Mass spectrometric analysis of cyp450s following mechanism based inactivation Lightning 2012

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Mass spectrometric analysis of cyp450s following mechanism based inactivation Lightning 2012

  1. 1. Mass Spectrometric Analysis of CYP450s Following Mechanism-Based Inactivation Luke Lightning, PhD Alquest Therapeutics San Francisco, CA Genentech Presentation 7/11/2012
  2. 2. Outline• Brief Introduction• LC/MS Analysis of Intact CYP450s• Identification of Peptide Adducts• Other Studies• Conclusions and Future Directions
  3. 3. Known Pathways of CYP450 Mechanism-Based Inactivation MBI* N Fe N N N Fe MBI MBI* N N N N Fe N Fe NN N N N N N Cys
  4. 4. Purification and Mass Spectral Analysis of P450s MM 1A2 2A6 2C9 2C9 3A4 P450 2C9 C175RkDa 55,578.6 Da 94 67 ESI-MS 43 30 20 • specific contents = 14.3-16.6 nmol/mg • Charge States = 25 • Predicted MM = 55,575.1 Da • Error = 0.006% (3.5 Da)
  5. 5. Standard Inactivation Experimental Protocol• CYP450:reductase:cytochrome b5 (various ratios, 1 nmol CYP450)• 50 mM potassium phosphate buffer• Dialyze overnight at 4ºC to remove glycerol and/or detergent• 50 µg DLPC, 2000 U/mL catalase added• Set on ice for 60 min• Preincubate at 30ºC for 2 min with inactivator (1% MeOH maximum)• Initiate reaction with NADPH and incubate for 60 min• Set on ice until LC/MS/MS analysis of intact protein or incubate with protease or CNBr (reaction times vary) for peptide digests• Can pre-treat the intact protein mixture with denaturants (e.g. Guanidinum HCl, urea)
  6. 6. Standard LC/MS/MS Analysis• HPLC column: Poros R2 perfusion column (4.6 x 100 mm) from Applied Biosystems (Cambridge, MA)• Buffer A – 0.05% TFA (pH 3.0), Buffer B – 0.05% TFA in 95:5% ACN:H2O• Flow rate: 3 mL/min with 50 µL diverted to the MS• 300 pmol of spectrally detectable CYP450 or 1 nmol of inactivated CYP450 and components was injected onto the system• VG Quattro II triple quad ESI-MS running MassLynx software• Data acquisition from m/z 200-2000Da• Improvements with time: – Can purchase purified CYP450s, CYP450 reductase, and cytochrome b5 – Mass spectrometers and software (e.g. SEQUEST) – 2.1 x 30 mm columns  lower flow rates, less protein required
  7. 7. CYP450 2B1 + 8-MOP: HPLC Separation 250 200 CYP450 reductase CYP2B1Absorbance 150 (214 nm) 100 50 b5, Heme 0 0 Time (min) 8.0 400 300 Binding StoichiometryRadioactivity (dpm) = 0.7 ± 0.1 200 100  8-MOP binding is specific for the apoprotein of 2B1 short HPLC run time (8-10 min)  hydrolysis of the label occurs (e.g. HPLC, SDS-PAGE, & enzymatic digest) Biochemistry 37, 13184-13193 (1998)
  8. 8. Methods in Enzymology, Volume 357, page 296-300 (2002)
  9. 9. Mechanism-based Inactivators of CYP450s 8-MOP 2A6 & 2B1 O O O OCH3 O S X 2C9Tienilic Acid Cl O OCH2COOH Cl Nu: CYP450 N OH OH 3A4 L-754,394 H O N N N O O NH
  10. 10. CYP2C9 + Tienilic Acid Monoadduct 55,578.6 Da (+) 344 ± 1 2C9 Diadduct CYP2C9 (+) 694 ± 4 + (+) GSH 2C9 monoadduct O STienilic Acid Cl OCH2COOH Cl  (+) 344 and (+) 694 Da correlate w/ addition of TA-OH to 2C9  suggested thiophene epoxide mechanism is operative Biochemistry 38, 2312-2319 (1999)
  11. 11. CYP2B1 + 8-MOP: LC/MS Analysis CYP2B1 CYP2B1/8-MOP + CYP2B1 56,163.6 Da 55,925.7 Da CYP2B1 + 8-MOP• predicted = 56,933.8 Da •  = 237.9 Da (furanoepoxide = 234.2 Da)• error = 8.1 Da (0.01%) • similar results obtained with CYP450 2A6 Biochemistry 37, 10047-10061 (1998)
  12. 12. Methods in Enzymology, Volume 357, page 296-300 (2002)
  13. 13. MM 2B1 + MM kDa 8-MOP kDa 200 8-MOP CYP2B1-8-MOP 116 97 66 N 55 N Fe N N 37 31 22 CNBr 17 14 14 11 8 6 3.5 2.5 Radioactivity 800 CNBr Peptide 600Radioactivity (dpm) 400 200 MALDI-MS 0 0 10 20 30 40 Time (min)
  14. 14. MALDI-MS: CYP2B1 + 8-MOP 35000 2722.1 30000 25000Counts 20000 15000 10000 5000 0 2500 2600 2700 2800 2900 3000 Mass (m/z) 2721.9 ± 0.1 290-ISLLSLFFAGTETSSTTLRYGFLLM-314 (2721.2 Da) SRS-4 (I helix)
  15. 15. CYP2B1 + 8-MOP 8-MOP
  16. 16. CYP3A4 + L-754,394 SDS-PAGE HPLC MALDI-MSBiochemistry 39, 4276-4287 (1999)
  17. 17. MALDI-MS: CYP3A4 + L-754,394 4000 4705.2 3000Counts 2000 1000 0 4000 4300 4600 4900 5200 5500 Mass (m/z) 4704.2 ± 1.0 275-IDSQNSKETESHKALSDLELVAQSIIFIFAGYETTSSVLSFIM-317 (4703.2 Da) SRS-4 (I-helix)
  18. 18. CYP3A4 + Raloxifene
  19. 19. raloxifene- adductedPIA adducted CYP3A4 peptide, (Cys-reacting) 237-NICVFPR-243. Cys239 No radiolabel MS2 adducted peptide from proteinase K digest MS3
  20. 20. Cys2393 peptide from tryptic digest Tyrosine 75
  21. 21. 20112011 CYP2B6 CYP2B6 + clopidogrel CYP2B6+ clopidogrel+ DTT
  22. 22. Other studies2004 2005 2006
  23. 23. Other studies 2010 2011 2012
  24. 24. Conclusions• LC/MS analyses of intact CYP450s is possible – Short run times, accurate• Adducted intact proteins and peptide adducts can be identified without use of a radiolabeled molecule
  25. 25. Future Directions?• Crystal structures of adducted proteins• Proteomic analysis and scanning for adducts? – Microsomes, hepatocytes – Supersomes – Labeled compounds or isotope labeled proteins – Affinity purification techniques – Potential issues with ESI • Amount of protein required  clogging of columns • Stability of adducts to workup conditions – MALDI
  26. 26. Future Directions – MALDI?• Tumor specific protein signals were detected • Proteomic information was extracted • Try with HLMs
  27. 27. Thank you!! Luke Lightning, PhD Alquest Therapeutics llightning@alquest.us http://www.meetup.com/BayAreaLifeTech/Next event: Happy Hour in SF, Thursday 7/12/2012

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