Q exactive focus

Electrospray ionization (ESI) mass spectrometric analyses were carried out on a Q Exactive^TM Focus, high-resolution and high-mass accuracy, hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Spectra were acquired in the 200–2000 m/z range. Samples were dissolved in a mixture of MeCN/water (1:1, v/v) and 0.1% formic acid. Liquid chromatography–mass spectrometry (LC–MS) was carried out on an UltiMate 3000 UHPLC system (Thermo Fisher Scientific) coupled to the same spectrometer. Compounds were separated on a Supelco Ascentis C18 column (90 Å, 150 × 2.1 mm, 3 µm) (Hesperia, CA, USA), using a linear gradient from 20–90% B in 20 min (eluent A: ddH₂O, 0.1% HCOOH; eluent B: 80% MeCN, 0.1% HCOOH at a flow rate of 0.2 mL/min) and the column temperature was set to 40 °C. High-resolution mass spectra were acquired in the 200–2000 m/z range. LC–MS data were analysed by the Xcalibur^TM software (Thermo Fisher Scientific).

The identification of the peptide analogues and conjugates was achieved by electrospray ionization mass spectrometry (ESI-MS) on a Bruker Daltonics Esquire 3000 Plus (Bremen, Germany) ion trap mass spectrometer, operating in continuous sample injection at 4 µL/min flow rate. Samples were dissolved in ACN-water (50:50 v/v%) mixture containing 0.1 v/v% AcOH. Mass spectra were recorded in positive ion mode in the m/z 50–2000 range.
For the stability and metabolism studies of the conjugates, liquid chromatography–mass spectrometry (LC-MS) analyses were performed on a Q ExactiveTM Focus, high resolution and high mass accuracy, hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) using on-line UHPLC coupling. UHPLC separation was performed on a Dionex 3000 UHPLC system using a Supelco Ascentis C18 column (2.1 × 150 mm, 3 µm). Linear gradient elution (0 min 2% B, 1 min 2% B, 17 min 90% B) with eluent A (0.1% HCOOH in water, v/v) and eluent B (0.1% HCOOH in acetonitrile/water, 80:20, v/v) was used at a flow rate of 0.2 mL/min at 40 °C. High-resolution mass spectra were acquired in the 200–1600 m/z range. LC-MS data were analyzed by XcaliburTM software (Thermo Fisher Scientific) and with Origin Pro 8 (OriginLab Corp., Northampton, MA, USA).

The identification of the peptide derivatives and their DSPE-PEG conjugates was achieved by electrospray ionization mass spectrometry (ESI-MS) on a Bruker Daltonics Esquire 3000 Plus (Bremen, Germany) ion trap mass spectrometer, operating in continuous sample injection with 4 µL/min flow rate. Samples were dissolved in CH₃CN-water mixture (50:50 vol/vol%) containing 0.1 vol/vol% AcOH. Mass spectra were recorded in positive ion mode in the m/z 50–2000 range.
Before the biological characterization, the purity of the conjugate as the final product was investigated on a Q ExactiveTM Focus, high resolution and high mass accuracy, hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) using an online UHPLC coupling. UHPLC separation was performed on a Dionex 3000 UHPLC system using a Supelco Ascentis C18 column (2.1 × 150 mm, 3 µm). Linear gradient elution (0 min 2% B, 1 min 2% B, 17 min 90% B) with eluent A (0.1% HCOOH in water, vol/vol) and eluent B (0.1% HCOOH in acetonitrile/water, 80:20, vol/vol) was used at a flow rate of 0.2 mL/min at 40 °C. High-resolution mass spectra were acquired in the 200–1600 m/z range. LC-MS data were analyzed by XcaliburTM software (Thermo Fisher Scientific) and with Origin Pro 8 (OriginLab Corp., Northampton, MA, USA).

Samples were analyzed by HPLC-MS/MS using a Dionex UltiMate 3000 system coupled with a Q Exactive^TM Focus, high resolution and high mass accuracy, hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) using a Supelco Ascentis C18 column (2.1 × 150 mm, 3 µm). Linear gradient elution (0 min 2% B, 1 min 2% B, 11 min 90% B, 11.5 min 90% B, 12 min 2% B, 15 min 2% B) with eluent A (0.1% HCOOH in water, vol/vol%) and eluent B (0.1% HCOOH in acetonitrile/water, 80:20, vol/vol%) was used at a flow rate of 0.2 mL/min at 40 °C. Detection of daunomycin was in ESI + mode using Parallel Reaction Monitoring (PRM) at a resolution of 17,500 FWHM. The precursor ion (m/z: 528.19) was selected for analysis. The isolation window with was set to 2 m/z. Normalized collision energy (NCE) was 15%. LC-MS/MS data were visualized and analyzed by Xcalibur^TM software (Thermo Fisher Scientific). Peak area from the Extracted Ion Chromatograms (EIC) of the ion transition m/z 528.19 -> 321.07 (±0.5 Da) were used to calculate relative daunomycin concentration.

Chromatographic separation was accomplished in an Thermo Ultimate 3000 system equipped with an ACQUITY UPLC^® HSS T3 (150×2.1 mm, 1.8 μm, Waters) column maintained at 40°C. The temperature of the autosampler was 8°C. Gradient elution was carried out with 0.1% formic acid in water (C) and 0.1% formic acid in acetonitrile (D) or 5 mM ammonium formate in water (A) and acetonitrile (B) at a flow rate of 0.25 mL/min. Injection of 2 μL of each sample was done after equilibration. An increasing linear gradient of solvent B (v/v) was used as follows: 0~1 min, 2% B/D; 1~9 min, 2%~50% B/D; 9~12 min, 50%~98% B/D; 12~13.5 min, 98% B/D; 13.5~14 min, 98%~2% B/D; 14~20 min, 2% D-positive model (14~17 min, 2% B-negative model).
The ESI-MSn experiments were executed on the Thermo Q Exactive Focus mass spectrometer with a spray voltage of 3.5 kV and -2.5 kV in positive and negative modes, respectively. Sheath gas and auxiliary gas were set at 30 and 10 arbitrary units, respectively. The capillary temperature was 325°C. The orbitrap analyzer scanned over a mass range of m/z 81-1 000 for a full scan at a mass resolution of 70,000. Data-dependent acquisition (DDA) MS/MS experiments were performed with a HCD scan. The normalized collision energy was 30 eV. Dynamic exclusion was implemented to remove some unnecessary information in MS/MS spectra.

For method development, a Thermo Scientific UltiMate 3000 LC-system coupled to a Thermo Scientific Q Exactive focus hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used. In order to detect the PlTXs, electrospray ionisation in positive was used. The spray voltage in positive ionisation mode was set at 3.5 kV. The capillary temperature was set at 260 °C. For infusion experiments, PlTX was infused directly into the Mass spectrometer together with a flow of 0.1 mL min⁻¹ mobile phase containing 50% acetonitrile and additives. A full MS scan event of 500 to 1400 m/z with a resolution of 70,000 full width at half maximum (FWHM) was acquired. In order to obtain fragmentation spectra of PlTX, fragmentation spectra were also acquired. The so called MS² scans were obtained by selecting mass 898 ([M + H + 2Li]³⁺) with an isolation window of 4 Da. As collision gas nitrogen was used. The normalized collision energy (NCE) was set at 20 during fragmentation. Then after fragmentation, the ions were scanned from 187 to 1850 m/z with a resolution set at 70,000 FWHM. The automatic gain control representing the maximum capacity of the C-trap was set at a maximum of 10⁶ ions or a maximum injection time of 200 ms for the full scan and a maximum of 2 × 10⁵ ions or a maximum injection time of 200 ms for the MS² scans were allowed.