1 Presented By Malaika Argade Department of Medicinal Chemistry Virginia Commonwealth University Email argademdvcuedu Date 25 th March 2011 LCMS 2 LIQUID CHROMATOGRAPHY MASS SPECTROMETRY ID: 1040066
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1. Applications of LC-MS in Chemical and Biochemical Sciences1Presented By : Malaika ArgadeDepartment of Medicinal ChemistryVirginia Commonwealth UniversityEmail : argademd@vcu.eduDate : 25th March 2011
2. LC-MS?2LIQUID CHROMATOGRAPHYMASS SPECTROMETRYSeparates componentsIdentification from retention time is difficult Component identification is superiorBut, interference from other ionsArdrey, R. E. Liquid Chromatography Mass spectrometry: an introduction, Wiley, West Sussex, England, 2003
3. HISTORY1906 : Mikhail Tsvet invented chromatography1930 : Edgar Lederer, Chromatographic separation of carotenoids1960s : Csaba Horvath, developed the first HPLC1990s: Engineering developments in HPLC2004: UPLC and monolithic columns3Henry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p. 21-49
4. Mass Spectrometry1897- Sir “J.J.” Thomson, charge/mass of electron 1919- Francis W. Aston with Thomson developed a “mass spectrometer” to separate isotopes of elements1970s- Interfacing LC with MS1977-1980- Moving Belt Interface & Direct Liquid Interface1989- Electro Spray Ionization, John B. Fenn4Watson, J. T. et al. In Introduction to mass spectrometry, 3rd ed, Wiley: Wiltshire, 2007.
5. COMPONENTS OF LC5LIQUID RESERVOIRPUMPSAMPLE INJECTORCOLUMNDETECTORRECORDERHenry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p. 21-49
6. COLUMNS 6 Backpressure overcome by,Elevated temperaturesMonolithic columnsSwartz, M.E. J. Liq. Chromatogr. R. T. 2005, 28, 1253-1263.HeightHeight equivalent to theoretical plate(HETP)
7. 7Basic components of MSION SOURCEFrit-Fast Atom Bombardment(Frit FAB),Electro Spray Ionization (ESI)MASS ANALYSERDETECTORElectron Multiplier tubeTime-of-Flight,Quadrupole,Magnetic Sectorhttp://www.epa.gov/esd/chemistry/org-anal/reports/phthalates/Fig1phms.png (accessed on 3/23/2011)
8. MASS SPECTROMETER8 A mixture of molecules.Different molecular weights and sizes.Sorted by the mass spectrometer according to abundance and m/z. http://www.asms.org/Portals/0/Concept3.gif (accessed on 3/21/2011)
9. 9SAMPLEMS 1Precursor ionMS 2Product ionTANDEM MS or MS/MSDETECTED!ESI,FABCIDSelected Reaction Monitoring (SRM)Multiple Reaction Monitoring (MRM)
10. APPLICATIONSIn areas such as,Organic chemistryArchaeological scienceToxicology studiesForensic sciences and urinanalysisImpurity detection or identificationNatural product dereplicationIdentification of metabolitesEnzyme inhibition studies10
11. APPLICATIONSLC-MS has wide applications in,Screening botanical extracts.11
12. DISCOVERING INHIBITORS FROM BIOLOGICAL EXTRACTS12Biological extracts are complex mixtures of compounds.Difficult to isolate a particular compound.Problems of co-extraction and interference. So Ultrafiltration with LC-MS
13. INHIBITORS OF QR-2Resveratrol, a natural product inhibitor of Quinone Reductase 2.13IncubationQR-2ExtractRemoval of unbound compoundsDissociationLC-MS2UltrafiltrationLiu, D. et al. Anal.Chem. 2007, 79, 9398-9402.
14. Resveratrol14Test: Resveratrol + active QR-2 (solid line)Control: Resveratrol + denatured QR-2 (dotted line)m/z : 227 Difference indicates active binding.Choi, Y. et al. Anal. Chem. 2011, 83, 1048-1052.
15. Test : Extract of Actinomyces +active QR-2 Control : Extract + denatured QR-2m/z of 317 Structure of TME determined by NMRActinomyces sp. EXTRACT15Choi, Y. et al. Anal. Chem. 2011, 83, 1048-1052.
16. HOPS EXTRACT16Test : Extract + active QR-2Control: Extract + denatured QR-2m/z : 353 & 369Structure confirmed by LC-MS, co-elution with standard.But are they binding at the same pocket as that of Resveratrol ? Choi, Y. et al. Anal. Chem. 2011, 83, 1048-1052.
17. COMPETITIVE BINDING STUDIES17Difference in the peaks obtained indicate active binding.Extract + QR-2 + ResveratrolChoi, Y. et al. Anal. Chem. 2011, 83, 1048-1052.
18. RESULTSTME, xanthohumol and xanthohumol D bind at QR-2 and compete with resveratrol. Enzyme inhibition assay determined, X-ray Crystallography confirmed xanthohumol and X-D binding to active pocket of QR-2. 18CompoundIC50 Resveratrol5.1μMTetrangulol methyl ether0.16μM (most potent)Xanthohumol196μMXanthohumol D110μMChoi, Y. et al. Anal. Chem. 2011, 83, 1048-1052.
19. APPLICATIONSLC-MS has wide applications in,Identification of natural productsStructural characterization of peptides19
20. ESI-MS of Bovine Serum Albumin20Molecular weight by ESI-MS : 66465.8 DaAverage molecular weight calculated from 582 residues: 66267.1 DaDifference: 198.7 Da An undetected residue?Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
21. Determining Amino Acid Sequence At DNA levelBase sequencing techniqueChain terminators usedErrorsTime consumingAt Amino Acid LevelEdman degradationCleaving of peptide from N-terminal sideOne peptide at a timeNot for more than 50 amino acidsFrit-FAB LC-MS/MSMS/MS gives valuable daughter ion informationVery quick21
22. 22COMPARISON WITH HSA AND RSA94 95156
23. PROCEDURE 23BSA+TrypsinHPLCCleaved sequenceFrit-FAB MSHirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
24. RESULTS FROM HPLC 2475 peaks foundHirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
25. Two cases encountered during comparison,25Peaks from HPLCMatching Tryptic SequencesPeak 12Arg144 – Tyr147Gln195 – Arg198Peak 36Ala128 – Lys136Glu564 – Lys573Peak 66Unmatched.Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
26. Peak 12 : Daughter ions from HPLC-MS/MS indicating RHYP sequence .y3≈416.3a3≈303.3a2≈266.326Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
27. Two cases encountered during comparison,27Peptides Matching Sequences and ResultsPeak 12Arg144 – Tyr147Confirmed.Similarly,Peak 36 Found to contain –VEG- Glu564 – Lys573Confirmed.Peak 66Edman degradation confirmed sequenceFYAPELLYY 148-156 sequenceConfirmed by MS/MSY detected in 156th position!!!Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
28. POSITION 94th AND 95thSome peptides were not identified by Frit-FAB LC-MS after trypsin digestion like sequence 82 - 98 So, BSA was digested with lysyl endopeptidase which matched the sequences, VASLRETYGDMADC*C*EK QEPERNEC*FLSHKGlu82 to Arg98 was established94th and 95th : -QE- was established2877-9394-106Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
29. COMPARISON OF RESULTSBSAPrevious findingsNew findings Molecular weight 66267.1 Da66430.3 Da156th positionNo residueTyrosineResidue on 94th and 95th position-EQ--QE- Total amino acid residues582 by Edman Degradation58329
30. APPLICATIONSLC-MS has wide applications in,Identification of natural productsStructural characterization of peptidesMeasuring enzyme activity30
31. Angiotensin Converting EnzymeACE is a target for anti-hypertensive drugs because,31Angiotensin 1Angiotensin 2ACEVasoconstrictionBradykininVasodilatationACEANDGeng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317.
32. DETECTING ACE ACTIVITYACE activity is usually determined by formation of a product from a substrate.Hippuric acid formed indicates ACE activity.32Hippuryl-Histidine-LeucineACEHippuric acidGeng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317
33. METHOD33HHLHA Standard solutions of HA were analyzed by UPLC-MS and peak area plotted against known concentration. Inhibitors were added with HHL and ACE.After incubation HA was analyzed by UPLC-MS and compared with standard solutions.Geng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317
34. RESULTS Where, C0 = HA concentration without inhibitor C = HA concentration with inhibitorAdvantages;Quick screeningLower limits of detectionLesser analysis time 34ConcentrationPeak areaStandard. HA curveGeng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317
35. APPLICATIONSLC-MS has wide applications in,Identification of natural productsStructural characterization of peptidesMeasuring enzyme activityForensic analysis35
36. Detection Of SteroidsFor drug-free competitionsTo avoid false positivesSensitive method to detect drugs in small amounts from hair samplesE.g. Stanzolol and Nandrolone. ELISATraditional method for steroid detection.Based on competition between drug and drug-enzyme conjugate.36Deshmukh, N. et al. Steroids. 2010, 75, 710-714.
37. ELISA37Y Y Y YY Y Y YY Y Y YY Y Y YY Y Y YSSSSLessdrugMoredrugVoller, A. et al. J. Clin. Pathol. 1978, 31, 507-520Tetra methylbenzidineY – Antibody - Drug - Drug enzyme conjugate
38. 38NandroloneStanzololUPLC-MS/MSRESULTSDeshmukh, N. et al. Steroids. 2010, 75, 710-714.m/z transition 275.2 109.2m/z transition329.2 81.1
39. ELISA Vs. UPLC-MS/MS39METHODNANDROLONESTANZALOLELISA316UPLC-MS/MS112UPLC-MS/MS more sensitive than ELISANumber of participants : 160Deshmukh, N. et al. Steroids. 2010, 75, 710-714.
40. APPLICATIONSLC-MS has wide applications in,Identification of natural productsStructural characterization of peptidesForensic analysisMeasuring enzyme activityWine Chemistry40
41. CONTENTS OF SHEDEHOf religious importance in Ancient EgyptBlood of God Osiris, symbolizes rebirth of the deadContents were unknownVery small samples LC-MS/MS in MRM mode: highly specific.41Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2004, 33, 98-101.
42. DETECTIONTartaric acid: Wine markerSyringic acid: Red wine markerSo, wine it is!White or red ?42Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2006, 33, 98-101Guasch-Jané, M.R. et al. Anal. Chem. 2004, 76, 1672-1677
43. Why alkaline fusion?43Malvidin-3-glucosideSyringic acidAlkalinefusionGuasch-Jané, M.R. et al. J. Archaeol. Sci. 2006, 33, 98-101
44. RESULTSShedeh is indeed red wine.Successful detection of syringic and tartaric acid in trace amounts.MRM mode is a confirmation in itself.44
45. SummaryLC-MS applications are wideOver the years, MS has been replaced by MS/MS and even MSn ; LC by UPLC .The technique offers a lot of flexibility and adaptability. Each engineering aspect plays an important role.45
46. ACKNOWLEDGEMENTSDr. Umesh DesaiThe Desai GroupDepartment of Medicinal Chemistry, School of PharmacyVirginia Commonwealth UniversityFriends and family46