Tof ultraflex 2 mass spectrometer

The selected protein spots were excised from silver-stained gels, cut into small pieces, and dehydrated in 50 µl acetonitrile (ACN) for 5 min at room temperature. The gel pieces were dried following removal of the acetonitrile and incubated in 50 µl 10 mM DTT at 56°C for 1 h, followed by an alkylating incubation in 50 µl 55 mM iodoacetamide in the dark. Subsequently, the spots were dehydrated with 50 µl ACN, rehydrated in 5 µl trypsin for 30 min, and then 10 µl 25 mM ammonium bicarbonate was added. Proteolysis continued overnight at 37°C and was then stopped by adding 10 µl 2% formic acid and desalted using C18 ZipTips (EMD Millipore). The resulting peptides were concentrated, mixed with α-cyano-4-hydroxycinnamic acid (α-HCCA), deposited on a 384-well MALDI target and air-dried. All samples were analyzed in the positive-ion, reflectron mode on a TOF Ultraflex II mass spectrometer (Bruker Corporation). The accelerating potential was 20 kV with eight shots per second. Trypsin autodigestion peaks were used for internal calibration.

All the starting materials were purchased from Aladdin or Sangon Biotech. Commercial reagents were directly used, unless otherwise noted. All chemicals are in reagent grade or better. NTR was purchased from Sigma-Aldrich [1 U is defined as the enzyme activity that cleaves 1 μmol of the standard substrate per minute in the presence of menadione and NADH at 37°C]. A 300-MHz Bruker AV 300 spectrometer was used to obtain the ¹H nuclear magnetic resonance (NMR) and ¹³C NMR spectra. Electrospray ionization–MS spectra were obtained on an LCQ Advantage MAX ion trap mass spectrometer (Thermo Fisher Scientific). MALDI ionization–time-of-flight (TOF)/TOF mass spectra were obtained on a TOF Ultraflex II mass spectrometer (Bruker Daltonics). An Agilent 1200 HPLC system, which was equipped with a G1322A pump, an in-line diode array ultraviolet detector, and an Agilent Zorbax 300SB-C18 RP column, was used for HPLC analyses. TEM observations were conducted on a JEM-2100F electron microscope with a working acceleration voltage of 100 kV. Normal HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) containing 10% fetal bovine serum at 37°C, 5% CO₂, and humid atmosphere. Hypoxic HeLa cells were cultured in DMEM (HyClone) with 10% fetal bovine serum at 37°C, 1% O₂, and 5% CO₂.