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B Day 1_0Gy Day 1_8Gy DIM 1 DIM 2 -0.06 -0.04 -0.02 -0.00 0.02 0.04 0.00 0.02 0.04-0.02 -0.04 Top 50 Ions From RF – 100% Accuracy AC -0.2 -0.1 0.0 Upregulated.

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Presentation on theme: "B Day 1_0Gy Day 1_8Gy DIM 1 DIM 2 -0.06 -0.04 -0.02 -0.00 0.02 0.04 0.00 0.02 0.04-0.02 -0.04 Top 50 Ions From RF – 100% Accuracy AC -0.2 -0.1 0.0 Upregulated."— Presentation transcript:

1 B Day 1_0Gy Day 1_8Gy DIM 1 DIM 2 -0.06 -0.04 -0.02 -0.00 0.02 0.04 0.00 0.02 0.04-0.02 -0.04 Top 50 Ions From RF – 100% Accuracy AC -0.2 -0.1 0.0 Upregulated in 8Gy Group S Plot p[1]P(Loadings) p(corr)[1]P (Correlation) 0.10.2 Downregulated in 8Gy Group 0.0 0.2 0.4 0.6 0.8 1.0 -0.8 -0.6 -0.4 -0.2 Day1_0Gy Day1_8Gy 0.2 0.6 0.2 1 Colour Key

2 Supplementary Figure S1: UPLC-ESI-TOFMS GI metabolomics 1 day post-radiation exposure: CD2F1 mice were either sham irradiated or exposed to study 8Gy of γ radiation. The mice were euthanized 1 day, post-radiation exposure for blood and organ collection. Comparative metabolomic profiling of GI tissue was performed as described (see methods). Panel A. OPLS loadings S-plot comparing features from control GI (sham) with irradiated group. Panel B. Two dimensional accuracy plot for top 50 features interrogated using Random Forests. The X-axis denotes the interclass separation while the Y-axis displays the intra-class variability. Panel C. Heat map visualization of the feature rankings comparing relative levels in control and irradiated GI samples. Each row represents a unique feature with a characteristic mass to charge and retention time value.

3 Supplementary Figure S2. Determination of chemical structure of metabolite (m/z 203.11) in GI tissue by tandem mass spectrometry. Top panel shows MS/MS fragmentation of the ion with m/z 203.11 from GI tissue extract, while the bottom panel shows the fragmentation for standard D-Tryptophan. Tissue Extract TOF MS/MS 203.11 ES- D-Tryptophan TOF MS/MS 203.11 ES-

4 Supplementary Figure S3. Determination of the chemical structure of metabolite (m/z = 150.10) in GI tissue by tandem mass spectrometry in electrospray positive mode. Top panel shows MS/MS fragmentation spectrum of the parent ion GI tissue extract while the bottom panel shows the fragmentation for standard methionine. Tissue extract standard Tissue Extract TOF MS/MS 150.10 ES+ Methionine TOF MS/MS 150.10 ES+

5 Supplementary Figure S4. Determination of the chemical structure of metabolite (m/z=148.10) in GI tissue by tandem mass spectrometry. Top panel shows MS/MS fragmentation spectrum of the parent ion from GI tissue extract while the bottom panel shows the fragmentation for standard Glutamic acid. Tissue Extract TOF MS/MS 148.10 ES+ Glutamic acid TOF MS/MS 148.10 ES+

6 Supplementary Figure S5. Determination of the chemical structure of metabolite (m/z= 179.049) in GI tissue by tandem mass spectrometry. Top panel shows MS/MS fragmentation of the parent ion from GI tissue extract while bottom panel shows the fragmentation for standard Cys-Gly. Tissue Extract TOF MS/MS 179.049 ES+ Cys-Gly TOF MS/MS 179.049 ES+

7 Supplementary Figure S6. Determination of the chemical structure of lipid (PS(16:0/0:0)) with m/z 496.275 and retention time of 1.0176 minutes in GI tissue extracts by matching the fragmentation pattern with that obtained from the standard. Matched Unmatched m/z Intensity 255.2361 409.2421 496.2751

8 140.0131 196.0404 Matched Unmatched m/z Intensity Supplementary Figure S7. Determination of the chemical structure of lipid (PE(16:0/0:0)) in GI tissue by SimLipid 3.0 (Waters). tandem mass spectrometry with m/z 452.285 and RT 1.2883. 452.2851

9 Supplementary Figure S8. Determination of the chemical structure of lipid (PE(20:1/0:0)) in GI tissue by SimLipid 3.0 (Waters). tandem mass spectrometry with m/z 506.334 and RT 1.3263. 140.0131 196.0404 Matched Unmatched m/z Intensity 506.3342

10 S. Nom/z RT (minutes) ESI Mode Fold Change (Rad/Sham) Radiation Dose P-Value 1 425.91900.30 POS4 Gy0.01 2 299.28014.86 POS4 Gy0.01 3 564.83129.39 POS4 Gy0.05 4 507.86228.43 POS4 Gy0.002 5 682.79910.25 POS4 Gy0.001 6 332.01320.31 NEG4 Gy0.002 7 835.57339.24 NEG4 Gy0.04 8 632.47507.25 NEG4 Gy0.03 9 469.78439.48 NEG4 Gy0.05 10 411.78729.52 NEG4 Gy0.05 11 614.28802.94 POS8Gy0.03 12 804.55257.54 POS8Gy0.01 13 198.10031.61 POS8Gy0.01 14 527.39848.42 POS8Gy0.03 15 493.31996.17 NEG8Gy0.04 16 761.50668.87 NEG8Gy0.002 17 189.98801.18 NEG8Gy0.004 18 809.18611.21 NEG8Gy0.05 19 371.10521.40 NEG8Gy0.05 20 436.86989.39 NEG8Gy0.01 Supplementary Table ST1: Unidentified putative markers of IR exposure at Day 1.

11 Supplementary Figure S9. Box-and-whisker plots of putative biomarkers of radiation injury of GI tissue in CD2F1 mice. The features that were significantly altered in the irradiated GI tissue (4 & 8Gy) at day 1 were shortlisted via multivariate data analysis, putatively identified by accurate mass based search using Madison Metabolomics Consortium Database (MMCD) and confirmed by matching fragmentation pattern with standard compounds. Day 1_0Gy Day 1_8Gy LMGP01011254 (502.315_0.6916) Day 1_0Gy Day 1_4Gy LMGP01010612 (538.387_2.2977) Day 1_0Gy Day 1_8Gy LMGP03050002 (496.275_1.0176) Day 1_0Gy Day 1_4Gy LMGP02050002 (452.285_1.2883) Day 1_0GyDay 1_8Gy Methionine (150.059_0.3822) Glutamic acid (148.061_0.3308) Day 1_0Gy Day 1_8Gy Cys-Gly (179.049_0.3636) Day 1_0Gy Day 1_4Gy D-Tryptophan (203.082_1.4182) Day 1_0Gy Day 1_8Gy LMGP02050020 (506.334_1.3263) Day 1_0Gy Day 1_8Gy

12 B Day 4_8Gy Day 4_0Gy DIM 1 DIM 2 -0.06 -0.04 -0.02 -0.00 0.02 0.04 0.00 0.02 0.04-0.02 -0.04 Top 50 Ions From RF – 100% Accuracy AC -0.2 -0.1 0.0 Upregulated in 8Gy Group S Plot p[1]P(Loadings) p(corr)[1]P (Correlation) 0.10.2 Downregulated in 8Gy Group 0.0 0.2 0.4 0.6 0.8 1.0 -0.8 -0.6 -0.4 -0.2 Day4_0Gy Day4_8Gy

13 Supplementary Figure S10: UPLC-ESI-TOFMS based GI metabolomics at 4 days post-IR exposure: CD2F1 mice were either sham irradiated or exposed to study 8Gy of γ radiation and euthanized after 4 days. Comparative metabolomic profiling of GI tissue was performed as described (see methods). Panel A. OPLS loadings S-plot comparing features from sham with irradiated group. Panel B. Two dimensional accuracy plot for top 50 features interrogated using Random Forests. The X-axis denotes the interclass separation while the Y-axis displays the intra-class variability. Panel C. Heat map visualization of the feature rankings comparing relative levels in control and irradiated GI samples. Each row represents a unique feature with a characteristic mass to charge and retention time value.

14 Supplementary Figure S11. Determination of the chemical structure of metabolite (m/z = 146.20) by tandem mass spectrometry. Top panel shows MS/MS fragmentation spectrum of the parent ion from GI tissue extract while the bottom panel shows the fragmentation for standard spermidine. Tissue Extract TOF MS/MS 146.20 ES+ Spermidine TOF MS/MS 146.20 ES+

15 Supplementary Figure S12. Determination of the chemical structure of metabolite (m/z = 311.16) by tandem mass spectrometry. Top panel shows MS/MS fragmentation spectrum of the parent ion with m/z 311.169 while the bottom panel shows the fragmentation for standard Eicosenoic acid. Tissue Extract TOF MS/MS 311.16 ES+ Eicosenoic acid TOF MS/MS 311.16 ES+

16 Supplementary Figure S13. Determination of the chemical structure of metabolite (m/z = 606.0739) by tandem mass spectrometry. Top panel shows MS/MS fragmentation of the parent ion while the bottom panel shows the fragmentation pattern for standard UDP-N-Acetyl-glucosamine. Tissue Extract TOF MS/MS 606.06 ES- UDP-N-Acetyl-glucosamine TOF MS/MS 606.06 ES-

17 Supplementary Figure S14. Determination of the chemical structure of metabolite (m/z = 514.27) by tandem mass spectrometry. Top panel shows MS/MS fragmentation of the parent ion while the bottom panel shows fragmentation pattern of standard Taurocholic acid. Tissue Extract TOF MS/MS 514.27 ES- Taurocholic acid TOF MS/MS 514.27 ES-

18 Supplementary Figure S15. Determination of the chemical structure of lipid (PE(O-20:0/20:5)) in GI tissue by SimLipid 3.0 (Waters). tandem mass spectrometry with m/z 778.547 and RT 5.0338. Matched Unmatched m/z Intensity 742.5486 778.5473

19 241.0149 255.5363 279.237 553.2868 833.5335 Matched Unmatched m/z Intensity Supplementary Figure S16. Determination of the chemical structure of lipid (PI(18:2/16:0)) with m/z 833.534 and retention time of 4.3 minutes in GI tissue extracts by matching the fragmentation pattern with that obtained from the standard.

20 Supplementary Figure S17. Determination of the chemical structure of lipid (PI(18:1/18:1)) in GI tissue by SimLipid 3.0 (Waters). tandem mass spectrometry with m/z 861.565 and RT 4.7912. 241.0153 281.2524 Matched Unmatched m/z Intensity 861.5652

21 Supplementary Figure S18. Determination of the chemical structure of lipid (PI(18:0/0:0)) with m/z 599.331 and retention time of 1.4 minutes in GI tissue extracts by matching the fragmentation pattern matching with the standard. 283.2679 437.2751 599.3305 Matched Unmatched m/z Intensity

22 S. Nom/zRTMode Fold Change (Rad/Sham) Radiation Dose P-Value 1 83.02150.28 POS4 Gy0.05 2 441.07530.45 POS4 Gy0.03 3 111.11751.90 POS4 Gy0.001 4 231.02987.08 POS4 Gy0.05 5 736.55438.25 POS4 Gy0.05 6 834.24720.48 NEG4 Gy0.03 7 162.02140.44 NEG4 Gy0.002 8 364.17993.13 NEG4 Gy0.05 9 227.98670.30 NEG4 Gy0.001 10 445.02600.45 NEG4 Gy0.05 11 197.12032.14 POS8Gy0.05 12 811.53829.41 POS8Gy0.001 13 562.00880.36 POS8Gy0.03 14 431.09080.36 POS8Gy0.003 15 71.050670.32 POS8Gy0.02 16 630.10260.40 NEG8Gy0.04 17 708.12681.17 NEG8Gy0.05 18 457.07170.42 NEG8Gy0.003 19 112.03930.34 NEG8Gy0.001 20 469.78439.48 NEG8Gy0.004 Supplementary Table ST2: Unidentified putative markers of IR exposure at Day 4.

23 Supplementary Figure S19: Box-and-whisker plots of putative biomarkers of IR injury of GI tissue in CD2F1 mice. The features that were significantly altered after 4 days of IR exposure were shortlisted via multivariate data analysis, putatively identified by accurate mass based search using Madison Metabolomics Consortium Database (MMCD) and confirmed by matching fragmentation pattern with standard compounds. Day 4_0Gy Day 4_8Gy LMGP02050011 (476.287_1.0965) Day 4_0Gy Day 4_8Gy LMGP06010002 (861.565_4.7912) Day 4_0Gy Day 4_8Gy Taurocholic acid (514.285_3.1882) Day 4_0Gy Day 4_4Gy Spermidine (146.166_0.2437) Day 4_0Gy Day 4_4Gy LMGP02020084 (778.574_5.0338) Day 4_0Gy Day 4_4Gy LMGP06010847 (833.534_4.2999) UDP (606.077_0.3992) Day 4_0Gy Day 4_8Gy Eicosenoic acid (311.169_0.1542) Day 4_0Gy Day 4_4Gy LMGP06050004 (599.331_1.4042) Day 4_0Gy Day 4_8Gy

24 Supplementary Figure S20. Global metabolome visualization of GI tissue in sham or IR treated CD2F1 mice. Panels A and B. Self-organizing Maps (SOMs) reveal the effect of IR exposure after 1 and 4 days respectively. The data were acquired by UPLC- QTOF MS, pre-processed using XCMS and the feature intensities were normalized to internal standards and to total protein concentration. Top 100 features were used to construct SOMs which create a series of coherent mosaic heat maps representing overall ion profile in each sample. Tiles containing the highest-abundance ions are shaded in deep red, while those containing low abundance ions are shaded deep blue. The areas highlighted in black boxes show the change in the intensity of metabolites in GI tissue in a time and dose dependent manner. Panel A: Day 1 Panel B: Day 4 0GY 4GY 8GY 0GY 4GY 8GY

25 Supplementary Table ST3: Functional pathway analysis of canonical pathways enriched in response to IR exposure

26 B Day 1_4Gy_Kidney Day 1_4Gy_GI DIM 1 DIM 2 -0.06 -0.04 -0.02 -0.00 0.02 0.04 0.00 0.02 0.04-0.02 -0.04 Top 50 Ions From RF – 100% Accuracy AC Day1_4Gy_GI Day1_4Gy_Kidney 0.2 0.6 0.2 1 Colour Key Number of variables RF Accuracy [%] 0 20 40 60 80 100 60 80 10040 20

27 Supplementary Figure S21: UPLC-ESI-TOFMS based comparative metabolomic profiling of GI and Kidney tissue at 4 days, post-IR exposure: CD2F1 mice were either sham irradiated or exposed to 4Gy of γ radiation and euthanized after 4 days. Panel A. Two dimensional separation plot for top 50 features interrogated using Random Forests. Panel B. Normalized accuracy plot showing unambiguous separation of the two groups Panel C. Heat map visualization of feature rankings comparing relative levels in the irradiated GI and Kidney tissue. Each row represents a unique feature with a characteristic mass to charge and retention time value.

28 Supplementary Table ST4. Putative serum biomarkers of radiation injury in C57BL6 mice irradiated with 4 & 8 Gy gamma radiation after 1 and four days respectively.


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