applications of lc-ms in chemical and biochemical sciences 1 presented by : malaika argade...

Applications of LC-MS in Chemical and Biochemical Sciences

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Presented By : Malaika ArgadeDepartment of Medicinal ChemistryVirginia Commonwealth UniversityEmail : [email protected] : 25th March 2011

LC-MS?

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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

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

3Henry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p. 21-49

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

4Watson, J. T. et al. In Introduction to mass spectrometry, 3rd ed, Wiley: Wiltshire, 2007.

COMPONENTS OF LC

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LIQUID RESERVOIR PUMP SAMPLE

INJECTOR

COLUMNDETECTORRECORDER

Henry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p. 21-49

COLUMNS

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Backpressure overcome by,•Elevated temperatures•Monolithic columns

Swartz, M.E. J. Liq. Chromatogr. R. T. 2005, 28, 1253-1263.

Height

Height equivalent to theoretical plate

(HETP)

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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

http://www.epa.gov/esd/chemistry/org-anal/reports/phthalates/Fig1phms.png (accessed on 3/23/2011)

MASS SPECTROMETER

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• 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)

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SAMPLE

MS 1Precursor ion

MS 2Product ion

TANDEM MS or MS/MS

DETECTED!

ESI,FAB CID

Selected Reaction Monitoring (SRM)Multiple Reaction Monitoring (MRM)

APPLICATIONSIn 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

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APPLICATIONSLC-MS has wide applications in,

• Screening botanical extracts.

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DISCOVERING INHIBITORS FROM BIOLOGICAL EXTRACTS

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•Biological extracts are complex mixtures of compounds.

•Difficult to isolate a particular compound.

•Problems of co-extraction and interference.

•So Ultrafiltration with LC-MS

INHIBITORS OF QR-2• Resveratrol, a natural product inhibitor of Quinone

Reductase 2.

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IncubationQR-2

Extract

Removal of unbound compounds

Dissociation

LC-MS

2

Ultrafiltration

Liu, D. et al. Anal.Chem. 2007, 79, 9398-9402.

Resveratrol

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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.

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

15Choi, Y. et al. Anal. Chem. 2011, 83, 1048-1052.

HOPS EXTRACT

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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 ?


COMPETITIVE BINDING STUDIES

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Difference in the peaks obtained indicate active binding.

Extract + QR-2 + Resveratrol


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.

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Compound IC50

Resveratrol 5.1μM

Tetrangulol methyl ether 0.16μM (most potent)

Xanthohumol 196μM

Xanthohumol D 110μM



• Identification of natural products• Structural characterization of peptides

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ESI-MS of Bovine Serum Albumin

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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

Determining Amino Acid SequenceAt 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

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COMPARISON WITH HSA AND RSA10 20 30 40 50 60 70 80

. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |B 1 D T H K S E I A H R F K D L G E E H F K G L V L I A F S Q Y L Q Q C P F D E H V K L V N E L T E F A K T C V A D E S H A G C E K S L H T L F G D E L C K V A S LH 1 D A H K S E V A H R F K D L G E E N F K A L V L I A F A Q Y L Q Q C P F E D H V K L V N E V T E F A K T C V A D E S A E N C D K S L H T L F G D K L C T V A T LR 1 E A H K S E I A H R F K D L G E Q H F K G L V L I A F S Q Y L Q K C P Y E E H I K L V Q E V T D F A K T C V A D E N A E N C D K S I H T L F G D K L C A I P K L

90 100 110 120 130 140 150 160. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

B 81 R E T Y G D M A D C C E K E Q P E R N E C F L S H K D D S P D L P K L - K P D P N T L C D E F K A D E K K F W G K Y L Y E I A R R H P Y F Y A P E L L - Y A N KH 81 R E T Y G E M A D C C A K Q E P E R N E C F L Q H K D D N P N L P R L V R P E V D V M C T A F H D N E E T F L K K Y L Y E I A R R H P Y F Y A P E L L F F A K RR 81 R D N Y G E L A D C C A K Q E P E R N E C F L Q H K D D N P N L P P F Q R P E A E A M C T S F Q E N P T S F L G H Y L H E V A R R H P Y F Y A P E L L Y Y A E K

170 180 190 200 210 220 230 240. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

B 159 Y N G V F Q E C C Q A E D K G A C L L P K I E T M R E K V L A S S A R Q R L R C A S I Q K F G E R A L K A W S V A R L S Q K F P K A E F V E V T K L V T D L T KH 161 Y K A A F T E C C Q A A D K A A C L L P K L D E L R D E G K A S S A K Q R L K C A S L Q K F G E R A F K A W A V A R L S Q R F P K A E F A E V S K L V T D L T KR 161 Y N E V L T Q C C T E S D K A A C L T P K L D A V K E K A L V A A V R Q R M K C S S M Q R F G E R A F K A W A V A R M S Q R F P N A E F A E I T K L A T D V T K

250 260 270 280 290 300 310 320. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

B 239 V H K E C C H G D L L E C A D D R A D L A K Y I C D N Q D T I S S K L K E C C D K P L L E K S H C I A E V E K D A I P E N L P P L T A D F A E D K D V C K N Y QH 241 V H T E C C H G D L L E C A D D R A D L A K Y I C E N Q D S I S S K L K E C C E K P L L E K S H C I A E V E N D E M P A D L P S L A A D F V E S K D V C K N Y AR 241 I N K E C C H G D L L E C A D D R A E L A K Y M C E N Q A T I S S K L Q A C C D K P V L Q K S Q C L A E I E H D N I P A D L P S I A A D F V E D K E V C K N Y A

330 340 350 360 370 380 390 400. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

B 319 E A K D A F L G S F L Y E Y S R R H P E Y A V S V L L R L A K E Y E A T L E E C C A K D D P H A C Y S T V F D K L K H L V D E P Q N L I K Q N C D Q F E K L G EH 321 E A K D V F L G M F L Y E Y A R R H P D Y S V V L L L R L A K T Y E T T L E K C C A A A D P H E C Y A K V F D E F K P L V E E P Q N L I K Q N C E L F E Q L G ER 321 E A K D V F L G T F L Y E Y S R R H P D Y S V S L L L R L A K K Y E A T L E K C C A E G D P P A C Y G T V L A E F Q P L V E E P K N L V K T N C E L Y E K L G E

410 420 430 440 450 460 470 480. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

B 399 Y G F Q N A L I V R Y T R K V P Q V S T P T L V E V S R S L G K V G T R C C T K P E S E R M P C T E D Y L S L I L N R L C V L H E K T P V S E K V T K C C T E SH 401 Y K F Q N A L L V R Y T K K V P Q V S T P T L V E V S R N L G K V G S K C C K H P E A K R M P C A E D Y L S V V L N Q L C V L H E K T P V S D R V T K C C T E SR 401 Y G F Q N A V L V R Y T Q K A P Q V S T P T L V E A A R N L G R V G T K C C T L P E A Q R L P C V E D Y L S A I L N R L C V L H E K T P V S E K V T K C C S G S

490 500 510 520 530 540 550 560. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

B 479 L V N R R P C F S A L T P D E T Y V P K A F D E K L F T F H A D I C T L P D T E K Q I K K Q T A L V E L L K H K P K A T E E Q L K T V M E N F V A F V D K C C AH 481 L V N R R P C F S A L E V D E T Y V P K E F N A E T F T F H A D I C T L S E K E R Q I K K Q T A L V E L V K H K P K A T K E Q L K A V M D D F A A F V E K C C KR 481 L V E R R P C F S A L T V D E T Y V P K E F K A E T F T F H S D I C T L P D K E K Q I K K Q T A L A E L V K H K P K A T E D Q L K T V M G D F A Q F V D K C C K

570 580. . . . | . . . . | . . . . | . . . . | . . . . |

B 559 A D D K E A C F A V E G P K L V V S T Q T A L A -H 561 A D D K E T C F A E E G K K L V A A S Q A A L G LR 561 A A D K D N C F A T E G P N L V A R S K E A L A -

94 95 156

PROCEDURE

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BSA

+ Trypsin

HPLC

Cleaved sequence

Frit-FAB MS


RESULTS FROM HPLC

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75 peaks found


• Two cases encountered during comparison,

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Peaks from HPLC Matching Tryptic Sequences

Peak 12 Arg144 – Tyr147

Gln195 – Arg198

Peak 36 Ala128 – Lys136

Glu564 – Lys573

Peak 66 Unmatched.


H2N

HN

HN

H2C

H2C

H2C

HC C

NH2

OHN C

H

CH2

NHN

C

O

N

C O

NH

HC COOH

CH2

OH

Peak 12 : Daughter ions from HPLC-MS/MS indicating RHYP sequence .

y3≈416.3a3≈303.3a2≈266.3

26Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646

• Two cases encountered during comparison,

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Peptides Matching Sequences and Results

Peak 12 Arg144 – Tyr147

Confirmed.

Similarly,Peak 36 Found to contain –VEG-

Glu564 – Lys573

Confirmed.

Peak 66Edman degradation confirmed sequenceFYAPELLYY 148-156 sequenceConfirmed by MS/MSY detected in 156th position!!!


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

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77-93

94-106


COMPARISON OF RESULTSBSA Previous findings New findings

Molecular weight

66267.1 Da 66430.3 Da

156th position No residue Tyrosine

Residue on 94th and 95th position

-EQ- -QE-

Total amino acid residues

582 by Edman Degradation

583

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• Identification of natural products• Structural characterization of peptides• Measuring enzyme activity

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Angiotensin Converting Enzyme

ACE is a target for anti-hypertensive drugs because,

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Angiotensin 1 Angiotensin 2ACE

Vasoconstriction

Bradykinin Vasodilatation

ACE

AND

Geng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317.

DETECTING ACE ACTIVITY

• ACE activity is usually determined by formation of a product from a substrate.

• Hippuric acid formed indicates ACE activity.

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Hippuryl-Histidine-LeucineACE

Hippuric acid

Geng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317

METHOD

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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.


RESULTS

Where,

C0 = HA concentration without inhibitor

C = HA concentration with inhibitor

Advantages;• Quick screening• Lower limits of detection• Lesser analysis time

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Concentration

Pea

k ar

ea

Standard. HA curve



• Identification of natural products• Structural characterization of peptides• Measuring enzyme activity• Forensic analysis

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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.

36Deshmukh, N. et al. Steroids. 2010, 75, 710-714.

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ELISA

Y Y Y YY Y Y Y Y Y Y Y

Y Y Y YY Y Y Y

SS

SS

Lessdrug

Moredrug

Voller, A. et al. J. Clin. Pathol. 1978, 31, 507-520

Tetra methylbenzidine

Y – Antibody - Drug - Drug enzyme conjugate

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Nandrolone

Stanzolol

UPLC-MS/MSRESULTS

Deshmukh, N. et al. Steroids. 2010, 75, 710-714.

m/z transition 275.2 109.2

m/z transition329.2 81.1

ELISA Vs. UPLC-MS/MS

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METHOD NANDROLONE STANZALOL

ELISA 3 16

UPLC-MS/MS 1 12

UPLC-MS/MS more sensitive than ELISA

Number of participants : 160

Deshmukh, N. et al. Steroids. 2010, 75, 710-714.


• Identification of natural products• Structural characterization of peptides• Forensic analysis• Measuring enzyme activity• Wine Chemistry

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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.

41Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2004, 33, 98-101.

DETECTIONTartaric acid: Wine marker

Syringic acid: Red wine

marker

So, 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

• Why alkaline fusion?

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Malvidin-3-glucoside Syringic acidAlkalinefusion

Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2006, 33, 98-101

RESULTS

• Shedeh is indeed red wine.

• Successful detection of syringic and tartaric acid in trace amounts.

• MRM mode is a confirmation in itself.

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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.

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ACKNOWLEDGEMENTS

• Dr. Umesh Desai• The Desai Group• Department of Medicinal Chemistry,

School of Pharmacy• Virginia Commonwealth University• Friends and family

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applications of lc-ms in chemical and biochemical sciences 1 presented by : malaika argade...

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