Jet stream dual electrospray

The separation and analysis of samples were performed with a HPLC-MS system consisting of an Agilent 1290 Infinity II Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an Automated Multisampler module and a High-Speed Binary Pump and connected to an Agilent 6550 Q-TOF Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA) using an Agilent Jet Stream Dual electrospray (AJS-Dual ESI) interface.
Standards or samples (20 uL) were injected onto an Agilent Zorbax Eclipse Plus C18 (2.1 × 100 mm, 1.8 um) HPLC column, at a flow rate of 0.4 mL/min. The column was equilibrated at 40 °C. Solvents A (MilliQ water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid) were used for the compound separation with the following elution program: 2 min at 3% B, linear gradient from 3 to 100% B in 9 min, and 1 min at 100% B.
The mass spectrometer was operated in the positive mode. Extracted ion chromatograms of the following compounds were analyzed: 273.1849 > 255.1749 m/z for β-estradiol (E2), 315.2319 > 109.0660 for P4, 330.0566 > 136.0623 for AMPc and 346.0547 > 152.05686 for GMPc.

Proteomics was performed on 50 grown oocytes/group recovered by slicing ovaries in PBS. ZP was solubilized by incubation at 75°C for 45 min (Izquierdo-Rico et al., 2009 (link)) and analyses were carried out on one technical replicate with a HPLC/MS system consisting of an Agilent 1290 Infinity II Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an Automated Multisampler module and a High Speed Binary Pump, and connected to an Agilent 6550 Q-TOF Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA) using an Agilent Jet Stream Dual electrospray (AJS-Dual ESI) interface. Experimental parameters for HPLC and Q-TOF were set in MassHunter Workstation Data Acquisition software (Agilent Technologies, Rev. B.08.00). Data processing and analysis was performed on Spectrum Mill MS Proteomics Workbench (Rev B.06.00.201, Agilent Technologies, Santa Clara, CA, USA).