Dr Berwyck Poad Compiled: 3 February 2017 Updated: 28 March 2017 Data disclosure for: High-pressure ozone-induced dissociation for lipid structure elucidation on fast chromatographic timescales Dr Berwyck Poad Compiled: 3 February 2017 Updated: 28 March 2017

Based on the instrument diagram in the Waters Synapt G2-Si HDMS hardware manual. Processing software: GLE 4.2.4c and Adobe Illustrator CS6 Figure 1: Overview of the experimental setup required to perform OzID in the ion-mobility spectrometry (IMS) cell of a Waters SYNAPT G2-Si. 2-20% ozone in oxygen (w/w) is generated externally and delivered to the IMS cell through a needle valve, yielding a pressure of ~3 mBar in the cell. Unused ozone is converted to oxygen by a catalytic destruction unit and exhausted from the laboratory. An ambient ozone monitor is interlocked to the ozone generator and stops ozone production if the ambient level rises above 75 ppb.

Data file used: OzID_005.RAW, Acquired 30/6/2016 Software: Chemdraw 15 (structure), MassLynx 4 (spectrum export) GLE 4.2.4c (plotting spectrum), Adobe Illustrator CS6 (final image composition) Figure 2: OzID spectrum of the [M+H]+ precursor ion of the synthetic phosphatidylcholine, PC 18:1(n-9)/18:1(n- 9). These data were obtained with an external ozone concentration of 185 g Nm-3, a reaction time of ~20 ms, and pre- and post-IMS activation energies of 4 eV and 2 eV. The molecular structure of the ionized lipid is indicated and shows representative bond cleavages arising from CID and OzID (both primary and secondary).

Data file used: OzID_067.RAW, Acquired 5/7/2016 Software: MassLynx 4 (spectrum export) GLE 4.2.4c (plotting spectrum), Adobe Illustrator CS6 (final image composition) Figure 3: (a) UPLC chromatogram of a mixture of synthetic PC 18:1(n-9)/18:1(n-9) and PC 18:1(n-12)/18:1(n- 12) isomers. Ion current arising from the [M+H]+ precursor ions at m/z 786 is indicated by the black trace. XICs are shown for OzID product ions from oxidative cleavage of n-9 (green squares) and of n-12 (red diamonds) double bonds and CID product ion m/z 184 (blue line). Representative OzID mass spectra of the chromatographic features between (b) 7.20 and 7.25 mins and (c) 7.32 and 7.40 mins. The [M+H+O3]+ adduct ion is marked with an asterisk (*).These data were obtained with an external ozone concentration of 30 g Nm-3, a reaction time of ~200 ms, and pre- and post-IMS activation energies of 24.2 eV and 4.0 eV, respectively. Plots of these spectra showing m/z annotations of all peaks of interest are provided as Supporting Information (Figure S2).

Data file used: OzID_069.RAW, Acquired 5/7/2016 Software: MassLynx 4 (spectrum export) GLE 4.2.4c (plotting spectrum), Adobe Illustrator CS6 (final image composition) Figure 4: LC-MS analysis of a commercially available phosphatidylcholine fraction from chicken egg yolk. (a) Chromatograms obtained from the precursor ion at m/z 786 corresponding to the [M+H]+ of PC 36:2 (black line). XICs for the CID product ion at m/z 184 (blue trace) and OzID product ions corresponding to cleavage of a monounsaturated n-9 double bond (green squares), monounsaturated n-7 double bond (purple circles) and polyunsaturated n-6 and n-9 double bonds (yellow triangles). OzID mass spectra obtained by integrating between (b) 7.25 - 7.32 and (c) 7.32 - 7.40 min. In both mass spectra, the [M+H+O3]+ ion is indicated by an asterisk (*). These data were obtained with an external ozone concentration of 30 g Nm-3, a reaction time of ~200 ms, and pre- and post-IMS activation energies of 24.2 eV and 4.0 eV, respectively. Plots of these spectra showing m/z annotations of all peaks of interest are provided as Supporting Information (Figure S3).

Data file used: OzID_073. RAW (panels a & b) OzID_074 Data file used: OzID_073.RAW (panels a & b) OzID_074.RAW (panel c), Acquired 30/6/2016 Software: MassLynx 4 (spectrum export) GLE 4.2.4c (plotting spectrum), Adobe Illustrator CS6 (final image composition) Figure 5: LC-MS analysis of a commercially available phosphatidylcholine fraction from chicken egg yolk. (a) Chromatogram obtained from the precursor ion at m/z 760.6 corresponding to the [M+H]+ of PC 34:1 (black line). XICs for the CID product ion at m/z 184.1 (blue trace) and OzID product ions arising from cleavage of n-9 (green squares) and n-7 (purple circles) double bonds. (b) OzID mass spectrum for the [PC 34:1+H]+ precursor ion at m/z 760.6 obtained by integrating between 7.15 - 7.30 min. (c) The analogous OzID mass spectrum for the [PC34:1 + Na]+ precursor ion at m/z 782.6 acquired in a separate LC experiment. [M+H+O3]+ ions are indicated by an asterisk (*). All data were obtained with an external ozone concentration of 30 g Nm-3, a reaction time of ~200 ms, and pre- and post-IMS activation energies of 24.2 eV and 4.0 eV, respectively.

Data file used: Panel a – OliveOil_TAG_OzID_016 Data file used: Panel a – OliveOil_TAG_OzID_016.RAW, Acquired 30/6/2016 at Waters Wilmslow facility Software: MassLynx 4 (spectrum export) Panel b – 20160720_TAG_OzID_03604-15.wiff (scan #5), acquired at CARF QUT 20/7/2016 Software: Analyst 1.6.3 (spectrum export) Panel c – TAG18-1_18-1_18-1_run1_0020ms.raw, acquired at CARF QUT 20/7/2016 Software: Xcalibur 3 (spectrum export) Final Figure: GLE 4.2.4c (plotting spectrum), Adobe Illustrator CS6 (final image composition) Figure S1: Comparison of OzID spectra obtained for the sodium adduct ion of trioleylglycerol, TG(54:3), using a 20 ms reaction time on three different mass spectrometer geometries: (a) Waters SYNAPT quadrupole time-of-flight mass spectrometer with ozone present in the IMS cell at ca. 1016 molecules cm-3, (b) SCIEX 6500 QTRAP hybrid triple quadrupole with ozone present in the collision cell (q2) at ca. 1013 molecules cm-3, (c) Thermo Fisher Scientific LTQ-XL linear ion trap with ozone present in the segmented trap at ca. 1010 molecules cm-3 in the helium buffer gas. The other experimental parameters used to obtain these spectra are provided in the manuscript (SYNAPT) or as Supporting Information (QTRAP and LTQ-XL).

Software: ChemDraw 15 (structures), Adobe Illustrator CS6 (final image composition) Figure S2: Proposed mechanism and structures of the OzID ions present in Figure 2 of the manuscript. Precursor [M+H]+ ions at m/z 786.6 form a primary ozonide (m/z 834.6) which fragments to form either an aldehyde (m/z 692.4) or Criegee ion (m/z 676.4). These ions may then undergo further ozonolysis of the remaining double bond, yielding m/z 598.3, 582.3 and 566.3. We note that the indicated carbonyl oxide (zwitterion) structures may undergo rearrangement to more stable carboxylic acid or vinyl hydroperoxide moieties.

Data file used: OzID_067.RAW, Acquired 5/7/2016 Software: MassLynx 4 (spectrum export) GLE 4.2.4c (plotting spectrum), Adobe Illustrator CS6 (final image composition) Figure S3: Mass spectra shown in Figure 3 (main text) with full m/z annotation of peaks. OzID mass spectra of the [M+H]+ ions at m/z 786.6 from chromatographically separated lipid standards (a) PC 18:1(n-9)/18:1(n-9) and (b) PC 18:1(n-12)/18:1(n-12).

Data file used: OzID_069.RAW, Acquired 5/7/2016 Software: MassLynx 4 (spectrum export) GLE 4.2.4c (plotting spectrum), Adobe Illustrator CS6 (final image composition) Figure S4: Mass spectra obtained from LC-OzID analysis of chicken egg yolk extract. Spectra are identical to those shown in Figure 4 (main text) but with m/z annotation of peaks and different magnifications. These OzID spectra were obtained on precursor ions of m/z 786.6 by integrating across retention times (a) 7.25 - 7.32 and (c) 7.32 - 7.40 min.

Data file used: OzID_005.RAW, Acquired 30/6/2016 Software: DriftScope 4 (spectrum), Adobe Illustrator CS6 (final image composition) Figure S5: Drift time mobility of PC 18:1(n-9)/18:1(n-9) taken from the same file used to produce Figure 2 in the manuscript. Clear differences can be observed in the arrival times of the OzID fragment ions m/z 692.4 and 676.4 compared to the [M+H]+ precursor ion at m/z 786.6

Software: ChemDraw 15 (structures), Adobe Illustrator CS6 (final image composition) Figure S6: Proposed mechanism and structures for the diagnostic CID/OzID ions present in Figure 5c of the manuscript.