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
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-1 2 1 % 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 65-166 Sigma ) PBS 1.5 1 ( ) 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 5 2 21, 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
Agilent 126 Infinity (1:1 ) LC MS 4 GHz 1 1.5 1 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 5.5 1 %B 1 %B MS 1 RF.4 ml/min.6 ml/min.1 1 1 5 C 15 L ESI 25 C 1 L/min 45 psi,5 V 6 V m/z 5 1,7 m/z 119.6 9.16 2. CAS RT ( ) L- C 5 H 9 NO 4 56-6- 2.26 L- C 4 H 7 NO 4 56-4- 2.29 L- C H 6 O 79--4 2.9 C 2 H 2 N 6 O 12 S 2 2725-41- 4.4 C 4 H 6 O 4 11-15-6 5.5 2- C 5 H O 5 29-1- 5.11 C 4 H 4 O 4 11-17- 5.1 L- C 4 H 6 O 5 97-67-6 5.1 ( -KG) C 5 H 6 O 5 2-5-7 5.24 (GDP) C 1 H 15 N 5 O 11 P 2 146-91- 5. C 1 H 15 N 5 O 1 P 2 5-64- 5.9 C 6 H 6 O 6 499-12-7 6.1 - ( ) C 6 H O 7 661-97- 6.1 ( -) C 6 H O 7 77-92-9 6.1 (GTP) C 1 H 16 N 5 O 14 P 6-1-1 6.57 C 1 H 16 N 5 O 1 P 97-65-5 6.6
Profinder -KG Profinder % 1 2-HG 2-HG M+ 2-HG M+5 2-HG 1. Agilent MassHunter Profinder 2-HG 4
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
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) + +1 +2 + +4 +5 +6 4. CS-1 U- 1 C-Gln Hours.5 1 2E5 4E5 6E5 E5 1E6 1.2E6 1.4E6 Unlabeled 6
5 () Profinder 1 2 Gln- 5-1 2 Gln- ADP GDP ATP GTP ( 6) Gln 2- - % 1 6 4 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
( 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
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(), 6-52. 2. Susa, M.; et al.alendronate inhibits growth of high-grade chondrosarcoma cells.anticancer Res.29, 29, 179 1. 9
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