Improved Sensitivity Mass Spectrometric Detection of Eicosanoids by Charge

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1 Supplemental Material Improved Sensitivity Mass Spectrometric Detection of Eicosanoids by Charge Reversal Derivatization James G. Bollinger, Wallace Thompson, Ying Lai 2, Rob C. Oslund, Teal S. Hallstrand 2, Martin Sadilek, Frantisek Turecek, and Michael H. Gelb,3 * Department of Chemistry, Medicine 2, and Biochemistry 3, University of Washington, Seattle, WA 9895 *Address correspondence to Michael H. Gelb, Depts. of Chemistry and Biochemistry, Campus Box 35, University of Washington, Seattle, WA 9895 USA; (office ; fax , gelb@chem.washington.edu). This work was supported by grants from the National Institutes of Health (HL54 to MHG and HL8925 to TSH) and by grants from National Science Foundation (CHE and CHE to FT).

2 . Exploration of sample work up prior to LC/ESI-MSMS. Inferior recoveries of eicosanoids were obtained using the following conditions: ) Liquid-liquid extraction of serum with MeOH/CHCl 3 (2/) or with t-butyl methyl ether/hexane (3/) or with ethyl acetate/hexane (/) without solid phase extraction. 2) Treatment of serum with volumes of CH 3 CN followed by centrifugation to remove precipitated protein (no solid phase extraction). 3) Elution of the solid phase extraction cartridge with MeOH/. formic acid instead of MeOH.

3 . Supplemental Material Table. Autosampler (2795 Alliance HT LC) and ESI-MSMS (Waters Quattro Micro) data collection parameters. Injection type Fill mode Sequential Partial loop Pre-sample airy boundary 2 µl Post-sample air boundary 2 µl Post-injection wash frequency Flush time Wash time cycles 6 sec 5 sec Secondary wash volume 6 µl Negative Mode Positive Mode Capillary Voltage (kv) (-) 2.8 (+) 3.5 Extractor (V) 3 3 RF Offset.2.2 Dwell Time (msec) 5 5 Inter-Scan Delay (msec) Inter-Channel Delay (msec) Source Temp ( o C) 3 3 Desolvation Temp ( o C) 5 5 Desolvation Gas (L/Hr) Cone Gas (L/Hr) Collision Cell Entrance (V) -2-2 Collision Cell Exit (V)

4 . Supplemental Material Table 2. Autosampler (Water ACQUITY UPLC) and ESI-MSMS (Waters Quattro Premier) data collection parameters. Injection type Fill mode Sequential Partial loop with needle overfill Weak wash volume (H 2 O) 6 µl Strong wash volume (5:5 ACN:MeOH) 2 µl Pre-Aspirate air gap Post-Aspirate air gap Syringe draw rate Needle placement Automatic Automatic Automatic Automatic Negative Mode Positive Mode Capillary Voltage (kv) Extractor (V) 5 5 RF Offset.. Dwell Time (msec) 5 5 Inter-Scan Delay (msec) 5 5 Inter-Channel Delay (msec) Source Temp ( o C) 3 3 Desolvation Temp ( o C) Desolvation Gas (L/Hr) 9 9 Cone Gas (L/Hr) Collision Cell Entrance (V) -5-5 Collision Cell Exit (V)

5 . Supplemental Material Figure. B3LYP/6-3G* calculated potential energy profile for the N CH 2 benzylic bond dissociation in the charge tag. See main text for further discussion. B3LYP/6-3G* Ion H 2 C N HN H 3 C O

6 . Supplemental Material Figure 2. Standard curves for LC/ESI-MSMS analysis of eicosanoid AMPP amides. See main text for more information. (A) Data obtained with the Waters Quattro Micro. (B) Data obtained with the Waters Quattro Premier. Figure is shown in the pages below.

7 6-Keto PGF a y =.677x TxB 2 y =.36x R 2 = R 2 = Ratio Ratio PGF 2a y =.53x R 2 =.9967 PGE 2 y =.76x R 2 = Ratio.6 Ratio PGD 2 y =.683x LTB 4 y =.93x R 2 = R 2 = Ratio.8.6 Ratio

8 .4 5-Hete y =.62x +.2 R 2 = Hete y =.83x -.44 R 2 = Ratio Ratio Hete y =.72x +.22 R 2 = Hete y =.76x R 2 = Ratio 4 Ratio Hete y =.4x +.4 R 2 = Ratio

9 .2 6-Keto PGF α y =.493x R 2 = TxB2 y =.3583x +.22 R 2 = Analyte/IS Ratio Analyte/IS Ratio PGF 2α y =.42x +.96 R 2 =.9993 PGE 2 y =.365x +.85 R 2 = Analyte/IS Ratio Analyte/IS Ratio PGD 2 y =.377x R 2 =.9947 LTB 4 y =.968x +.47 R 2 = Analyte/IS Ratio Analyte/IS Ratio

10 5(S)HETE y =.426x +.85 R 2 =.995 8(S)Hete y =.787x +.56 R 2 = Analyte/IS Ratio Analyte/IS Ratio (S)HETE y =.588x +.65 R 2 = (S)HETE y =.354x +.76 R 2 = Analyte/IS Ratio Analyte/IS Ratio (S)HETE y =.228x +.88 R 2 =.996 Arachidonic Acid y =.52x +.39 R 2 = Analyte/IS Ratio Analyte/IS Ratio

11 . Supplemental Material Figure 3. LC/ESI-MSMS analysis of the full set of eicosanoids in mouse serum. See main text for full discussion. The figure is shown after the standard curve figures.

12 6-Keto 6-Keto e > e d4 6-Keto > d4 6-Keto 2.999e e2 d4 6-Keto e2 d4 6-Keto e TXB > e+5 TXB e4 TXB e d4 TXB > e+3 d4 TXB e2 d4 TXB e PGF2a 52.5 > e+3 PGF2a e Alternate PGF > e d4 PGF2a > e+3 d4 PGF2a e d4 PGF2a e PGE > e PGD > e Alternate PGE > e d4 PGE > d4 PGE e e d4 PGD > 3.35 d4 PGD2 3.39e e2 d4 PGD e

13 LTB4 F2:MRM of 3 channels,es > e+4 LTB e3 LTB e3 LTB e3 d4 LTB4 F2:MRM of 3 channels,es > e+3 d4 LTB e d4 LTB e LTE F2:MRM of 3 channels,es > e HHTrE F2:MRM of 3 channels,es+ 2 HHTrE > e e3 5-Hete F2:MRM of 3 channels,es > Hete 4.53e e4 8-Hete F2:MRM of 3 channels,es > Hete.547e e Hete F2:MRM of 3 channels,es > e+5.4 Alternate -Hete F2:MRM of 3 channels,es > Alternate -Hete 3.77e e5 2-Hete F2:MRM of 3 channels,es > Hete 9.282e e Hete F2:MRM of 3 channels,es > Hete 3.375e e4 d8 5-Hete F2:MRM of 3 channels,es > d8 5-Hete.887e e

14 . Supplemental Material Figure 4. LC/ESI-MSMS analysis of the full set of eicosanoids in calcium ionophore stimulated airway epithelial cells. See main text for full discussion. The figure is shown after the serum eicosanoid figure.

15 6Keto PGF 3.47 X > e d4-txb2.47 X 6 + d4-pgf2.47 X 6 + d4-pge2.47 X >34.3 d4-txb e d4-txb e e >24.23 d4-pgf2.29e e >24.23 d4-pge2.657e e d4-6keto PGF.47 X 6 + PGF2.47 X >24.23 d4-6keto PGF 6.89e e >239.2 PGF2.923e e PGD/PGE alternate X > PGD/PGE alternate 4.534e e PGD2.47 X > e TXB2.47 X PGF2 alternate.47 X 6 + PGE X 6 + d4-pgd2.47 X > e >37.24 PGF2 alternate 4.353e e >239.2 PGE2.76e e >3.26 d4-pgd2.32e e

16 LTB4.47 X 6 + F2:MRM of 2 channels,es+ 53.3> e d4-ltb4.47 X 6 + d4-ltb e F2:MRM of 2 channels,es > e (S)Hete.47 X F2:MRM of 2 channels,es > e d8-5(s)hete.47 X 6 + F2:MRM of 2 channels,es > e+3 8(S)Hete.47 X F2:MRM of 2 channels,es > e+3 (S)Hete.47 X 6 + F2:MRM of 2 channels,es > e Alternate (S) Hete 3.47 X F2:MRM of 2 channels,es > e (S)Hete.47 X 6 + F2:MRM of 2 channels,es > e (S)Hete.47 X F2:MRM of 2 channels,es > e Arachidonate 3 F3:MRM of 6 channels,es+.47 X >239.2 Arachidonate.292e e3 284 d8-arachidonate 3.47 X 6 + F3:MRM of 6 channels,es > e

17 Supplemental Figure 5. Proton NMR of AMPP

19 Supplemental Material Figure 6. Fragment ion spectra of eicosanoid AMPP amides. (A) PGD 2, PGE 2, PGF 2α ; (B) arachidonic acid, TxB 2, 6-keto-PGF α ; (C) 5(S)-, 8(S)-, (S)-HETE; (D) LTB 4, 5(S)- and 2(S)-HETE.

23 Supplemental Material Figure 7. Selected ion traces for LC/ESI-MSMS analysis of eicosanoids in mouse serum. Panel is for d 4 -TxB 2, panel 2 is for TxB 2, panel 3 is for d 4 - PGF 2α, and panel 4 is PGF 2α.

24 Supplemental Figure 8. AMPP LC/ESI-MSMS method cross-validation with commercial EIA kit.

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