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
1
LC-MS?
LIQUID CHROMATOGRAPHY
•Separates
components
•Identification from
retention time is
difficult
MASS SPECTROMETRY
• Component
identification is
superior
•But, interference
from other ions
Ardrey, R. E. Liquid Chromatography Mass spectrometry: an introduction,
Wiley, West Sussex, England, 2003
2
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
3
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, 3rd ed, Wiley:
Wiltshire, 2007.
4
COMPONENTS OF LC
LIQUID
RESERVOIR
RECORDER
PUMP
SAMPLE
INJECTOR
DETECTOR
COLUMN
Henry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana
Press: USA, 1981; p. 21-49
5
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.
6
Basic components of MS
ION
SOURCE
Frit-Fast Atom Bombardment
(Frit FAB),
Electro Spray Ionization (ESI)
MASS
ANALYSER
Time-of-Flight,
Quadrupole,
Magnetic Sector
DETECTOR
Electron Multiplier tube
http://www.epa.gov/esd/chemistry/org-anal/reports/phthalates/Fig1phms.png
(accessed on 3/23/2011)
7
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)
8
TANDEM MS or MS/MS
ESI,FAB
CID
SAMPLE
MS 1
Precursor
ion
Selected Reaction Monitoring (SRM)
Multiple Reaction Monitoring (MRM)
MS 2
Product
ion
DETECTED!
9
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
APPLICATIONS
LC-MS has wide applications in,
• Screening botanical extracts.
11
DISCOVERING INHIBITORS FROM
BIOLOGICAL EXTRACTS
•Biological extracts
compounds.
are
complex
mixtures
of
•Difficult to isolate a particular compound.
•Problems of co-extraction and interference.
•So Ultrafiltration with LC-MS
12
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.
13
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.
14
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.
15
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.
16
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.
17
RESULTS
• TME, xanthohumol and xanthohumol D bind at QR-2
and compete with resveratrol.
• Enzyme inhibition assay determined,
Compound
IC50
Resveratrol
5.1μM
Tetrangulol methyl ether
0.16μM (most potent)
Xanthohumol
196μM
Xanthohumol D
110μM
• X-ray Crystallography confirmed xanthohumol and XD binding to active pocket of QR-2.
Choi, Y. et al. Anal. Chem. 2011, 83, 1048-1052.
18
APPLICATIONS
LC-MS has wide applications in,
• Identification of natural products
• Structural characterization of peptides
19
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
20
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
COMPARISON WITH HSA AND RSA
B
H
R
1
1
1
10
. . . . | . . . . |
20
. . . . | . . . . |
30
. . . . | . . . . |
40
. . . . | . . . . |
50
. . . . | . . . . |
60
. . . . | . . . . |
70
. . . . | . . . . |
80
. . . . | . . . . |
DTHK S E I AHR
DAHK S EVAHR
EAHK S E I AHR
FKDL GE EHFK
F K D L GE EN F K
F K D L GEQH F K
GL V L I A F SQY
A L V L I A F AQ Y
GL V L I A F SQY
L QQ C P F D E HV
L QQ C P F E DHV
LQKCPY E EH I
K L VN E L T E F A
K L VN E V T E F A
K L VQ E V T D F A
K TC VADE SHA
K TCVADE SAE
K TC VAD ENA E
GC EK S L HT L F
NC DK S L HT L F
NC DK S I HT L F
GDE L C K VA S L
GDK L C TVA T L
GDK L C A I P K L
90
. . . . | . . . . |
B
H
R
81
81
81
R E T Y G DM A D C
R E T Y G EMA D C
R DN Y G E L A D C
170
. . . . | . . . . |
B
H
R
159
161
161
YNGV FQ EC CQ
Y KAAF T ECCQ
YN E V L TQC C T
250
. . . . | . . . . |
B
H
R
B
H
R
B
H
R
B
H
R
B
H
R
239
241
241
319
321
321
399
401
401
479
481
481
559
561
561
VHK EC C HGDL
VHT EC C HGDL
I NK EC C HGDL
100
. . . . | . . . . |
C EK EQP ERN E
C AKQ EP ERN E
C AKQ EP ERN E
180
. . . . | . . . . |
A EDK GAC L L P
AADKAAC L L P
E SDKAAC L TP
260
. . . . | . . . . |
L EC ADDRADL
L EC ADDRADL
L EC ADDRA E L
330
. . . . | . . . . |
340
. . . . | . . . . |
EAKDA F L GS F
E A K D V F L GM F
EAKDV F L GT F
L Y EY SRRHP E
L Y EY ARRHP D
L Y EY SRRHP D
410
. . . . | . . . . |
420
. . . . | . . . . |
Y G F QN A L I V R
Y K F QN A L L V R
Y G F QN A V L V R
C F L SHKDDSP
C F L Q H K D DN P
C F L Q H K D DN P
190
. . . . | . . . . |
K I E TMR E K V L
K L DE L RDEGK
K L DAVK EKA L
270
. . . . | . . . . |
A K Y I C DNQ D T
AK Y I C ENQD S
A K YMC E N Q A T
350
. . . . | . . . . |
Y AV SV L L R L A
VV L L L R L A
94YY SS95
V S L L L R L A
Y TRKVPQV S T
Y TKKVPQV S T
Y TQK APQV S T
490
. . . . | . . . . |
500
. . . . | . . . . |
L VN R R P C F S A
L VN R R P C F S A
L V ERRPC F SA
L TPDE TY VPK
L EVDE TY VPK
L TVDE TY VPK
570
. . . . | . . . . |
580
. . . . | . . . . |
ADDK EAC F AV
ADDK E TC F A E
A A D K DN C F A T
110
. . . . | . . . . |
EGP K L VV S TQ
EGK K L VAA SQ
EGPN L VAR S K
430
. . . . | . . . . |
P T L V EV SR S L
P T L V EV S RN L
P T L V EAARN L
510
. . . . | . . . . |
AFDEK L F T FH
E FNAE T F T FH
E FKAE T F T FH
120
. . . . | . . . . |
DL P K L - KP DP
N L PR L VRP EV
N L PP FQRP EA
200
. . . . | . . . . |
A S S ARQR L RC
A S SAKQR L KC
V A A V R Q RMK C
280
. . . . | . . . . |
I S SK L K ECCD
I S SK L K ECC E
I S SK LQACCD
360
. . . . | . . . . |
K EY EAT L E EC
K TY E T T L EKC
KKY EAT L EKC
440
. . . . | . . . . |
GK VGTRC C TK
GK VGS K C C K H
GRVGTK C C T L
520
. . . . | . . . . |
AD I C T L PDT E
AD I C T L S EK E
SD I C T L P DK E
130
. . . . | . . . . |
N T L CDE FKAD
D VMC T A F H DN
E AMC T S F Q E N
210
. . . . | . . . . |
AS I QK FGERA
AS LQK FGERA
S S MQ R F G E R A
290
. . . . | . . . . |
KP L L EK SHC I
KP L L EK SHC I
KP V LQK SQC L
370
. . . . | . . . . |
C AKDDP HAC Y
C AAADP HEC Y
C A EGDP P AC Y
450
. . . . | . . . . |
P E S E RMP C T E
P E A K RMP C A E
P E AQ R L P C V E
530
. . . . | . . . . |
KQ I KKQ TA L V
RQ I K KQ TA L V
KQ I KKQ TA L A
140
. . . . | . . . . |
E K K F WG K Y L Y
E E T F L KKY L Y
P T S F L GHY L H
220
. . . . | . . . . |
L K AW S V A R L S
F K AWA V A R L S
F K AWA V A RM S
300
. . . . | . . . . |
A EV EKDA I P E
A E V E N D EMP A
A E I E H DN I P A
380
. . . . | . . . . |
S TV F DK L KHL
AKV FDE FKP L
GTV L AE FQP L
460
. . . . | . . . . |
DY L S L I LNR L
DY L S VV L NQ L
DY L SA I LNR L
540
. . . . | . . . . |
E L L KHKP KA T
E L VKHKP KA T
E L VKHKP KA T
150
. . . . | . . . . |
E I ARRHP Y F Y
E I ARRHP Y F Y
EVARRHP Y F Y
230
. . . . | . . . . |
QK FPKAE FV E
QR FPKAE FAE
QR FPNAE FAE
310
. . . . | . . . . |
N L PP L TADFA
DL P S L AADFV
DL P S I AADFV
390
. . . . | . . . . |
156
V D E P QN L I KQ
V E E P QN L I KQ
V E EPKN L VK T
470
. . . . | . . . . |
CV L HEK TPV S
CV L HEK TPV S
CV L HEK TPV S
550
. . . . | . . . . |
E E Q L K T VM E N
K E Q L K A VMD D
E DQ L K T VMG D
160
. . . . | . . . . |
A P E L L - Y AN K
AP E L L F FAKR
AP E L L Y Y AEK
240
. . . . | . . . . |
V TK L V TDL TK
V SK L V TDL TK
I TK L A TDV TK
320
. . . . | . . . . |
EDKDVC KNYQ
E SKDVC KNY A
EDK EVC KNY A
400
. . . . | . . . . |
N C DQ F E K L G E
NC E L F EQ L GE
NC E L Y EK L GE
480
. . . . | . . . . |
EKV TKCC T E S
DRV TKC C T E S
EKV TKCC SGS
560
. . . . | . . . . |
F VA F VDKC C A
FAAFV EKCCK
F AQ F V DK C C K
. . . . |
TAL A AAL GL
EAL A -
22
PROCEDURE
+
BSA
Frit-FAB MS
Trypsin
Cleaved sequence
HPLC
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
23
RESULTS FROM HPLC
75 peaks found
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
24
• Two cases encountered during comparison,
Peaks from HPLC
Matching Tryptic Sequences
Peak 12
Arg144 – Tyr147
Gln195 – Arg198
Peak 36
Ala128 – Lys136
Glu564 – Lys573
Peak 66
Unmatched.
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
25
a2≈266.3
a3≈303.3
y3≈416.3
Peak 12 : Daughter
ions from HPLCMS/MS indicating
RHYP sequence .
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
26
• Two cases encountered during comparison,
Peptides
Matching Sequences and Results
Peak 12
Arg144 – Tyr147
Confirmed.
Similarly,
Glu564 – Lys573
Peak 36
Confirmed.
Found to contain –
VEGPeak 66
Edman degradation confirmed sequence
FYAPELLYY 148-156 sequence
Confirmed by MS/MS
Y detected in 156th position!!!
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
27
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,
77-93
VASLRETYGDMADC*C*EK
QEPERNEC*FLSHK
94-106
• Glu82 to Arg98 was established
• 94th and 95th : -QE- was established
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173, 639-646
28
COMPARISON OF RESULTS
BSA
Previous findings
New findings
Molecular
weight
66267.1 Da
66430.3 Da
156th position
No residue
Tyrosine
Residue on 94th -EQand 95th position
-QE-
Total amino acid 582 by Edman
residues
Degradation
583
29
APPLICATIONS
LC-MS has wide applications in,
• Identification of natural products
• Structural characterization of peptides
• Measuring enzyme activity
30
Angiotensin Converting Enzyme
ACE is a target for anti-hypertensive drugs because,
Angiotensin 1
ACE
Angiotensin 2
Vasoconstriction
AND
Bradykinin
Vasodilatation
ACE
Geng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317.
31
DETECTING ACE ACTIVITY
• ACE activity is usually determined by formation of a
product from a substrate.
ACE
Hippuryl-Histidine-Leucine
Hippuric acid
• Hippuric acid formed indicates ACE activity.
Geng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317
32
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.
Geng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317
HHL
HA
33
Peak area
RESULTS
Where,
C0 = HA concentration without inhibitor
C = HA concentration with inhibitor
Standard. HA curve
Concentration
Advantages;
• Quick screening
• Lower limits of detection
• Lesser analysis time
Geng, F. et al. Biomed. Chromatogr. 2010, 24, 312-317
34
APPLICATIONS
LC-MS has wide applications in,
•
•
•
•
Identification of natural products
Structural characterization of peptides
Measuring enzyme activity
Forensic analysis
35
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 drugenzyme conjugate.
Deshmukh, N. et al. Steroids. 2010, 75, 710-714.
36
Y – Antibody
- Drug
- Drug enzyme conjugate
ELISA
Y Y Y Y
Y Y Y Y
S
Tetra
methyl
benzidine
Less
drug
More
drug
Y Y Y Y
S
S
S
Y Y Y Y
Voller, A. et al. J. Clin. Pathol. 1978, 31, 507-520
Y Y Y Y
37
UPLC-MS/MS
RESULTS
Nandrolone
m/z transition
275.2
109.2
Stanzolol
m/z transition
329.2
81.1
Deshmukh, N. et al. Steroids. 2010, 75, 710-714.
38
ELISA Vs. UPLC-MS/MS
Number of participants : 160
METHOD
NANDROLONE
STANZALOL
ELISA
3
16
UPLC-MS/MS
1
12
UPLC-MS/MS more sensitive than ELISA
Deshmukh, N. et al. Steroids. 2010, 75, 710-714.
39
APPLICATIONS
LC-MS has wide applications in,
•
•
•
•
•
Identification of natural products
Structural characterization of peptides
Forensic analysis
Measuring enzyme activity
Wine Chemistry
40
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.
41
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
42
• Why alkaline fusion?
Malvidin-3-glucoside
Alkaline
fusion
Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2006, 33, 98-101
Syringic acid
43
RESULTS
• Shedeh is indeed red wine.
• Successful detection of syringic and tartaric
acid in trace amounts.
• MRM mode is a confirmation in itself.
44
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.
45
ACKNOWLEDGEMENTS
• Dr. Umesh Desai
• The Desai Group
• Department of Medicinal Chemistry,
School of Pharmacy
• Virginia Commonwealth University
• Friends and family
46