Appendix S1. Determination of lipid targets used in analysis
(Includes text related to Figure S4 and Tables S2, S3, and S4)
Lipid analytical targets for the direct-infusion MRM MS method were identified from previous
work and the literature (e.g., Stelmach et al., 2001; Welti et al., 2002; Andersson et al., 2006;
Buseman et al., 2006; Devaiah et al., 2006; Ibrahim et al., 2011; Wewer et al., 2011; Schrick
et al., 2012; Samarakoon et al., 2012; Vu et al., 2012; Li et al., 2014; Vu et al., 2014) and
from lipid discovery by precursor or neutral loss scanning with a triple quadrupole MS. For
structural lipids and storage lipids, representative molecular species were chosen for
analysis, while for lipid classes believed to be stress-induced, lipid coverage was more
extensive.
As a basis for defining target analytes, one approach was to identify fatty acyl
components and then use precursor or neutral loss scanning with a triple quadrupole MS to
“discover” the intact molecular species with the fatty acids. In particular, this approach was
used to identify intact lipids containing oxidized fatty acyl components. To identify fatty acyl
components of intact lipids to be used as “bait” in precursor scanning, one approach was
collision induced dissociation (CID) time-of-flight (TOF) MS (Buseman et al., 2006; Esch et
al., 2007).
CID-TOF MS is a non-targeted method that allows detection and identification (by accurate
mass) of fatty acid fragments from all complex lipids above a set mass threshold (here, 440
m/z). CID-TOF MS analysis of the first 10 samples in each treatment group is shown in
Figure S4, and the CID-TOF MS data are provided in Table S2. CID-TOF MS revealed the
complex lipid oxidized fatty acyl fragments observed to be induced by plant wounding. The
fragments, which may represent acyl anions or fragments of acyl anions due to water loss(es)
during CID, include 9:1-O, 16:4-O, 16:3-O, 18:5-O, 18:4-O, 18:2-O, 18:4-2O, and 18:3-2O.
Because CID-TOF MS has limited sensitivity, these may not represent all the induced acyl
chains, and, thus, these acyl anions do not correspond exactly to the ones used in precursor
scanning.
Table S3 shows the data from precursor ion scanning in negative mode, performed
for the purpose of discovering intact lipids that contain oxidized fatty acids. Table S3 lists
precursors of 15 acyl anions or fragments thereof, along with tentative annotations for many
“discovered” intact lipid species. A subset of the intact lipids listed in Table S3 was among
the target analytes in the current work. Table S4 provides interpretation of the fragments
used in the precursor scanning, which include fragments detected by accurate mass
analysis of intact lipid ions (CID-TOF, Table S2, and QTOF MS, summarized in Table S5)
and fragments reported in the literature.
Tentatively annotated oxidized acyl species identified from the literature or by QTOF
MS, but not detected by CID-TOF MS, perhaps due to its limited sensitivity, include 16:3-2O
(Buseman et al., 2006; Ibrahim et al., 2011), 18:3-O (Ibrahim et al., 2011; Vu et al., 2012),
18:5-2O (Ibrahim et al., 2011; Vu et al., 2012), 18:4-3O (Table S6), 18:3-3O (Table S6),
18:2-3O (Table S6), and 18:3-4O. Intact lipids containing these fatty acids are found in the
target list (Table S5).