Presentation on theme: "Applications of LC-MS in Chemical and Biochemical Sciences"— Presentation transcript:
1 Applications of LC-MS in Chemical and Biochemical Sciences Presented By : Malaika ArgadeDepartment of Medicinal ChemistryVirginia Commonwealth UniversityDate : 25th March 2011
2 LC-MS? Separates components Component identification is superior LIQUID CHROMATOGRAPHYMASS SPECTROMETRYSeparates componentsIdentification from retention time is difficultComponent identification is superiorBut, interference from other ionsArdrey, R. E. Liquid Chromatography Mass spectrometry: an introduction, Wiley, West Sussex, England, 2003
3 HISTORY 1906 : Mikhail Tsvet invented chromatography 1930 : Edgar Lederer, Chromatographicseparation of carotenoids1960s : Csaba Horvath, developed the first HPLC1990s: Engineering developments in HPLC2004: UPLC and monolithic columnsHenry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p
4 Mass Spectrometry 1897- 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 MSMoving Belt Interface & Direct Liquid Interface1989- Electro Spray Ionization, John B. FennWatson, J. T. et al. In Introduction to mass spectrometry, 3rd ed, Wiley: Wiltshire, 2007.
5 COMPONENTS OF LC LIQUID RESERVOIR SAMPLE INJECTOR PUMP RECORDER DETECTORCOLUMNHenry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p
6 COLUMNS Backpressure overcome by, Elevated temperatures Height equivalent to theoretical plate(HETP)HeightBackpressure overcome by,Elevated temperaturesMonolithic columnsSwartz, M.E. J. Liq. Chromatogr. R. T. 2005, 28,
7 Basic components of MS ION SOURCE MASS ANALYSER DETECTOR Time-of-Flight,Quadrupole,Magnetic SectorFrit-Fast Atom Bombardment(Frit FAB),Electro Spray Ionization (ESI)DETECTORElectron Multiplier tube(accessed on 3/23/2011)
8 MASS SPECTROMETER Different molecular weights and sizes. A mixture of molecules.Different molecular weights and sizes.Sorted by the mass spectrometer according to abundance and m/z.(accessed on 3/21/2011)
9 TANDEM MS or MS/MS SAMPLE MS 2 MS 1 Product Precursor ion ion ESI,FABCIDSAMPLEMS 2ProductionMS 1PrecursorionSelected Reaction Monitoring (SRM)Multiple Reaction Monitoring (MRM)DETECTED!
10 APPLICATIONS In areas such as, Organic chemistry Archaeological scienceToxicology studiesForensic sciences and urinanalysisImpurity detection or identificationNatural product dereplicationIdentification of metabolitesEnzyme inhibition studies
11 APPLICATIONS LC-MS has wide applications in, Screening botanical extracts.
12 DISCOVERING INHIBITORS FROM BIOLOGICAL EXTRACTS Biological 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 2.2ExtractUltrafiltrationRemoval of unbound compoundsIncubationQR-2LC-MSDissociationLiu, D. et al. Anal.Chem. 2007, 79,
14 Resveratrol Test: Resveratrol + active QR-2 (solid line) Control: Resveratrol + denatured QR-2 (dotted line)m/z : 227Difference indicates active binding.Choi, Y. et al. Anal. Chem. 2011, 83,
15 Actinomyces sp. EXTRACT Test : Extract of Actinomyces +active QR-2Control : Extract + denatured QR-2m/z of 317Structure of TME determined by NMRChoi, Y. et al. Anal. Chem. 2011, 83,
16 HOPS EXTRACT Test : Extract + active QR-2 Control: 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,
17 COMPETITIVE BINDING STUDIES Difference in the peaks obtained indicate active binding.Extract + QR-2 + ResveratrolChoi, Y. et al. Anal. Chem. 2011, 83,
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.CompoundIC50Resveratrol5.1μMTetrangulol methyl ether0.16μM (most potent)Xanthohumol196μMXanthohumol D110μMChoi, Y. et al. Anal. Chem. 2011, 83,
19 APPLICATIONS LC-MS has wide applications in, Identification of natural productsStructural characterization of peptides
20 ESI-MS of Bovine Serum Albumin Molecular weight by ESI-MS : DaAverage molecular weight calculated from 582 residues: DaDifference: DaAn undetected residue?Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,
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 quick
23 PROCEDURE + BSA Cleaved sequence Frit-FAB MS HPLC Trypsin Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,
24 RESULTS FROM HPLC 75 peaks found Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,
25 Two cases encountered during comparison, Peaks from HPLCMatching Tryptic SequencesPeak 12Arg144 – Tyr147Gln195 – Arg198Peak 36Ala128 – Lys136Glu564 – Lys573Peak 66Unmatched.Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,
26 Peak 12 : Daughter ions from HPLC-MS/MS indicating RHYP sequence . Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,
27 Two cases encountered during comparison, PeptidesMatching Sequences and ResultsPeak 12Arg144 – Tyr147Confirmed.Similarly,Peak 36Found to contain –VEG-Glu564 – Lys573Peak 66Edman degradation confirmed sequenceFYAPELLYY sequenceConfirmed by MS/MSY detected in 156th position!!!Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,
28 POSITION 94th AND 95thSome peptides were not identified by Frit-FAB LC-MS after trypsin digestion like sequenceSo, BSA was digested with lysyl endopeptidase which matched the sequences,VASLRETYGDMADC*C*EKQEPERNEC*FLSHKGlu82 to Arg98 was established94th and 95th : -QE- was established77-9394-106Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,
29 COMPARISON OF RESULTS BSA Previous findings New findings Molecular weightDaDa156th positionNo residueTyrosineResidue on 94th and 95th position-EQ--QE-Total amino acid residues582 by Edman Degradation583
30 APPLICATIONS LC-MS has wide applications in, Identification of natural productsStructural characterization of peptidesMeasuring enzyme activity
31 Angiotensin Converting Enzyme ACE is a target for anti-hypertensive drugs because,ACEAngiotensin 1VasoconstrictionAngiotensin 2ANDBradykininVasodilatationACEGeng, F. et al. Biomed. Chromatogr. 2010, 24,
32 DETECTING ACE ACTIVITY ACE activity is usually determined by formation of a product from a substrate.Hippuric acid formed indicates ACE activity.ACEHippuryl-Histidine-LeucineHippuric acidGeng, F. et al. Biomed. Chromatogr. 2010, 24,
33 METHODStandard 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.HHLHAGeng, F. et al. Biomed. Chromatogr. 2010, 24,
34 RESULTS Advantages; Quick screening Lower limits of detection Where,C0 = HA concentration without inhibitorC = HA concentration with inhibitorAdvantages;Quick screeningLower limits of detectionLesser analysis timePeak areaStandard. HA curveConcentrationGeng, F. et al. Biomed. Chromatogr. 2010, 24,
35 APPLICATIONS LC-MS has wide applications in, Identification of natural productsStructural characterization of peptidesMeasuring enzyme activityForensic analysis
36 Detection Of Steroids For drug-free competitions To 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.Deshmukh, N. et al. Steroids. 2010, 75,
37 ELISA Less drug More drug Y – Antibody - Drug - Drug enzyme conjugate Y Y Y YY Y Y YY Y Y YSSSTetra methylbenzidineSY Y Y YY Y Y YLessdrugMoredrugVoller, A. et al. J. Clin. Pathol. 1978, 31,
38 UPLC-MS/MS RESULTS Nandrolone Stanzolol m/z transition 275.2 109.2 NandroloneStanzololm/z transitionDeshmukh, N. et al. Steroids. 2010, 75,
39 ELISA Vs. UPLC-MS/MS METHOD NANDROLONE STANZALOL ELISA 3 16 UPLC-MS/MS Number of participants : 160METHODNANDROLONESTANZALOLELISA316UPLC-MS/MS112UPLC-MS/MS more sensitive than ELISADeshmukh, N. et al. Steroids. 2010, 75,
40 APPLICATIONS LC-MS has wide applications in, Identification of natural productsStructural characterization of peptidesForensic analysisMeasuring enzyme activityWine Chemistry
41 CONTENTS OF SHEDEH Of religious importance in Ancient Egypt Blood of God Osiris, symbolizes rebirth of the deadContents were unknownVery small samplesLC-MS/MS in MRM mode: highly specific.Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2004, 33,
42 DETECTION Tartaric acid: Wine marker Syringic acid: Red wine marker So, wine it is!White or red ?Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2006, 33,Guasch-Jané, M.R. et al. Anal. Chem. 2004, 76,
43 Why alkaline fusion? Syringic acid Alkaline Malvidin-3-glucoside Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2006, 33,
44 RESULTS Shedeh is indeed red wine. Successful detection of syringic and tartaric acid in trace amounts.MRM mode is a confirmation in itself.
45 Summary LC-MS applications are wide Over 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.
46 ACKNOWLEDGEMENTS Dr. Umesh Desai The Desai Group Department of Medicinal Chemistry, School of PharmacyVirginia Commonwealth UniversityFriends and family
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