Supporting information

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1 Supporting information Figure legends Supplementary Table 1. Specific product ions obtained from fragmentation of lithium adducts in the positive ion mode comparing the different positional isomers of NO 2 -CLA and NO 2 -LA. Structural analysis in the positive ion mode of mitochondrial lipid extracts and - synthetic standards of NO 2 -CLA generated by acidic nitration with NO 2 or [ 15 - N]O 2 were conducted by HPLC-ESI-MS/MS using a hybrid triple quadrupole mass spectrometer (API Qtrap 4000, Applied Biosystems, Framingham, MA). The following parameter settings were used: declustering potentials (DP) 90V, collision energy (CE) 30 ev and source temperature 600 C. NO 2 -FA were separated using a C18 reversed phase column (2 x 150 mm, 3 m, Phenomenex, Torrance, CA) at a 250 µl/min flow rate. NO 2 -FAs were eluted from the column using a solvent system consisting of A (H 2 O % acetic acid) and B (acetonitrile (ACN) % acetic acid) following an initial gradient condition of 35% B to 100% B in 45 min, held for 5 min at 100% and re-equilibrated to the initial condition (35 % B) for 10 min. Lithium acetate was infused post column at 2 µl/min to achieve a final concentration of 30 µm. Supplementary Table 2. CID fragmentation products obtained from the different positional isomers of NO 2 -(9,11)-CLA and NO 2 -(9,12)-LA in the negative ion mode. Collision-induced dissociation (CID) spectra were acquired in a LTQ Orbitrap Velos (Thermo Scientific, Waltham, MA) equipped with a HESI II electrospray source. Samples were analyzed using the following parameters: source temperature 400 C, capillary temperature 360 C, sheath gas flow 30, auxiliary gas flow 15, sweep gas flow 2, source voltage -3.3 kv, S-lens RF level 44 (%) and CE 30 ev. The instrument FT-mode was calibrated using the manufacturer recommended calibration solution with the addition of malic acid as a low calibration point in the negative ion mode. Specific multiple reaction monitoring (MRM) transitions corresponding to the most abundant isomers of CLA were generated from structural data obtained from MS 2 fragmentation analysis of biological samples. MRM transitions were acquired in a AB5000 triple quadrupole or API Qtrap 4000 mass spectrometer (Applied Biosystems, Framingham, MA) The following parameter settings were used: DP 90V and source temperature 550 C. CE of ev was used to obtain products ion containing structural information. LC separation method was performed as previously described. Product ions obtained from CID analysis of bis-allylic NO 2 -LA and conjugated diene NO 2 -CLA are listed. 1

2 Supplementary Fig. 1. Mass spectrometric characterization of mitochondrial-derived NO 2 - FA by lithium adducts. The unidentified NO 2 -FA species generated by mitochondria were structurally characterized by HPLC-ESI-MS/MS using a post-column infusion of lithium acetate. Positive charged Li + -adducts of the NO 2 -FA ([M+Li] +, m/z 332.3) were formed in the ion source, thus retaining the same chromatographic profile as in the negative ion mode. The MS 2 spectrum of [M+Li] + (m/z 332.2) shows the specific neutral loss of HNO 2 (47 amu) to form a common ion with m/z [M-HNO 2 +Li] + for both peaks at 36.3 and 37.4 min. The fragmentation of synthetic 9-,10-,12- or 13-NO 2 -(9Z,12Z)-LA isomers (RT 34.6 and 35.8 min) yields specific product ions at m/z 192, 179, 232 and 219 for the NO - 2 group at carbons C-9, C-10, C-12 and C- 13 respectively as previously showed in Supplementary Table 2. However, upon fragmentation of the mitochondrial derived NO 2 -FA two predominant product ions at m/z and were obtained (Supplementary Fig. 1a). Thus, in view of the mechanisms involving chain fragmentation and gas-phase formation of nitrosamine and aldehyde upon CID, we postulated that the NO 2 group, in the mitochondria-generated nitrolkenes, are at positions C-9 and C-12, with the double bonds located at carbon C-9 and C-11 on the 18:2 fatty acid configurations (Supplementary Fig. 1b). These, two peaks corresponding to 9-NO 2 -CLA and 12-NO 2 -CLA eluted at 37.5 and 36.4 min, respectively. Structures were confirmed using [ 15 N]O - 2, where products ions at m/z and were obtained. Thus, the MS-derived structural data indicated that the substrate for nitration was a conjugated diene of linoleic acid rather than the - bis-allylic diene. As a confirmation, incubation of pure (9Z,11E)-CLA with NO 2 under mild acidic conditions resulted in reaction products co-eluting with the peaks identified in mitochondria (Supplementary Fig. 1a, upper and second panel). Under the same conditions, the use of LA as substrate did not yield any detectable nitrated products. To a lower extent, the 10- NO 2 -CLA and 13-NO 2 -CLA isomers were also detected in mitochondria (1-5%) (Supplementary Fig. 1c-d). These minor nitrated products displayed characteristic product ions at m/z (10-NO 2 -CLA) and m/z (13-NO 2 -CLA) as previously discussed in Supplementary Table 2. Supplementary Fig. 2. Structural analysis of 10 and 13-NO 2 -(10,12)CLA by CID fragmentation in negative ion mode. a) MS profile comparison of synthetic NO 2 -(9,11)CLA (dashed line) with mitochondria-generated NO 2 -(10,12)CLA (solid line)(upper panel). b) MS analysis of acidic nitration products of commercial (10E,12Z)-CLA (NaNO 2 5 mm, ph 5) following the proposed product ion fragments for 10-NO 2 -(10,12)-CLA (MRM 324.2/182, 2

3 324.2/224, 324.2/238) and 13-NO 2 -(10,12)-CLA (MRM 324.2/171, 324.2/185, 324.2/227, 324.2/209) obtained using the gas phase fragmentation mechanism proposed for NO 2 -LA. c) MS profile comparison of the synthetic specific isomer, 9-NO 2 -CLA, with mitochondria-generated [- 15 N]O 2 -CLA. Supplementary Fig. 3. NMR spectrometric analysis of [ 15 N]O 2 -CLA. Nitration of CLA was performed in a biphasic system of hexane/sodium phosphate (50 mm, ph 2) 3:1 v/v containing CLA (200 mg) and placed in a stopper sealed round-bottom flask and purged with N 2 for 15 min. Then, a degassed solution of Na[ 15 N]O 2 (2 M, 0.5 ml) was added to achieve a final 50 mm concentration. The biphasic solution was kept under vigorous stirring and N 2 atmosphere at room temperature overnight. A second bolus of Na[ 15 N]O 2 (2 M, 0.5 ml) was added and incubated for 1 h before the upper organic layer was recovered and the solvent evaporated in vacuo and solvated in methanol for nitrated lipid purification. Nitrated CLA was purified by HPLC using a preparative C18 reversed phase column (250 x 21.2 mm, 5m, Luna, Phenomenex, Torrance, CA). Separation was performed as previously described by a gradient solvent system compose of A (H 2 O % acetic acid) and B (ACN % acetic acid) following an initial gradient condition of 35% B to 100% B in 45 min, held for 5 min at 100% and re-equilibrated to the initial condition (35 % B) for 10 min at 20 ml/min flow rate. Fractions were collected and absorbance followed at 234 and 310 nm for diene (CLA) and conjugated nitroalkene (NO 2 -CLA) containing compounds, respectively. Fractions containing NO 2 -CLA (verified using mass spectrometry) were pooled and extracted. The structure of synthetic [ 15 N]O 2 -CLA (a mixture of C-9 and C-13 regioisomers) was analyzed by 1 H NMR. [ 15 N]-9- and 13-Nitrooctadeca-9,11-dienoic acid: 1 H (600MHz, CDCl 3 ) δ 7.52 (dt, J = 7.6, 3.8, CH-CH=CNO 2 ), 6.32 (td, J = 11.4, 7.6, CH=CH- CH=CNO 2 ), 6.19 (td, J = 11.4, 3.8, CH=CH-CH=CNO 2 ), 2.64 (br m, CNO 2 -CH 2 -CH 2 ), 2.34 (td, J = 7.3, 2.5 Hz, CH 2 CO 2 H), 2.24 (q, J = 7.0 Hz, CH 2 -CH 2 -CH=CH), 1.63 (br m, 2H), 1.52 (q, J = 7.2, 2H), 1.46 (q, J = 7.3, 2H), 1.33 (br m, 12 H), 0.89 (dt, J = 6.6, 3.4 Hz, 3H). Supplementary Fig. 4. Detection of CLA in mitochondria. CLA detection and characterization was performed by LC-ESI-MS/MS in negative ion mode after derivatization with 4-phenyl-1,2,4 triazoline-3,5 dione (PTAD) to form the respective Diels-Alder adducts, in the presence of heptadecanoic acid as internal standard. a) Scheme of derivatization reaction of conjugated diene isomers (9Z,11E- and 10E,12Z-CLA) with the dienophile PTAD. b) Product ion MS 2 spectra of derivatized CLA isomers (PTAD-CLA) ((9,11)-CLA, lower panel and (10,12)-CLA, upper panel). MS 2 fragmentation of the heterocyclic Diels Alder reaction products at m/z renders isomerspecific product ions at m/z 238.2, 220.4, and corresponding to (10,12)-CLA and ions at m/z 224.2, 206.2, 191.2, and for (9,11)-CLA. c) MS chromatograms of CLA standard and mitochondrial lipid extracts display peaks corresponding to derivatized (9,11)-CLA and 3

4 (10,12)-CLA. d) MS acquisition in MRM scan mode using specific transitions for derivatized CLA positional isomers: (10,12)-CLA (MRM 454/182, 454.3/205, 454/220, 454/238) and (9,11)- CLA (MRM 454/168, 454/191, 454/206, 454/224). Total derivatized CLA was determined by scanning for the common product ion at m/z 454 (MRM 454/335). Quantification was performed using external standard curves of PTAD-CLA (0.5 nm-1 M) and heptadecanoic acid (1 M). Supplementary Fig. 5. Identification of NO 2 -CLA in the different in vitro and in vivo experimental models. Specific formation of 9- and 12-NO 2 -CLA is shown and compared between different chemical and biological settings. Displayed chromatograms show normalization to internal standards [ 13 C 18 ] NO 2 -LA to account for interday variation in retention times. The ischemia/reperfusion data was obtained as previously described in the manuscript. Supplementary Fig. 6. Internal standard curve for quantification of NO 2 -CLA in humans. Quantitative analysis of NO 2 -CLA was performed by addition of [ 15 N]O 2 -CLA to plasma samples previous to lipid extraction. The internal standard curve shows a linear range over 4 orders of magnitude. Supplementary Fig. 7. Acidic nitration of endogenous CLA by incubation with [ 15 N]O - 2. Plasma of healthy donors (1.5 ml) was incubated with 1 mm of [ 15 N]O - 2 at ph 5. Lipids were extracted using a mixture of hexane/propan-2-ol/formic acid. a) Chromatograms displayed peaks corresponding to the 9-[ 15 N]O 2 -CLA and 12-[ 15 N]O 2 -CLA isomers (upper panel) which were confirmed by CID fragmentation of ions m/z (1) and (2) (middle and lower panel, respectively). b) MS 2 analysis of [ 15 N]O 2 -CLA was performed using accurate mass and high resolution mass spectrometry. All data was acquired at the 3 ppm level using a Velos Orbitrap mass spectrometer. c) Control of artifactual formation of NO 2 -CLA during sample preparation. Lipid extraction of plasma, performed under acidic condition, was evaluated for lipid nitration artifacts by adding 10 M of Na[ 15 N]O 2 in the presence of 1 mm hexadiene (used as a reaction quencher) and [ 13 C 18 ]NO 2 -LA as internal standard. Chromatogram of endogenous NO 2 -CLA (MRM 324.2/46)(upper panel) compared to chromatogram showing no formation of [ 15 N]O 2 - CLA (MRM 325.2/47)(lower panel) during extraction. The concentration of Na[ 15 N]O 2 chosen to control for nitration artifacts is ~40 times higher than endogenous plasma NO - 2 levels. 4

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