SUPPLEMENTARY DATA. Materials and Methods

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1 SUPPLEMENTARY DATA Materials and Methods HPLC-UV of phospholipid classes and HETE isomer determination. Fractionation of platelet lipid classes was undertaken on a Spherisorb S5W 150 x 4.6 mm column (Waters Ltd. Herts, UK) with a gradient of % B over 25 min (A, hexane:propan-2-ol, 3:2; B, solvent A:water, 94.5:5.5) at a flow rate of 1.5 ml.min -1. Absorbance was monitored at 205 nm and products identified using a mixture of standard phospholipids (Sigma, Poole, UK). Fractions were collected at 30 sec intervals for subsequent analysis by ESI/MS/MS. Flow injection analysis of esterified HETEs was performed by injecting 10!l of each fraction under flow (1 ml.min -1 ) in methanol into the electrospray source, with specific MRM transitions monitored using m/z as the daughter ion, and areas for each transition determined by integration of the peaks. For determination of positional isomer composition in each phospholipid class, extracts were separated by normal phase HPLC, the PE and PC-containing fractions (6-8 min and min respectively) collected, saponified as described below, then resuspended in 50!l methanol, and HETE isomers analyzed by reverse phase LC/MS/MS as described in the main section of the paper. For chiral phase analysis, 12-HETE was collected from the reverse phase eluate, resuspended in chiral phase mobile phase (hexane:2- propanol:acetic acid, 95:5:0.1) and injected onto a Chiralcel OD 250 x 4.6mm column (Chiral Technologies Ltd., Exton, PA) with isocratic separation at 1 ml.min -1 and absorbance monitored at 235 nm. Saponification. Lipids were suspended in 1.5 ml of propan-2-ol, and then fatty acids released by base hydrolysis with 1.5 ml NaOH at 60 C for 30 min under N 2. Hydrolyzed samples were acidified to ph 3.0 with 0.5 M HCl, and fatty acids were extracted twice with 3 ml hexane. The combined hexane layers were dried, resuspended in 100!l MeOH and stored under N 2 at -80 C until analysis by LC/MS/MS. Oxidation of PE and PC using recombinant LOXs. 10mM brain L- -phosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL), 18:0a/20:4-PC or PE (Sigma) was incubated for 30 min at 37 C in 0.1 M KH 2 PO 4 buffer, ph 7.4, 0.2 % sodium cholate, with 100!l of I428A mutant rabbit 15-lipoxygenase, that oxidized primarily at C12 (6). Samples were then reduced using 1 mm SnCl 2 for 10 min, 20 C, and extracted as before for LC/MS/MS analysis. Results Structural characterization of esterified HETEs. Several approaches were utilized which are detailed below. These included normal phase separation of platelet phospholipid classes followed by LC/MS/MS to determine headgroup of each ion containing HETE, then generation of MS/MS spectra during online LC separation of each ion in negative or positive mode. 1. Normal phase separation of PL classes. To determine PL class for each ion, platelet lipid extracts were separated using normal phase HPLC, and fractions analyzed by MS/MS for the parent daughter transition. For 12-HETE analysis the parent - m/z.2 (specific 12-HETE daughter ion) transition was monitored. Most of the ions (m/z 738, 764, 766 and 782) eluted in the PE fraction, but a second with m/z 782 and also m/z 810 eluted within the PC fraction (Fig. S1). This indicates that there are four major PE and two major PC lipids containing 12-HETE, including a pair of isobaric ions comprising a member of each PL class. 2. Structural Determination of 12-HETE-PE and 12-HETE-PCs using LC/MS/MS. Lipids were separated using LC/MS/MS in MRM mode, and product ion spectra acquired at the apex of elution for each lipid by switching the MS to ion trap mode during the LC run. For structural identification, PE and PC standards were oxidized using a mutant rabbit LOX (Ile418Ala) that favors oxidation at C12 (6). The LC/MS/MS profile of m/z from human platelets shows two main ions at 23 and 25.3 min (Fig S2 A). Both also co-elute with the transition (shown in black on trace), indicating that these lipids generate both (HETE [M-H] - ) and (diagnostic for 12-HETE) daughter ions on CID. The negative MS/MS spectrum of the first lipid at 23.0 min (labeled i on Fig S2 A) shows several HETE ions including, and. (Fig S2 B). There is also a prominent ion at 255, indicating an acyl-linked 16:0 at sn-1. Based on this, the structure was predicted to be 16:0a/12-HETE-PC. Oxidation of 16:0/20:4-PC using the mutant

2 rabbit LOX, followed by SnCl 2 reduction, generated a species with identical LC retention time and MS/MS spectrum (Fig S2 C). In negative mode, PC appears as [M-15] -, thus in positive mode, [M+H] + will be 16 amu greater, appearing at m/z 798 with a characteristic daughter ion at m/z 184 (PC headgroup). For both platelet lipid extract and rabbit LOX generated 16:0a/12-HETE-PC, ions at m/z 798 " 184 appeared at 23 min, confirming the identity of the platelet lipid as 16:0a/12-HETE-PC (Fig S2 D,E). The second peak with MRM m/z 782 " also contained several HETE-related ions, including m/z,, and. The ion at m/z 283 indicates that the fatty acid at sn-1 is an acyl-linked 18:0 (Fig S2 F), and the structure is proposed as 18:0/12-HETE-PE. Oxidation of 18:0a/20:4-PE using mutant rabbit 12-LOX, followed by SnCl 2 reduction generates a lipid which elutes at the same retention time on LC/MS/MS and has the same MS/MS spectrum (Fig S2 G). Product ion spectra for the other three PE ions at m/z 738, 764 and 766 also show 12-HETE fragments at m/z,, and but no sn-1 ions indicating that they are plasmalogen or ether lipids (Fig S3 A-C, left insets). Oxidation of brain PE which contains a high proportion of plasmalogen but little ether PE using 12-LOX, generated ions with identical RT and MS/MS spectra (Fig S3 A-C, right insets). This identifies the lipids as 16:0p/12-HETE-PE, 18:1p/12-HETE-PE and 18:0p/12-HETE-PE respectively. The daughter ion at 281 in Panel A, right inset, originates from an isobaric contaminant present in the mutant rabbit LOX preparation that elutes at the same retention time (not shown). The ion at m/z 810, identified as PC has 12-HETE ions at, and and indicates an 18:0a fatty acid at m/z 283 (Fig S4). This was consistent with 18:0a/12-HETE-PC. Oxidation of 18:0a/20:4- PC using the mutant rabbit 12-LOX generated a lipid with identical RT and negative product ion spectrum (Fig S4). A series of ions showing in the platelet lipid MS/MS for m/z 810 that are absent in the standard (m/z 723, 437 and 419) originate from an isobaric phosphatidylserine (18:0/20:4-PS), also eluting at 24.9 min. The presence of this lipid in the MS/MS also explained the higher relative intensity of m/z 283 (18:0), relative to m/z. Finally, positive ion LC/MS/MS demonstrated a lipid with m/z 826"184, with identical RT and MS/MS spectrum, for both platelet lipid extract and mutant rabbit 12-LOX oxidized lipid (Fig S4 D,E). 3. Confirmation of isomeric composition of phospholipid-esterified HETE using chiral chromatography. Although detection of a daughter ion at m/z is diagnostic for the 12-HETE positional isomer, and fragment ions from other HETE positional isomers were absent from MS/MS spectra of all the platelet lipids (e.g. m/z 219 or 115; for 15- or 5-HETE respectively), further evidence that they contained primarily 12-HETE was sought using traditional chromatography techniques. PE and PC platelet lipids were purified using normal phase chromatography, saponified to yield free HETE, which was analyzed using LC/MS/MS. As shown, the 12-positional isomer predominated in both PE and PC from platelets (Fig S5 A,B). Chiral phase LC-UV analysis of 12-HETE derived from either platelet PE or PC indicated the majority of this present is the S-enantiomer (Fig S5 C, D). These data indicate that esterified HETEs are generated by platelet 12-LOX, specifically; 16:0p/12S-HETE-PE, 18:1p/12S-HETE-PE, 18:0p/12S-HETE- PE, 18:0a/12S-HETE-PE, 16:0a/12S-HETE-PC and 18:0a/12S-HETE-PC (Scheme 1). Figure Legends Figure S1. Characterizing phospholipid headgroups for esterified HETEs. Separation of esterified HETEs using normal phase LC. Lipid extracts from thrombin-activated platelets were separated on normal phase HPLC as described in Experimental procedures, with fractions collected at 30 sec intervals. 10!l of each fraction was analyzed by flow injection into the mass spectrometer for levels of specific negative ion parent!.2 MRM transitions. Phospholipid classes for each ion were identified based on retention time of phospholipid standards. Figure S2. Structural identification of m/z 782 as two separate lipids, 18:0a/12-HETE-PE and 16:0a/15-HETE-PC, using MS/MS. Panel A. Negative ion LC/MS/MS elution profile of m/z 782!. Lipid extracts from thrombin-activated platelets were separated as described in Experimental procedures, monitoring m/z 782!, or m/z 782!. Two separate lipids are identified. Panel B. Negative ion MS/MS spectrum of Ion (i) shown in Panel A eluting at 23 min, from thrombin-activated platelets. Panel C. Negative ion MS/MS spectrum for m/z 782 generated by 12-LOX oxidation of 16:0a/20:4-PC as described in Experimental procedures, eluting at 23 min. Panel D. Positive ion MS/MS of m/z 798, eluting at 23 min from thrombin activated platelet lipids. Panel E. Positive ion MS/MS of m/z 798 generated by

3 12-LOX oxidation of 16:0a/20:4-PC eluting at 23 min. Panel F. Negative ion MS/MS spectrum of m/z 782 eluting at 25.3 min (peak (ii)) from thrombin-activated platelet lipids. Panel G Negative ion MS/MS spectrum of m/z 782 generated by 12-LOX oxidation of 18:0a/20:4-PE. Figure S3. Structural identification of m/z 738, 764 and 766 as plasmalogen PEs, using MS/MS. Panel A. Negative LC/MS/MS of m/z 738! and 738! from thrombin-activated platelet lipid extract. Left inset: Negative ion MS/MS spectrum at 23.1 min from platelet lipid. Right inset: Negative ion MS/MS spectrum of m/z 738 generated by 12-LOX oxidation of brain PE, eluting at 23.1 min. Panel B. Negative ion LC/MS/MS of m/z 764! and 764! from thrombin-activated platelet lipid extract. Left inset: Negative ion MS/MS spectrum of m/z 764 at 23.4 min from platelet lipid. Right inset: Negative ion MS/MS spectrum of m/z 764 generated by 12-LOX oxidation of brain PE, eluting at 23.4 min. Panel C Negative ion LC/MS/MS of m/z 766! and 766! from thrombin-activated platelet lipid extract. Left inset: Negative ion MS/MS spectrum of m/z 766 at 25.4 min from platelet lipid. Right inset: Negative ion MS/MS spectrum of m/z 766 generated by 12-LOX oxidation of brain PE, eluting at 25.4 min. Figure S4. Structural identification of m/z 810 as 18:0a/12-HETE-PE, using MS/MS. Panel A. Negative ion LC/MS/MS elution profile of m/z 810!. Lipid extracts from thrombin-activated platelets were separated as described in Experimental procedures, monitoring m/z 810!, or m/z 810!. Panel B. Negative ion MS/MS spectrum of m/z 810 shown in Panel A eluting at 24.9 min, from thrombinactivated platelets. Panel C. Negative ion MS/MS spectrum for m/z 810 generated by 12-LOX oxidation of 18:0a/20:4-PC as described in Experimental procedures, eluting at 24.9 min. Panel D. Positive ion MS/MS of m/z 826, eluting at 24.9 min from thrombin activated platelet lipids. Panel E. Positive ion MS/MS of m/z 826 generated by 12-LOX oxidation of 18:0a/20:4-PC eluting at 24.9 min. Figure S5. Identification of 12S-HETE as the major oxidized AA enantiomer in PE and PC phospholipids of thrombin-activated platelets. Phospholipid classes from thrombin-activated platelets were separated using normal phase LC, saponified, then HETE positional isomers determined using LC/MS/MS as described in Experimental procedures. 12-HETE was analyzed using chiral phase LC/UV. Panel A. Comparison of HETE positional isomers in PE. Panel B. Comparison of HETE positional isomers in PC. Panel C. Chiral separation of 12-HETE from PE of platelet lipids. Panel D. Chiral separation of 12- HETE from PC of platelet lipids. For panels A, B, n = 3, mean +/- SEM

4 Analyte Area 9.0e4 6.0e4 3.0e4 PE PC Retention Time (min) Figure S1

5 A ii e3 5.0e3 m/z!"#$%$&'($ i e3 m/z!"#$%$'!($ B 3.5e4 1.5e4 255 Peak (i) C 6.0e5 3.0e5 Peak (i) E 3.0e6 5.0e5 184 Peak (i) +ve MS/MS F 8.0e6 1.0e6 184 Peak (i) +ve MS/MS Figure S2 F 2.0e5 1.2e5 4.0e4 283 Peak (ii) G 3.5e5 283 Peak (ii) e

6 1.8e4 4.5e e5 1.0e4 2.5e5 5.0e e5 6.0e e3 m/z!&"%&'( m/z!&"%'!( B 1.0e4 5.5e e e3 3.5e5 1.5e e e3 m/z!)*%&'( m/z!)*%'!( C 1.4e4 5.0e e5 x e4 6.0e3 3.0e e e Figure S3 2.0e3 m/z!))%&'( m/z!))%'!(

7 A 4.0e e3 m/z "'+%&'( 0 m/z "'+%'!( B 3.5e e D 2.4e ve MS/MS C 3.2e5 1.6e5 E ve MS/MS 1.2e e6 4.0e Figure S4

8 A 12 PE-Fractions Amount (ng/2x10 8 platelets) B 8 12-HETE 5-HETE 8-HETE 11-HETE 15-HETE HETE Isomer PC-Fractions Amount (ng/2x108 platelets) C Absorbance (235nm) 12-HETE 5-HETE 8-HETE 11-HETE 15-HETE HETE Isomer Racemic R Platelet 12-HETE S from PE fraction Figure S5 D Absorbance (235nm) Time (min) Racemic Platelet 12-HETE From PC fraction R S Time (min)