Lcq deca xp max

The ACPs produced from E. coli or from the phosphopantetheinylation reaction mixture were directly analyzed by LC-MS (Agilent 1200, Thermo Finnigan LCQ Deca XP MAX). LC separation was carried out on a Agilent SB-C18 column (3.5 μm, 80 Å, 2.1 × 150 mm, Agilent) at 35 °C. Solvent A consisted of 0.1% formic acid. Solvent B consisted of acetonitrile. The following binary gradient was used: 0–5 min, 5% B; 5–45 min, a linear gradient to 75% B; followed by 3 min isocratic elution of 75% B, and equilibrated to initial condition for 13 min at a flow rate of 0.2 ml/min. UV detection was performed at both 254 nm and 280 nm. MS equipped with ESI source was performed as follows: positive; source voltage, 2.5 kV; capillary voltage, 41 V; sheath gas flow, 45 arb; aux/sweep gas flow, 5 arb; capillary temperature, 330 °C.

Ten mg hydrocortisone
3-(O-carboxymethyl)oxime (Toronto Research Chemicals) and 10.4 mg
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU, Fluorochem) were dissolved in 2 mL DMF. Subsequently, 44 μL
diisopropylethylamine (DIPEA, Sigma) was added together with 16.5
mg 1-(2 aminoethyl)maleimide hydrochloride (Sigma) before the mixture
was stirred for 4 h at room temperature. DMF was removed using a Buchi
rotary evaporator and a diaphragm pump. The remaining compound was
dissolved in Milli-Q water with 25% HPLC-grade acetonitrile and 0.1%
formic acid. Cortisol-3-CMO-maleimide was purified with a preparative
LC-MS system comprising of an LCQ Deca XP Max (Thermo Finnigan) ion-trap
mass spectrometer equipped with a Surveyor photodiode detector array
(PDA) detector (Thermo Finnigan). The mixture was purified with a
Phenomex kinetex 2.6 μm EVO C18 50 × 2.1 mm column and
an isocratic acetonitrile gradient of 34%. Fractions with the correct
mass (E/Z isomers were not separated) were collected using a PrepFC
fraction collector (Gilson Inc.) and subsequently freeze-dried before
being dissolved in DMSO and stored at −30 °C (Figure S4).

The ESI mass spectrum data of small peptides and proteins were obtained from a Thermo Finnigan LCQ DECA XP MAX (ESI ion source, positive mode). MagTran 1.03 and ESIProt 1.0 software were used for data deconvolution.

The metabolites were separated by an HPLC system (Thermo Finnigan, Waltham, MA, USA) using 0.1% (V/V) formic acid in water (buffer A) and 100% acetonitrile (buffer B) as mobile phases at a flow rate of 0.15 ml/min. The HPLC separation was carried out using a reversed-phase column (ZORBAX 300SB-C18, 2.1 × 150 mm, 3.5 μm; Agilent, Santa Clara, CA, USA). The gradient profile was 8% B for 2 min, increased to 20% B in 38 min, then to 100% B in 12 min and maintained for 10 min, and decreased to 8% B in 2 min and maintained for 10 min. The acquisition time was 55 min and delay time was 5 min per spectrum. The metabolites were detected by monitoring the absorption at 280 and 340 nm.
The metabolites were identified using an ion trap mass spectrometer (LCQ DECA XP MAX) coupled with an ESI source (Thermo Finnigan). The MS parameters for analysis were: capillary temperature 280 °C, spray voltage 4.5 kV, sheath gas (nitrogen) flow rate 40 arb and aux/sweep gas (nitrogen) flow rate 10 arb. Collision energy and other tune parameters were optimized for dissociation of parent ions into product ions for each metabolite. The mass spectrometer was acquired in data-dependent MS/MS mode: each full MS scan (in the range 100–220 m/z) was followed by four MS/MS of selected ions including substrate, intermediate product, end product and internal standard.

Identification of phenolic compounds present in the different HPLC separated fractions was carried out by ESI-ITMSⁿ using a Finnigan LCQ DECA XP Max ion trap mass spectrometer (Thermo Finnigan, San Josè, CA, USA), equipped with Xcalibur® system manager data acquisition software (Thermo Finnigan, San José, CA, USA). Mass spectra were recorded from mass-to-charge ratio (m/z) 80 to 1800 both in negative and in positive ionization mode. The capillary voltage was set at −28 V, the spray voltage was at 3 kV and the tube lens offset was at −10 V in negative ion mode, while the capillary voltage was set at 34 V, the spray voltage was at 3.5 kV and the tube lens offset was at 55 V in positive ion mode. The capillary temperature was 275 °C. Data were acquired in MS, MS/MS and MSⁿ scanning mode.

Ovaries were weighted and put into a tube with 1ml chloroform-methanol (2:1, V/V) solution. After thoroughly homogenized, the substances were removed by centrifugation and the fluid was dried using nitrogen gas (N₂). To get total cholesterol, 1U cholesterol esterase (Worthington) were added and the reaction was proceeded at 37°C for 1 hour. 1ml hexane was used to extract the total lipid. The hexane phase was dried and re-dissolved in 50μl acrylonitrile- isopropanol (4:1). 20μl fluid was used to detect total cholesterol content. Standard curve were done by accurate weighting and gradient diluting the standard cholesterol (Sigma). All of the HPLC analyses used an Agilent 1200 system. The reactions were analyzed using an Agilent Extend-C18 column (5um, 4.6×250mm) thermostatted at 35°C, a solvent system of methanol: Acetonitrile (9:1, v/v) and a flow rate of 0.8ml/min. The mass detection was carried out by a Thermo Finnigan LCQ Deca XP MAX. The source type was Atmospheric Pressure Chemical Ionization (APCI) in positive polarity mode. The capillary temperature and APCI vaporizer temperature was 150°C and 450°C respectively.