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 26 525-845; fax 26 685-8665, email 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- 7548 and CHE-349595 to FT).
. 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.
. 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) 25 25 Collision Cell Entrance (V) -2-2 Collision Cell Exit (V)
. 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) -2.8 +3.25 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) 45 45 Desolvation Gas (L/Hr) 9 9 Cone Gas (L/Hr) Collision Cell Entrance (V) -5-5 Collision Cell Exit (V)
. 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
. 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.
6-Keto PGF a y =.677x +.349 TxB 2 y =.36x +.554.4 R 2 =.9846 2.5 R 2 =.987.2 2.8.5 Ratio Ratio.6.4.5.2 2 4 6 8 2 4 6 8 2 2 4 6 8 2 4 6 8 2 PGF 2a y =.53x +.782 R 2 =.9967 PGE 2 y =.76x +.64.2 2.5 R 2 =.9949 2.8.5 Ratio.6 Ratio.4.2.5 2 4 6 8 2 4 6 8 2 2 4 6 8 2 4 6 8 2 PGD 2 y =.683x +.766 LTB 4 y =.93x -.645.6 R 2 =.9899.8 R 2 =.9749.4.6.2.4.2 Ratio.8.6 Ratio.8.6.4.4.2.2 2 4 6 8 2 4 6 8 2 2 4 6 8 2 4 6 8 2
.4 5-Hete y =.62x +.2 R 2 =.9634.6 8-Hete y =.83x -.44 R 2 =.9984.2.4.2.8 Ratio Ratio.8.6.6.4.4.2.2 2 4 6 8 2 4 6 8 2 2 4 6 8 2 4 6 8 2 -Hete y =.72x +.22 R 2 =.9992 2-Hete y =.76x +.222 R 2 =.9995 8 8 7 7 6 6 5 5 Ratio 4 Ratio 4 3 3 2 2 2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 5-Hete y =.4x +.4 R 2 =.999 4.5 4 3.5 3 Ratio 2.5 2.5.5 2 3 4 5 6 7 8 9
.2 6-Keto PGF α y =.493x +.494 R 2 =.9825 8 TxB2 y =.3583x +.22 R 2 =.9997 7 6 Analyte/IS Ratio.8.6.4 Analyte/IS Ratio 5 4 3 2.2 2 4 6 8 2 4 6 8 2 2 4 6 8 2 4 6 8 2 PGF 2α y =.42x +.96 R 2 =.9993 PGE 2 y =.365x +.85 R 2 =.999.9.8.8.7.7.6 Analyte/IS Ratio.6.5.4.3 Analyte/IS Ratio.5.4.3.2.2.. 2 4 6 8 2 4 6 8 2 2 4 6 8 2 4 6 8 2 PGD 2 y =.377x +.233 R 2 =.9947 LTB 4 y =.968x +.47 R 2 =.9985.8 2.7.8.6.6.4 Analyte/IS Ratio.5.4.3 Analyte/IS Ratio.2.8.6.2.4..2 2 4 6 8 2 4 6 8 2 2 4 6 8 2 4 6 8 2
5(S)HETE y =.426x +.85 R 2 =.995 8(S)Hete y =.787x +.56 R 2 =.9853.9.8.8.7.7.6 Analyte/IS Ratio.6.5.4.3 Analyte/IS Ratio.5.4.3.2.2.. 2 4 6 8 2 4 6 8 2 2 3 4 5 6 7 8 9 (S)HETE y =.588x +.65 R 2 =.9879 2(S)HETE y =.354x +.76 R 2 =.9949.2.8.7.6 Analyte/IS Ratio.8.6.4 Analyte/IS Ratio.5.4.3.2.2. 2 4 6 8 2 4 6 8 2 2 4 6 8 2 4 6 8 2 5(S)HETE y =.228x +.88 R 2 =.996 Arachidonic Acid y =.52x +.39 R 2 =.9963.5.2.45.4.35.8 Analyte/IS Ratio.3.25.2 Analyte/IS Ratio.6.5.4..2.5 2 4 6 8 2 4 6 8 2 2 4 6 8 2 4 6 8 2
. 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.
6-Keto 6-Keto 5.64 356.28 3.56e2 4.49 5.6 537.5 > 239.25 6.29.97e+4 6.5 6.44 6.49 6.64 6.89 7.7 8.36 4. 6. 8. d4 6-Keto 54.55 > 24.27 d4 6-Keto 2.999e+3 5.3 75.4.75e2 d4 6-Keto 5.3 75.4.75e2 d4 6-Keto 5.3 75.4.75e2 6.7 6.38 6.89 8.37 4. 6. 8. TXB2 537.5 > 337.27.7e+5 TXB2 6.5 46786.54 4.68e4 TXB2 6.5 46786.54 4.68e4 4. 6. 8. d4 TXB2 54.55 > 34.32.877e+3 d4 TXB2 6.6 637.83 6.38e2 d4 TXB2 6.6 637.83 6.38e2 4.68 4.34 6.79 7.99 4.4 8.8 4. 6. 8. PGF2a 52.5 > 239.25 6.9e+3 PGF2a 6.7 323.73 3.24e2 5.92 5.79 3.68 4.77 4.6 7.7 7.53 7.8 4. 6. 8. 8.33 Alternate PGF2 3.2 4.4 5.4 52.5>293.27.99e+3 7.73 7.53 7.47 5.25 6.73 4. 6. 8. 8. 8.23 8.36 d4 PGF2a 3.8 4.38 4.9 525.55 > 24.27 3.76e+3 d4 PGF2a 6.67 23.5 2.3e2 6.37 d4 PGF2a 6.67 23.5 2.3e2 8.8 4. 6. 8. 8.3 PGE2 4.53 6.8 5.47 59.5 > 239.25.699e+4 7.77 7.5 6.55 6.5 4. 6. 8. 8.34 PGD2 59.5 > 37.28 7.29.38e+3 7.8 6.46.77 4.3 4.59 4.23 5.35.67 7.38 8.8 4. 6. 8. 8.33 Alternate PGE2 4.63 3.99 59.5 >293.27.297e+3 7.85 5.75 5.4 7.4 7.78 4. 6. 8. 8. 8.33 d4 PGE2 523.55 > 24.27 d4 PGE2 2.986e+3 6.75 278.56 7. 2.79e2 7.8 4.48 3.8 4.9 5.7 7.59 8.3 4. 6. 8. d4 PGD2 3.42 523.55 > 3.35 d4 PGD2 3.39e+3 7. 23.97 2.3e2 d4 PGD2 7. 23.97 6.79 2.3e2 4.74 5.2 7.67 8.9 4. 6. 8.
LTB4 F2:MRM of 3 channels,es+ 53.45 > 323.27 8.9e+4 LTB4 9.78 925. 9.3e3 LTB4 9.78 925. 9.3e3 LTB4 9.78 925. 9.3e3 d4 LTB4 F2:MRM of 3 channels,es+ 57.5 > 325.3 2.52e+3 d4 LTB4 9.74 88.63 8.86e 8.6 8.75 9.36 d4 LTB4 9.74 88.63 8.86e.4.4.52 LTE4 9.48 9.2 8.64 9.25 F2:MRM of 3 channels,es+ 588.52 > 24.27 9.58 8.3e+2 9.98.55 9.95.43 9.89.59 9.. 9.. 9.. 2 HHTrE F2:MRM of 3 channels,es+ 2 HHTrE 445.49 > 239.27.2 6.526e+4 52.3 5.2e3 5-Hete F2:MRM of 3 channels,es+ 487.48 > 283.25 5-Hete 4.53e+5.37 3223. 3.22e4 8-Hete F2:MRM of 3 channels,es+ 487.48 > 323.27 8-Hete.547e+5.24 2..e4 9.78.52 9.. 9.. 9.. -Hete F2:MRM of 3 channels,es+ 487.48 > 335.3 6.42e+5.4 Alternate -Hete F2:MRM of 3 channels,es+ 487.48>293.25 Alternate -Hete 3.77e+6.7 286.9 2.8e5 2-Hete F2:MRM of 3 channels,es+ 487.48 > 347.3 2-Hete 9.282e+6.5 6934.63 6.9e5 9.. 9.. 9.. 5-Hete F2:MRM of 3 channels,es+ 487.48 > 387.34 5-Hete 3.375e+5.8 957.88.95e4 d8 5-Hete F2:MRM of 3 channels,es+ 495.55 > 284.27 d8 5-Hete.887e+3.36 39.36.39e2 9.. 8.62 9.24 9.2 9.53 9.95.5.6 9..
. 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.
6Keto PGF 3.47 X 6 + 537.34>239.2 3.734.2 4.2 3.64e+3 4.33 4.92 3.64 5.2 5.27 3.62 6.3 5.33 6.48 d4-txb2.47 X 6 + d4-pgf2.47 X 6 + d4-pge2.47 X 6 + 4. 5. 6. 4.8 3.74 4. 5. 6. 54.36>34.3 d4-txb2 9.994e+3 4.7 d4-txb2 3549.2 4.7 3.55e3 3549.2 9777 3.55e3 9777 5.94 525.34>24.23 d4-pgf2.29e+4 5.33 46.49.46e3 235 3.75 3.9 4.84 6.2 4. 5. 6. 523.34>24.23 d4-pge2.657e+4 5.47 234.92 2.3e3 597 5.87 3.76 3.9 4.3 4.75 4. 5. 6. d4-6keto PGF.47 X 6 + PGF2.47 X 6 + 54.36>24.23 d4-6keto PGF 6.89e+3 4.2 842.36 8.42e2 6485 4.78 5.6 5.2 5.78 4. 5. 6. 4. 5. 6. 52.32>239.2 PGF2.923e+4 5.36 2494.8 2.49e3 833 3.79 3.48 4.3 4.39 5.68 6. PGD/PGE alternate 4. 5. 6..47 X 6 + 59.32>293.23 PGD/PGE alternate 4.534e+3 5.49 445.69 4.46e2 4244 5.88 3. 4.65.2 3.72 6.36 PGD2.47 X 6 + 3.6 59.32>37.24 3.2e+3 5.3 4.85 4.54.46 5.49 4. 5. 6. 6.5 6.47 TXB2.47 X 6 + 3.763.84 3.64 4.35 3.2 PGF2 alternate.47 X 6 + PGE2 3.47.47 X 6 + d4-pgd2.47 X 6 + 537.34>337.28 6.2.599e+3 5.62 4. 5. 6. 4. 5. 6. 6. 6.43 52.32>37.24 PGF2 alternate 4.353e+3 5.37 459.9 4.59e2 42 4.26 3.99 5.6 6.3 6.3 6.39 4. 5. 6. 59.32>239.2 PGE2.76e+4 5.49 232.49 2.3e3 72 3.73 5.23 4.23 5.88 523.34>3.26 d4-pgd2.32e+4 5.86 75.4.72e3 295 5.48 3.74 4.9 4.37 6.36 4. 5. 6.
LTB4.47 X 6 + F2:MRM of 2 channels,es+ 53.3>323.24 7.62.827e+3 9.4.9 7.54 8.92 9. 8..6 9.75 7.36 8.. d4-ltb4.47 X 6 + d4-ltb4 7.79 43.86 4.3e2 6452 F2:MRM of 2 channels,es+ 57.32>325.24 6.72e+3 7.57 6.57 8.7 9.7 9.27 9.37.45 8.. 5(S)Hete.47 X 6 + 7.67 7.57 7.2 F2:MRM of 2 channels,es+ 487.32>283.24 7.74 9.454e+2 8.26 8.72 9. 9.8.27 9.79.4 8.. d8-5(s)hete.47 X 6 + F2:MRM of 2 channels,es+ 495.22>284.7 8.527e+3 8(S)Hete.47 X 6 + 7.6 F2:MRM of 2 channels,es+ 487.32>295.24.784e+3 (S)Hete.47 X 6 + F2:MRM of 2 channels,es+ 487.32>335.28 9.68e+2 7.58 6.65 6.96 7.66 8.8 9.3 9.83 9.57.6 8.68 8. 8.23 6.79 9.62.35 8.45 8.93 9.6 8.. 8.. 8.. Alternate (S) Hete 3.47 X 6 + 7.69 8.32 7.59 7.5 7.95 6.84 F2:MRM of 2 channels,es+ 487.32>293.24 2.55e+3 9.68.9 2(S)Hete.47 X 6 + F2:MRM of 2 channels,es+ 487.32>347.28 6.87 7.58 8.39e+2 7.68 6.99 7.8 8.73 9.83 8.26.39 5(S)Hete.47 X 6 + 7.9 7.55 8.32 7.5 7.2 F2:MRM of 2 channels,es+ 487.32>387.29.238e+3 9.93 9.8.6 8.. 8.. 8.. Arachidonate 3 F3:MRM of 6 channels,es+.47 X 6 + 47.28>239.2 Arachidonate.292e+5 2.56 84.23 8.4e3 284 d8-arachidonate 3.47 X 6 + F3:MRM of 6 channels,es+ 479.3>239.2 3.78 4.569e+6 2.32
Supplemental Figure 5. Proton NMR of AMPP
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.
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.
Supplemental Figure 8. AMPP LC/ESI-MSMS method cross-validation with commercial EIA kit.