1 / 46

Applications of LC-MS in Chemical and Biochemical Sciences

Applications of LC-MS in Chemical and Biochemical Sciences. Presented By : Malaika Argade Department of Medicinal Chemistry Virginia Commonwealth University Email : argademd@vcu.edu Date : 25 th March 2011. LC-MS?. LIQUID CHROMATOGRAPHY. MASS SPECTROMETRY. Separates components

smolina
Download Presentation

Applications of LC-MS in Chemical and Biochemical Sciences

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  2. LC-MS? LIQUID CHROMATOGRAPHY MASS 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 Henry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p. 21-49

  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 elements • 1970s- Interfacing LC with MS • 1977-1980- Moving Belt Interface & Direct Liquid Interface • 1989- Electro Spray Ionization, John B. Fenn Watson, J. T. et al. In Introduction to mass spectrometry, 3rded, Wiley: Wiltshire, 2007.

  5. COMPONENTS OF LC LIQUID RESERVOIR SAMPLE INJECTOR PUMP RECORDER DETECTOR COLUMN Henry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p. 21-49

  6. COLUMNS Height equivalent to theoretical plate (HETP) Height • Backpressure overcome by, • Elevated temperatures • Monolithic columns Swartz, M.E. J. Liq. Chromatogr. R. T. 2005, 28, 1253-1263.

  7. Basic components of MS ION SOURCE MASS ANALYSER Time-of-Flight, Quadrupole, Magnetic Sector Frit-Fast Atom Bombardment (Frit FAB), Electro Spray Ionization (ESI) DETECTOR Electron Multiplier tube http://www.epa.gov/esd/chemistry/org-anal/reports/phthalates/Fig1phms.png (accessed on 3/23/2011)

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

  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

  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-2 • Resveratrol, a natural product inhibitor of Quinone Reductase 2. 2 Extract Ultrafiltration Removal of unbound compounds Incubation QR-2 LC-MS Dissociation Liu, D. et al. Anal.Chem. 2007, 79, 9398-9402.

  14. Resveratrol • 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, 1048-1052.

  15. Actinomyces sp. EXTRACT • Test: Extract of Actinomyces + • active QR-2 • Control : Extract + denatured QR-2 • m/z of 317 • Structure of TME determined by NMR Choi, Y. et al. Anal. Chem. 2011, 83, 1048-1052.

  16. HOPS EXTRACT • 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, 1048-1052.

  17. COMPETITIVE BINDING STUDIES • Difference in the peaks obtained indicate active binding. • Extract + QR-2 + Resveratrol Choi, Y. et al. Anal. Chem. 2011, 83, 1048-1052.

  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-2. Choi, Y. et al. Anal. Chem. 2011, 83, 1048-1052.

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

  20. ESI-MS of Bovine Serum Albumin • Molecular weight by ESI-MS : 66465.8 Da • Average molecular weight calculated from 582 residues: 66267.1 Da • Difference: 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 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

  22. COMPARISON WITH HSA AND RSA 156 94 95

  23. PROCEDURE + Trypsin BSA Cleaved sequence Frit-FAB MS HPLC Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006,173, 639-646

  24. RESULTS FROM HPLC 75 peaks found Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006,173, 639-646

  25. Two cases encountered during comparison, Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006,173, 639-646

  26. a2≈266.3 y3≈416.3 a3≈303.3 Peak 12 : Daughter ions from HPLC-MS/MS indicating RHYPsequence . Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006,173, 639-646

  27. Two cases encountered during comparison, Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006,173, 639-646

  28. POSITION 94th AND 95th • Some 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*FLSHK • Glu82 to Arg98 was established • 94th and 95th : -QE- was established 77-93 94-106 Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006,173, 639-646

  29. COMPARISON OF RESULTS

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

  31. Angiotensin Converting Enzyme ACE is a target for anti-hypertensive drugs because, ACE Angiotensin 1 Vasoconstriction Angiotensin 2 AND Bradykinin Vasodilatation ACE Geng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317.

  32. DETECTING ACE ACTIVITY • ACE activity is usually determined by formation of a product from a substrate. • Hippuric acid formed indicates ACE activity. ACE Hippuryl-Histidine-Leucine Hippuric acid Geng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317

  33. METHOD • 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. HHL HA Geng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317

  34. RESULTS Peak area Where, C0 = HA concentration without inhibitor C = HA concentration with inhibitor Advantages; • Quick screening • Lower limits of detection • Lesser analysis time Standard. HA curve Concentration Geng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317

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

  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 • Traditionalmethod for steroid detection. • Based on competition between drug and drug-enzyme conjugate. Deshmukh, N. et al. Steroids. 2010, 75, 710-714.

  37. Y – Antibody - Drug - Drug enzyme conjugate ELISA Y Y Y Y Y Y Y Y Y Y Y Y S S S Tetra methyl benzidine S Y Y Y Y Y Y Y Y Less drug More drug Voller, A. et al. J. Clin. Pathol. 1978, 31, 507-520

  38. UPLC-MS/MS RESULTS m/z transition 275.2 109.2 Nandrolone Stanzolol m/z transition 329.2 81.1 Deshmukh, N. et al. Steroids. 2010, 75, 710-714.

  39. ELISA Vs. UPLC-MS/MS Number of participants : 160 UPLC-MS/MS more sensitive than ELISA Deshmukh, N. et al. Steroids. 2010, 75, 710-714.

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

  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. Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2004,33, 98-101.

  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, 98-101 Guasch-Jané, M.R. et al. Anal. Chem. 2004,76, 1672-1677

  43. Alkaline fusion • Why alkaline fusion? Malvidin-3-glucoside Syringic acid Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2006, 33, 98-101

  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 Pharmacy • Virginia Commonwealth University • Friends and family

More Related