight at 30 °C. The digests were analyzed via LC-MSMS, using an Agilent 1200 LC system, an Agilent Chip-cube interface and an Agilent 6520 Q-TOF tandem mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Data files were transferred to an Agilent workstation equipped with Spectrum Mill software (Agilent Technologies, Santa Clara, CA, USA). The raw MS/MS data files were extracted, sequenced, and searched against the seven amino acid sequences shown in Figure 1. The LC-MSMS data is shown in Figure S1.