Synapt g2 s mass spectrometer

We analyzed
all samples, except PLA 3, using ultrahigh performance LC-QTOF-MS/MS
with an Acquity UPLC Waters liquid chromatography system coupled to
a SYNAPT G2-S mass spectrometer (both Waters Norge, Oslo, Norway).
The analytical method has been described in the study of Zimmermann
et al.,^{30 (link),31 (link)} and a brief description as well as information
about the data analysis and compound identification can be found in
the Supporting Information (Nontarget Chemical
Analysis).

Nano electrospray ionization mass spectrometry (nanoESI MS) of gangliosides was performed using a SYNAPT G2-S mass spectrometer (Waters, Manchester, UK) equipped with a Z-spray source as previously described [22 (link),23 (link),24 (link)]. Gangliosides were extracted with methanol from silica gel that has been scraped from choleragenoid positive areas of the TLC plate, and extracts were directly applied to MS analysis. MS¹ spectra were recorded in the negative ion mode with the following source settings: temperature 80 °C, capillary voltage 0.8 kV, sampling cone voltage 20 V, and offset voltage 50 V.

Pellets of isolated mitochondria (corresponding to 10 µg of total protein) were lyzed in 5 µL of 10% SDS at 95 °C for 5 min, followed by sonification in a Bioruptor (Diagenode) for 15 min, and digested using an optimized SP3 protocol as described [32 (link)]. Digested peptides (200 ng) were separated by reversed-phase nanoUPLC on a 75 μm × 250 mm HSS-T3 column (Waters, Eschborn Germany) and analyzed using ion-mobility enhanced data-independent acquisition [33 (link)] on a Waters Synapt G2-S mass spectrometer in three technical replicates. Raw data processing, database search, and label-free quantification was performed as described before [34 (link)].

Mass spectrometry analyses were performed using Synapt G2-S mass spectrometer (Waters) equipped with the electrospray ion source and quadrupole-Time-of-flight mass analyzer. Sample was dissolved in methanol and injected directly into the electrospray ion source.Methanol was used as a solvent with the flow rate 100 μl/min. The measurement was performed in positive ion mode with the resolving power of the TOF analyzer 20000 FWHM. The instrument worked with external calibration on sodium formate in the mass range of m/z = 50–2000. The lock spray spectrum of the leucine-enkephalin was generated by the lock spray source and correction was done for every spectrum. The exact mass measurements for all peaks were performed within 3 mDa mass error. The nitrogen was used as desolvation and cone gas and their flow values were set to 861 L/h and of 222 L/h respectively.The desolvation gas flow was and temparature 150°C. The nebulizer gas pressure was set to 5.8 bar. The capillary voltage was set to 3.8 kV, and the sampling cone voltage and source offset were set to 20 V. The instrument was controlled and recorded data were processed using the MassLynx V4.1 software package (Waters). The quantity of compound 6, used for NMR studies was 7 mg.

H/DX-MS experiments were performed on a fully automated system equipped with a Leap robot (HTS PAL; Leap Technologies, NC), a Waters ACQUITY M-Class UPLC, a H/DX manager (Waters Corp., Milford, MA) and a Synapt G2-S mass spectrometer (Waters Corp., Milford, MA), as described elsewhere (Zhang et al., 2014 (link)). The protein samples were diluted in a ratio of 1:20 with deuterium oxide containing PBS buffer (pH 7.4) and incubated for 0 s, 10 s, 1 min, 10 min, 30 min or 2 hr. The exchange was stopped by diluting the labeled protein 1:1 in quenching buffer (200 mM Na₂HPO₄ × 2 H₂O, 200 mM NaH₂PO₄ × 2H₂O, 250 mM Tris (2-carboxyethyl)phosphine, 3 M GdmCl, pH 2.2) at 1°C. Digestion was performed on-line using an immobilized Waters Enzymate BEH Pepsin Column (2.1 × 30 mm) at 20°C. Peptides were trapped and separated at 0°C on a Waters AQUITY UPLC BEH C18 column (1.7 µm, 1.0 × 100 mm) by a H₂O to acetonitrile gradient with both eluents containing 0.1% formic acid (v/v). Eluting peptides were subjected to the Synapt TOF mass spectrometer by electrospray ionization. Samples were pipetted by a LEAP autosampler (HTS PAL; Leap Technologies, NC). Data analysis was conducted with the Waters Protein Lynx Global Server PLGs (version 3.0.3) and DynamX (Version 3.0) software package.

Ethanol extracts were diluted in acetonitrile and directly perfused into an electrospray ionization source on a Waters Synapt G2S mass spectrometer. The MS conditions were initially optimized to erythromycin, as well as a synthetic mycolactone standard before applying them to the ethanol extracts. The MS conditions were: flow rate 50 µL/min over a 2 min period; capillary voltage 2.98 kV; cone voltage 75 V; desolvation temperature 550 °C; desolvation gas 945 L/h; source temperature 150 °C; acquisition range 100–2000 m/z. Mycolactone A/B was detected by the presence of the more abundant sodium adduct [M + Na]⁺ (m/z 765.5); the protonated molecular ion [M + H]⁺ (m/z 743.5), and the dehydrated protonated molecular ion [M + H − H₂O]⁺ (m/z 725.5) as described previously [2 (link)].

MS¹ and MS² analysis of Gb3Cer was performed using a SYNAPT G2-S mass spectrometer (Waters, Manchester, UK) equipped with a Z-spray source as previously described^{45 (link),47} . Purified neutral GSLs from ACHN and Caki-2 cells were analysed in the positive ion sensitivity mode. Structures of individual GSLs were detected as singly charged monosodiated [M + Na]⁺ ions and structures were deduced from CID spectra.