JAJP.indd

Size: px

Start display at page:

Download "JAJP.indd"

ともみやぶき
5 years ago
Views:

1 Agilent VistaFlux 1 C- Hui Liu, Weige Qin, Hardik Shah, Bhavapriya Vaitheesvaran, Vladimir Yong, and Justin R. Cross Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center Lucia Salamanca-Cardona and Kayvan R. Keshari Department of Radiology and Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center ( ) ( 1 C 15 N 2 H ) () Agilent MassHunter VistaFlux TOF Q-TOF LC/MS

2 1 C 5 - (U- 1 C-Gln) Agilent LC/TOF MassHunter VistaFlux TCA 2- (2-HG) 2 (IDH2) IDH (IDH1/2) 2- (2-HG) 2-HG IDH 2-HG DNA TET mrna FTO ALKBH5 C (PHD) - ( -KG) (HIF) 1 IDH IDH 2-HG -KG TCA CS % FBS 25 mm 4 mm L- 1 units/ml 1 ug/ml DMEM 5 % CO 2 7 C 6-, / 2 DMEM 1 % FBS 4 mm [U- 1 C]-L- (Isotec Sigma ) PBS ( ) 1 % FBS 4 mm L- ( ) DMEM U- 1 C-Gln 1 ml/well % (v/v) - C 1 21, x g 15 4 C (Genevac ) PBS PBS PBS LC/MS 12 µl , x g 15 4 C 1 µl QC 1 µl 6 Agilent LC/MS Agilent 62 TOF Agilent 129 Infinity UHPLC Agilent 129 Infinity Agilent 129 Infinity HTS 2

3 Agilent 126 Infinity (1:1 ) LC MS 4 GHz TCA Agilent MassHunter Pathways to PCDL BioCyc (TCA) (PCDL) (.cdb) 2-HG Agilent METLIN PCDL PCDL Agilent PCDL Manager PCDL Agilent MassHunter Profinder ( 2) 1 C Profinder Omix Premium Profinder (.PFA) (.csv) 1. LC/MS LC Waters Cortecs C1+ 15 mm 2.1 mm 2.7 µm A) 5 mm N,N- ph 5.5 B) 5 mm N,N-9 % ph %B 1 %B MS 1 RF.4 ml/min.6 ml/min C 15 L ESI 25 C 1 L/min 45 psi,5 V 6 V m/z 5 1,7 m/z CAS RT ( ) L- C 5 H 9 NO L- C 4 H 7 NO L- C H 6 O C 2 H 2 N 6 O 12 S C 4 H 6 O C 5 H O C 4 H 4 O L- C 4 H 6 O ( -KG) C 5 H 6 O (GDP) C 1 H 15 N 5 O 11 P C 1 H 15 N 5 O 1 P C 6 H 6 O ( ) C 6 H O ( -) C 6 H O (GTP) C 1 H 16 N 5 O 14 P C 1 H 16 N 5 O 1 P

4 Profinder -KG Profinder % 1 2-HG 2-HG M+ 2-HG M+5 2-HG 1. Agilent MassHunter Profinder 2-HG 4

5 Profinder BioCyc Omix Premium TCA ( ) ADP GDP ATP GTP TCA Profinder Omix Premium ( 2) Component View Data Manager Profinder Omix Premium 2 ( ) 2. Agilent MassHunter Profinder Omix Premium 5

6 2-HG ( A ) ( B) C 4 y x ( ) Profinder U- 1 C-Gln 2-HG M+5 IDH2- CS-1 2-HG A B C 2-Hydroxyglutarate 2-Hydroxyglutarate 2-Hydroxyglutarate. 2-HG A) B) C) CS-1 U- 1 C-Gln Hours.5 1 2E5 4E5 6E5 E5 1E6 1.2E6 1.4E6 Unlabeled 6

7 5 () Profinder 1 2 Gln Gln- ADP GDP ATP GTP ( 6) Gln 2- - % Glutathione, oxidized Isotopologue abundances (corrected) m+ m+1 hours.5 hours 1 hour hours hours m+2 m+ m+4 m+5 m+6 m+7 m+ m+9 m+1 m+11 m+12 m+1 5. ADP Hours.5 1 5E5 1E6 1.5E6 2E6 ATP Hours 5E5 1E6 1.5E6 2E6 2.5E6 E6.5 1 Unlabeled Unlabeled GDP Hours 5E4 1E5 1.5E5.5 1 GTP Hours 2E4 4E4 6E4 E4 1E5 1.2E5.5 1 Unlabeled Unlabeled 6. ADP GDP ATP GTP 7

8 ( 7) BioCyc 7 ( ) t p <.5 U- 1 C-Gln 2-HG (Glu) 2-HG IDH2 Glu -KG ( ) 2-HG U- 1 C-Gln 5 1 C 2-HG TCA U- 1 C-Gln ( ) M+4 (OAA) LC/MS OAA M+4 L- (Asp) 7. 2-HG TCA U- 1 C-Gln

9 M+ 2-HG M+4 OAA -CoA 2 M+4 M+ IDH ( ) IDH M+5 TCA Gln Asp Glu Agilent MassHunter VistaFlux U- 1 C-Gln CS-1 Gln- -KG 2-HG IDH2 2-HG IDH M+5 2-HG IDH2 R172S 2-HG 2-HG 2-HG TET DNA C -KG DNA TCA U- 1 C-Gln VistaFlux 1. Losman, J. A.; Kaelin, W. G. Jr. What a difference a hydroxyl makes: mutant IDH, (R)-2- hydroxyglutarate, and cancer.genes Dev.21, 27(), Susa, M.; et al.alendronate inhibits growth of high-grade chondrosarcoma cells.anticancer Res.29, 29,

10 Agilent Technologies, Inc. 216 Printed in Japan, May 6, JAJP

Metabolomic Profiling_JA.qxp

Metabolomic Profiling_JA.qxp 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