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1 LC/MS-TOF LC/Q-TOF GeneSpring MS BLB: Bacterial Leaf Blight Steven Fischer Senior Application Chemist Agilent Technologies Santa Clara, California U.S.A Theodore Sana Senior Research Scientist Agilent Laboratories Santa Clara, California U.S.A ( Oryza sativa Oryza glaberrima) Xathomonas oryzae pv. oryzae (Xoo) (BLB) 5% BLB Xoo Xoo BLB Xoo PXO99 TP39 TP39-Xa2 2 PXO99 raxst TP39-Xa2 2 LC/MS Time-of-Flight (TOF) (MS) Quadrupole Time-of- Flight (Q-TOF) MS/MS /

2 O N C H N S C O H P C N S H C O METABOLOMICS (Oryza sativa Oryza glaberrima) % 25 3 Xanthomonas oryzae pv. oryzae (Xoo) ( ) BLB Xoo BLB Xa2 Taipei 39 (TP39) Xa2 Xa2 - (avr) ( Xoo AvrXa2 ) - 2 Xoo TP39-Xa2 AvrXa2 Xoo raxst AvrXa2 2 Xoo PXO99 raxst AvrXa2 ( 2) raxst PXO99-raxST AvrXa2 PX99 PX99-raxST - avrxa2 2. Xoo raxst TP39-Xa2 AvrXa2 2 PXO99 raxst AvrXa2 raxst PXO99- raxst raxst AvrXa2 BLB LC/MS Agilent 2 LC Agilent 62 Time-of-Flight LC/MS LC/MS 2 ppm Agilent GeneSpring MS multi-class data. ( ) ( ) 2

3 LC/MS-TOF LC/Q-TOF GeneSpring MS BLB: Bacterial Leaf Blight Agilent 2 LC Agilent 65 Quadrupole Time-of- Flight LC/MS Agilent LC/MS MS/MS 2 METLIN ID 2 (TP39 TP39-Xa2) 2 (PXO99 PXO99-raxST ) 7 LC/MS TP39 TP39-Xa2 2. TP39 TP39-Xa2 () ( ) ( ) PXO99 ( ) 6 6 Mock 6 6 (NT) 6 6 PXO99-raxST NA 6 (raxst ) Xoo (PXO99 PXO99-raxST ) (Tsuchiya 982 ) 3 C 72 6 (TP39 TP39-Xa2) 9 /ml (Kauffman 973 ) () 4 cm Weckwerth 3 2 mg 5 mm 2 ml ( 3) MM3 Retsch Mill 25 Hz 3 ml ( / / 2:3:3 v/v/v ) 2 C LC/MS GC/MS LC/MS ZORBAX SB-Aq 2. x 5 mm Agilent 2 LC ml 2 L.3 ml/min 46 / 2% 98% 54.9.% 3

4 O N C H N S C O H P C N S H C O METABOLOMICS Retsch Mill 2 o C DNA 3. (ESI) Agilent 62 Time-of-Flight LC/MS ESI m/z 5 95 MS / ESI Agilent 65 Quadrupole Time-of-Flight LC/MS MS/MS ESI MS Agilent MassHunter MassHunter Feature extraction (correlation algorithm) (co-variant) LC/TOF MS LC : ZORBAX SB-Aq 2. x 5 mm 3.5 µm : A =.% B =.% : 2% B 98% B 46 98%B % B 55 MS stop : 54.9 LC stop : 55 : 2 C :.3 ml/min : 2 µl + 3 MS : : * : L/min : 25 C : 35 psi : m/z 5 95 : 7 V : 4, V : m/z : µl/min * 4

5 LC/MS-TOF LC/Q-TOF GeneSpring MS BLB: Bacterial Leaf Blight LC/Q-TOF MS/MS LC : ZORBAX SB-Aq 2. x 5 mm 3.5 µm : A =.% B =.% : 2% B 98% B 46 98% B % B 55 MS stop : 54.9 LC stop : 55 LC : 7 : 2 C :.3 ml/min : 2 µl MS : : : L/min : 25 C : 4 psig MS: m/z, 25 ms/ MS/MS: m/z, 25 ms/ : 5 x +ev : : 7 V : 65 V RF : 75 V : 4, V : m/z : µl/min : 5 psig (Na + K + ) [M+H + ] Agilent GeneSpring MS MassHunter / ( 4 ):. Alignment (normalization) 2. (clustering) 3. (-way analysis) (2-way analysis) (ANOVA) 4. (PCA) 5. (fold changes) 6. METLIN Q-TOF LC/MS MS/MS 7. Q-TOF 5

6 O N C H N S C O H P C N S H C O METABOLOMICS Bacterial features 22 Immunity features Infected features TP39 TP 39 Xa2 + Group Sample Number File Name Rice Lines 7 Rice line features Y Infected wild and transgenic Z Uninfected wild Uninfected transgenic X MS/MS Fold-Change filtering PCA 4. MS GeneSpring MS / TP39 PXO99 PXO99-raxST ( 5) TP39 Xoo PXO99 PXO99 PXO99-raxST- PXO99-raxST- TP39-Xa2 PXO99 Xa2 PXO99 AvrXa2 TP39-Xa2 PXO99-raxST PXO99- raxst AvrXa2 TP39-Xa2 5. TP39 TP39-Xa2 6

7 LC/MS-TOF LC/Q-TOF GeneSpring MS BLB: Bacterial Leaf Blight (ANOVA) PXO99 ANOVA 3 (pair-wise comparison) : t- t-testanova TP39-Xa2 PXO99 vs. TP39-Xa2 Mock t- 7 TP39 PXO99 vs. TP39 Mock 2 t- TP39 Mock vs. TP39-Xa2 Mock t- I ( ) ( 7) way) ANOVA : -way) ANOVA I (42) t- (22) 6 Tukey (post-hoc test) ANOVA (P <.5) (25) (7) 6. MS ANOVA (P <.5) () () TP39-Xa2 Mock TP39-Xa2 PXO

8 O N C H N S C O H P C N S H C O METABOLOMICS TP39-Xa2 PX99 vs. TP39-Xa2 Mock Immunity features 39 Bacterial features 7 42 TP39 PX99 vs. TP39 Mock Infected features (PCA,Principle component analysis) PCA ( ) GeneSpring MS PCA TP39 TP39-Xa2 ( 8a) 2 ANOVA -way ANOVAPCA ( 8b) 7 Rice line features TP39 Mock vs. TP39-Xa2 Mock 7. 3 ANOVA TP39 a. PCA b. (-way) ANOVA PCA Y Y TP39-Xa2 TP39 Infected TP39-Xa2 8. PCA -way) ANOVA (b) Z X Z TP39 (Uninfected) (Uninfected) TP39-Xa2 X TP39 TP39-Xa2 TP39 Mock TP39-Xa2 Mock TP39 PXO99 TP39-Xa2 PXO99 TP39-Xa2 PXO99-raxST - 8

9 LC/MS-TOF LC/Q-TOF GeneSpring MS BLB: Bacterial Leaf Blight ANOVA(2-way ANOVA) ANOVA ANOVA ( 9): Z (2-way) ANOVA PCA Y TP39 Infected TP39-Xa2 X TP39 Uninfected TP39-Xa2 Uninfected () Rice line features PCA 3 PXO99-raxST TP39-Xa2 TP39-Xa2 TP39 XO99 ( ) 6 2 () 3 Class features Rice line() Class() 9. ANOVA 3 TP39 TP39 Mock TP39 PXO99 TP39-Xa2 TP39-Xa2 mock TP39-Xa2 PXO99 TP39-Xa2 PXO99-raxST -. ANOVA 3 PCA (Fold-change filtering) (visualizaton) ( ) ( ) (Mock Infection) GeneSpring MS GeneSpring MS Volcano Volcano t- 9

10 O N C H N S C O H P C N S H C O METABOLOMICS. ANOVA ( ) (Feature inspection) GeneSpring MS 2 mass inspector (view) 2 Microsoft Excel (mass inspector view) GeneSpring MS ( 2 ) / / t- 2. GeneSpring MS ( ) t- Microsoft Microsoft Corporation

11 LC/MS-TOF LC/Q-TOF GeneSpring MS BLB: Bacterial Leaf Blight Excel GeneSpring MS Up-regulated Downregulated ( 2 3) 2 () / 2. TP39-Xa2 XO99 () Up-regulated ( ) (u) Up-regulated 2 METLIN ± ppm 2 ppm 62 TOF 5 ppm 4 3. TP39-Xa2 XO99 () Down-regulated ( ) (u)

12 O N C H N S C O H P C N S H C O METABOLOMICS 4. METLIN METLIN ( 5 6) ID ID 5, METLIN 5. METLIN TP39-Xa2 PXO99 () Up-regulated METLIN ( ) (u) () C27H68NO3P C7HNO8F2S C38H6N7O C6H7N4O C24H4N4O C29H72N7OP C8H33NO C9H39NO C9H3N9O C32H65N7OS C53H79N2OP C25H62N9O C4HN6O6F6PS C58H76N8O C6H7N5O C34H63N9O3S C25H45N4O9PS 75 Harderoporphyrin C47H7N6O7P erythromycin ethylsuccinate C5H7NO C7H6O2 2 benzoic acid C3H33N2O2P C6H79N7OS C5H5N C7H8N6OPS C3HN3OPS C24H57NO4P 3 Vitamin K C4HF6PS 4 2

13 LC/MS-TOF LC/Q-TOF GeneSpring MS BLB: Bacterial Leaf Blight 6. METLIN TP39-Xa2 PXO99 () Down-regulated METLIN ( ) (u) () none C23H44N6OS C22H57N2O3PS 4 Violaxanthin, Neoxanthin CHNF2P none none C3H48N6O none C29H59N3PS none C29H47N7OP 32 2 Up-regulated 5 m/z Q-TOF LC/MS TOF MS/MS MS/MS ( 3) ( - CH 2 O 2 ) 2 CO METLIN ( 4) MS/MS 6 2 ( ) 2 LC 3

14 O N C H N S C O H P C N S H C O METABOLOMICS x m/z MS/MS CH 2 H + N -CO H + N O -CH 2 O 2 O H + NH O C OH m/z 3. m/z MS/MS (CH 2 O 2 ) CO METLIN METLIN u 6 4

15 LC/MS-TOF LC/Q-TOF GeneSpring MS BLB: Bacterial Leaf Blight (BLB) 2 LC/MS Time-of- Flight (TOF) (MS) Quadrupole Time-of-Flight (Q-TOF) MS/MS 7 / 2 PCA (ANOVA) 2 Agilent GeneSpring MS 2 TOF METLIN () ID () Agilent 65 Q-TOF LC/MS MS/MS 2 Agilent 62 Time-of-Flight LC/MS MS/MS LC (PCA) 5

16 O N C H N S C O H P C N S H C O METABOLOMICS. Song et. al. (995) Science 27: Sang-Wong Lee et. al. PNAS, 3(49) , Weckworth et. al. Proteomics 24, 4, : Agilent Technologies, Inc. 27 Printed in Japan May 2, JAJP

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