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Applications of LC-MS in Chemical and Biochemical Sciences 1 Presented By : Malaika Argade Department of Medicinal Chemistry Virginia Commonwealth University.

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Presentation on theme: "Applications of LC-MS in Chemical and Biochemical Sciences 1 Presented By : Malaika Argade Department of Medicinal Chemistry Virginia Commonwealth University."— Presentation transcript:

1 Applications of LC-MS in Chemical and Biochemical Sciences 1 Presented By : Malaika Argade Department of Medicinal Chemistry Virginia Commonwealth University Date : 25 th March 2011

2 LC-MS? 2 LIQUID CHROMATOGRAPHYMASS SPECTROMETRY Separates components Identification from retention time is difficult Component identification is superior But, interference from other ions Ardrey, R. E. Liquid Chromatography Mass spectrometry: an introduction, Wiley, West Sussex, England, 2003

3 HISTORY 1906 : Mikhail Tsvet invented chromatography 1930 : Edgar Lederer, Chromatographic separation of carotenoids 1960s : Csaba Horvath, developed the first HPLC 1990s: Engineering developments in HPLC 2004: UPLC and monolithic columns 3 Henry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p

4 Mass Spectrometry Sir “J.J.” Thomson, charge/mass of electron Francis W. Aston with Thomson developed a “mass spectrometer” to separate isotopes of elements 1970s- Interfacing LC with MS Moving Belt Interface & Direct Liquid Interface Electro Spray Ionization, John B. Fenn 4 Watson, J. T. et al. In Introduction to mass spectrometry, 3 rd ed, Wiley: Wiltshire, 2007.

5 COMPONENTS OF LC 5 LIQUID RESERVOIR PUMP SAMPLE INJECTOR COLUMNDETECTORRECORDER Henry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p

6 COLUMNS 6 Backpressure overcome by, Elevated temperatures Monolithic columns Swartz, M.E. J. Liq. Chromatogr. R. T. 2005, 28, Height Height equivalent to theoretical plate (HETP)

7 7 Basic components of MS ION SOURCE Frit-Fast Atom Bombardment (Frit FAB), Electro Spray Ionization (ESI) MASS ANALYSER DETECTOR Electron Multiplier tube Time-of-Flight, Quadrupole, Magnetic Sector (accessed on 3/23/2011)

8 MASS SPECTROMETER 8 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 9 SAMPLE MS 1 Precursor ion MS 2 Product ion TANDEM MS or MS/MS DETECTED! ESI,FABCID Selected Reaction Monitoring (SRM) Multiple Reaction Monitoring (MRM)

10 APPLICATIONS In areas such as, Organic chemistry Archaeological science Toxicology studies Forensic sciences and urinanalysis Impurity detection or identification Natural product dereplication Identification of metabolites Enzyme inhibition studies 10

11 APPLICATIONS LC-MS has wide applications in, Screening botanical extracts. 11

12 DISCOVERING INHIBITORS FROM BIOLOGICAL EXTRACTS 12 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-2 Resveratrol, a natural product inhibitor of Quinone Reductase Incubation QR-2 Extract Removal of unbound compounds Dissociation LC-MS 2 Ultrafiltration Liu, D. et al. Anal.Chem. 2007, 79,

14 Resveratrol 14  Test: 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,

15  Test : Extract of Actinomyces + active QR-2  Control : Extract + denatured QR-2  m/z of 317  Structure of TME determined by NMR Actinomyces sp. EXTRACT 15 Choi, Y. et al. Anal. Chem. 2011, 83,

16 HOPS EXTRACT 16  Test : Extract + active QR-2  Control: Extract + denatured QR-2  m/z : 353 & 369  Structure 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 17  Difference in the peaks obtained indicate active binding.  Extract + QR-2 + Resveratrol Choi, Y. et al. Anal. Chem. 2011, 83,

18 RESULTS TME, 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 CompoundIC 50 Resveratrol5.1μM Tetrangulol methyl ether0.16μM (most potent) Xanthohumol196μM Xanthohumol D110μM Choi, Y. et al. Anal. Chem. 2011, 83,

19 APPLICATIONS LC-MS has wide applications in, Identification of natural products Structural characterization of peptides 19

20 ESI-MS of Bovine Serum Albumin 20  Molecular weight by ESI-MS : Da  Average molecular weight calculated from 582 residues: Da  Difference: Da  An undetected residue? Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

21 Determining Amino Acid Sequence At DNA level Base sequencing technique Chain terminators used Errors Time consuming At Amino Acid Level Edman degradation Cleaving of peptide from N-terminal side One peptide at a time Not for more than 50 amino acids Frit-FAB LC-MS/MS MS/MS gives valuable daughter ion information Very quick 21

22 22 COMPARISON WITH HSA AND RSA

23 PROCEDURE 23 BSA + Trypsin HPLC Cleaved sequence Frit-FAB MS Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

24 RESULTS FROM HPLC peaks found Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

25 Two cases encountered during comparison, 25 Peaks from HPLCMatching Tryptic Sequences Peak 12Arg 144 – Tyr 147 Gln 195 – Arg 198 Peak 36Ala 128 – Lys 136 Glu 564 – Lys 573 Peak 66Unmatched. Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

26 Peak 12 : Daughter ions from HPLC- MS/MS indicating RHYP sequence. y3≈416.3 a3≈303.3 a2≈ Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

27 Two cases encountered during comparison, 27 PeptidesMatching Sequences and Results Peak 12Arg 144 – Tyr 147 Confirmed. Similarly, Peak 36 Found to contain – VEG- Glu 564 – Lys 573 Confirmed. Peak 66 Edman degradation confirmed sequence FYAPELLYY sequence Confirmed by MS/MS Y detected in 156 th position!!! Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

28 POSITION 94 th AND 95th Some peptides were not identified by Frit-FAB LC-MS after trypsin digestion like sequence So, BSA was digested with lysyl endopeptidase which matched the sequences, VASLRETYGDMADC*C*EK QEPERNEC*FLSHK Glu 82 to Arg 98 was established 94 th and 95 th : -QE- was established Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

29 COMPARISON OF RESULTS BSAPrevious findingsNew findings Molecular weight Da Da 156 th positionNo residueTyrosine Residue on 94 th and 95 th position -EQ--QE- Total amino acid residues 582 by Edman Degradation

30 APPLICATIONS LC-MS has wide applications in, Identification of natural products Structural characterization of peptides Measuring enzyme activity 30

31 Angiotensin Converting Enzyme ACE is a target for anti-hypertensive drugs because, 31 Angiotensin 1 Angiotensin 2 ACE Vasoconstriction Bradykinin Vasodilatation ACE AND Geng, 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. 32 Hippuryl-Histidine-Leucine ACE Hippuric acid Geng, F. et al. Biomed. Chromatogr. 2010, 24,

33 METHOD 33 HHL HA 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,

34 RESULTS Where, C 0 = HA concentration without inhibitor C = HA concentration with inhibitor Advantages; Quick screening Lower limits of detection Lesser analysis time 34 Concentration Peak area Standard. HA curve Geng, F. et al. Biomed. Chromatogr. 2010, 24,

35 APPLICATIONS LC-MS has wide applications in, Identification of natural products Structural characterization of peptides Measuring enzyme activity Forensic analysis 35

36 Detection Of Steroids For drug-free competitions To avoid false positives Sensitive method to detect drugs in small amounts from hair samples E.g. Stanzolol and Nandrolone. ELISA Traditional method for steroid detection. Based on competition between drug and drug- enzyme conjugate. 36 Deshmukh, N. et al. Steroids. 2010, 75,

37 ELISA 37 Y Y S S S S Less drug More drug Voller, A. et al. J. Clin. Pathol. 1978, 31, Tetra methyl benzidine Y – Antibody - Drug - Drug enzyme conjugate

38 38 Nandrolone Stanzolol UPLC-MS/MS RESULTS Deshmukh, N. et al. Steroids. 2010, 75, m/z transition m/z transition

39 ELISA Vs. UPLC-MS/MS 39 METHODNANDROLONESTANZALOL ELISA316 UPLC-MS/MS112 UPLC-MS/MS more sensitive than ELISA Number of participants : 160 Deshmukh, N. et al. Steroids. 2010, 75,

40 APPLICATIONS LC-MS has wide applications in, Identification of natural products Structural characterization of peptides Forensic analysis Measuring enzyme activity Wine Chemistry 40

41 CONTENTS OF SHEDEH Of religious importance in Ancient Egypt Blood of God Osiris, symbolizes rebirth of the dead Contents were unknown Very small samples LC-MS/MS in MRM mode: highly specific. 41 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 ? 42 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? 43 Malvidin-3-glucoside Syringic acid Alkaline fusion 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. 44

45 Summary LC-MS applications are wide Over the years, MS has been replaced by MS/MS and even MS n ; LC by UPLC. The technique offers a lot of flexibility and adaptability. Each engineering aspect plays an important role. 45

46 ACKNOWLEDGEMENTS Dr. Umesh Desai The Desai Group Department of Medicinal Chemistry, School of Pharmacy Virginia Commonwealth University Friends and family 46


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