Ltq orbitrap hybrid tandem mass spectrometer

The protein digests were analyzed in an Agilent 1,200 nanoflow HPLC system (Agilent Technologies, Santa Clara, CA, United States) connected to an LTQ-Orbitrap hybrid tandem mass spectrometer (ThermoFisher, Waltham, MA, United States). For the setting of LTQ-Orbitrap, multiple selective ion monitoring events with Orbitrap collecting various overlapping range of m/z was set up specifically, 300–800, 625–1,075, 925–1,375, 1,225–1,675, and 1,525–1975 m/z. Full scans with Orbitrap analyzes were collected in the range of 200 to 2000 m/z as the last event. To identify various peptides, the Dynamic Exclusion function in Data Dependent Settings was enabled, with the Repeat Count set to 2, Exclusion Duration set to 180 s, and Exclusion list Size set to 50 signals. Only charge 2 and charge 3 ions were not rejected after Charge State Rejection was enabled. The top three ions in the survey scan that fulfill the above criteria were analyzed for the MS/MS with LTQ collision cell/mass analyzer. File Converter in Xcalibur SR 2.0 (TheroFisher, United States) and in-house programs written in Visual Basic for Application (VBA) were used to extract the MS/MS and calculate the charge and masses of the precursor ions. All MS/MS data were utilized in subsequent searches by the TurboSEQUEST program (Thermo Electron) using the UniProt protein sequence database for mouse proteins.

The ESI(+)MS experiments were carried out using a LTQ-Orbitrap hybrid tandem mass spectrometer (ThermoFisher, Waltham, Massachusetts, USA) that was equipped with an electrospray ionization (ESI) source operating in positive ion mode and set up in line with an Agilent 1200 HPLC system. The HPLC column was an Agilent mRP-C18 High-Recovery Protein Column (length: 100 mm; internal diameter: 0.5 mm; bead size: 5 μm). The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. The parameters of the ESI(+) consisted of 4.0 kV ion spray voltage, 200 °C capillary temperature, and sheath gas flow rate at 3–5 arbitrary unit. Mass spectra were collected over the mass range of m/z 50–2000 at a resolving power of 30,000. After collection, the data were analyzed using Xcalibur software (ThermoFisher, Waltham, MA, USA). The molecular masses of the potential antibiotic molecules detected by ESI-MS were compared with those of known natural antibiotics.