Synapt g2 s hdms

Drift tube ion mobility-mass spectrometry (DT-IM-MS) and tandem mass spectrometry (MS/MS) were performed on a modified Synapt G2-S HDMS instrument (Waters Corporation, Manchester, UK) containing a drift tube instead of the commercial traveling wave cell^{39 (link)}. Glycolipid solutions (10 µM) were prepared as described in the previous section. In addition to protonated and sodiated glycolipids, silver adducts were also investigated. Silver adduction was shown to enable isomer distinction in several lipids by IM^{40 (link)}, and silver ion chromatography is commonly employed to separate lipids due to the preference of Ag⁺ ions to coordinate carbon–carbon double bonds in hydrocarbon chains^{41 (link),42 (link)}. Silver adducts were prepared by mixing a 17 mM solution of Ag[PF₆] in acetonitrile with 100 μM glycolipid solutions in a ratio of 1:10. Ions were generated by nano-electrospray ionization and drift times were converted into collision cross sections (CCS) using the Mason–Schamp equation^{43 (link)}. The measurements were repeated on three different days. The double standard deviation of the individual measurements is in all cases ≤1% of the absolute CCS. MS/MS spectra were obtained by collision-induced dissociation (CID) in the trap cell.

General reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or ACROS Organics (Belgium). Thin layer chromatography (TLC) was performed on alumina-backed silica gel 40 F254 plates (Merck) and illuminated under UV (254 nm) The melting points were determined on Optimelt MPA100 instrument (SRS, USA) and are uncorrected. Syntheses were performed on a CEM-DISCOVERY microwave reactor (CEM Corporation, Matthews, NC, USA) with temperature and pressure control. High resolution mass spectrometry (HRMS) analysis was performed for all new compounds on SYNAPT G2-S HDMS (Waters, USA). The purity of all compounds was assessed using a Agilent1260 equipped with a DAAD detector at 260 nm, RP-column: Eclipse plus C18 (3,5 μm); flow 0.5 ml/min. The time of each measurement was 21 min. Conditions: 0− 0.8 min (80% H₂O (0.1% TFA); 20% acetonitrile); 0.8−7 min (100% acetonitrile); 7−13 min (80% H₂O (0.1% TFA); 20% acetonitrile).
All ¹H NMR spectra were recorded on a Bruker AM-400 spectrometer (400 MHz) as well as Bruker AVANCE III (500 MHz). Chemical shifts are reported in ppm against the internal standard, Si(CH₃)₄. Easily exchangeable signals were omitted when diffuse. In general, thiosemicarbazones – 1a, 2a-b, 3a-c, Dp44mT and their thiosemicarbazide precursors were synthesized and characterized, as described previously [35 (link), 36 (link)]. Doxorubicin was purchased from Sigma-Aldrich.

A quantity of 4 μL samples (equivalent to ~1.4 μg total protein) were injected to nano Acquity UPLC (Ultra Performance Liquid Chromatography) ‐ system (Waters Corporation, MA, USA). TRIZAIC nanoTile 85 μm × 100 mm HSS‐T3u wTRAP was used for separation before mass spectrometer. Samples were loaded, trapped and washed for two minutes with 8.0 μL/min with 1% B. The analytical gradient used is as follows: 0–1 min 1% B, at 2 min 5% B, at 65 min 30% B, at 78 min 50% B, at 80 min 85% B, at 83 min 85% B, at 84 min 1% B and at 90 min 1% B with 450 nL/min. Buffer A: 0.1% formic acid in water and Buffer B: 0.1% formic acid in acetonitrile.
Data were acquired in DIA (data independent acquisition) fashion using HDMSE mode with Synapt G2‐S HDMS (Waters Corporation, MA, USA). The collected data range was 100–2000 m/z, scan time one‐second, IMS wave velocity 650 m/s, collision energy was ramped in trap between 20 and 60 V. Calibration was done with Glu1‐Fibrinopeptide B MS2 fragments and as a lock mass, Glu1‐Fibrinopeptide B precursor ion was used during the runs. The samples were run as triplicates and further analysis was done with, Progenesis QI for Proteomics – software (Nonlinear Dynamics, Newcastle, UK).

Four μL samples, equivalent to ~1.4μg total protein, were injected to nano Acquity UPLC (Ultra Performance Liquid Chromatography)—system (Waters Corporation, MA, USA). TRIZAIC nanoTile 85μm x 100 mm HSS-T3u wTRAP was used as separating device prior to mass spectrometer. Samples were loaded, trapped and washed for two minutes with 8.0 μL/min with 1% B. The analytical gradient used is as follows: 0–1 minutes 1% B, at 2 minutes 5% B, at 65 minutes 30% B, at 78 minutes 50% B, at 80 minutes 85% B, at 83 minutes 85% B, at 84 minutes 1% B and at 90 minutes 1% B with 450nL/min. Buffers were made to UPLC-grade chemicals (Sigma-Aldrich, MO, USA); Buffer A: 0.1% formic acid in water and Buffer B: 0.1% formic acid in acetonitrile.
The data was acquired in DIA (data independent acquisition) fashion using HDMSE-mode with Synapt G2-S HDMS (Waters Corporation, MA, USA). HDMSE mode included Ion mobility spectroscopy (IMS). The collected data range was 100–2000 m/z, scan time one second, IMS wave velocity 650 m/s, collision energy was ramped in trap between 20 to 60 V. Calibration was done with Glu1-Fibrinopeptide B MS2 fragments and as a lock mass, Glu1-Fibrinopeptide B precursor ion was used during the runs. The samples were run as triplicates and further analysis was done with, Progenesis QI for Proteomics–software (Nonlinear Dynamics, Newcastle, UK).