Fast and Direct Measurement of Reactants
in the Glycolysis Pathway
Using the RapidFire Mass Spectromety System
February 26, 2014
Glycolysis Pathway and Inhibition for Anticancer Treatment
Most cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation - “Warburg effect”
Targeting the glycolysis pathway may preferentially kill malignant cells.
2DG (Phase II for prostate cancer)
Lonidamine (Phase II/III for benign hyperplasia)
3PO (Preclinical)
TLN-232/CAP-232
(Phase II for MRC)
P. Front Pharmacol. (2011) Aug 25;2:49.
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February 26, 2014
Case Study - Measuring the Conversion
of F6P to F16bP by Phosphofructokinase
Conversion of F6P to F16bP
by Phosphofructokinase
A standard enzymatic assay for
phosphofructokinase involves
three additional enzymes and
measures NADH by absorbance
at 340 nm
Eur J Biochem. (1982) Dec;129(1):191-5.
• Phosphorylation of F6P to F16bP is the first “committed”, and rate limiting, step in glycolysis
• Phosphofructokinase is highly regulated to respond to the energy requirements of the cell
• The traditional assay to measure phosphofructokinase is complicated
• This reaction was therefore selected as a model system to illustrate the simplicity of measuring glycolysis reactants by SPE-MS/MS
• We chose the phosphofructokinase enzyme from potato tubers, which uses PPi (not ATP) as the phosphate donor
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February 26, 2014
The More Complex the Assay
The Higher the Potential for Data Artifacts
• UDP-glucose was thought to convert an ATP-dependent kinase into a PPi-dependent kinase
• Addition of PPi and UDP-glucose, instead of ATP, resulted in activity
FEBS Lett. (1984) 169:287-92.
Biochem. Biophys. Res. Comm. (1984) 121:842-47.
Phosphofructokinase
F6P
(dependent on ATP)
UTP
PPi + UDP-glucose
F16bP
Fructosebisphosphate
aldolase
G3P
Glyceraldehyde phosphate
dehydrogenase
Triosephosphate
isomerase
UDP-glucose
Pyrophosphorylase
(a contaminant from
Glyceraldehyde
phosphate
dehydrogenase)
13bPG
NADH
DHP
In actuality, when PPi and UDP-glucose were added to the reaction,
a contaminating enzyme produced UTP, which is a suitable
phosphate donor for the ATP-dependent phosphofructokinase
Plant. Physiol. (1985) 78:645-48.
Measuring reactions directly, using the RapidFire system, simplifies the assay ….
making the design and execution of experiments easier and the potential for data artifacts lower
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February 26, 2014
Measuring Glycolysis Reactants Using SPE-MS/MS
Rapid Fire Conditions
Solvent A
H2O + 6 mM
octylammonium acetate
Solvent B
80% acetonitrile in water
SPE Cartridge
RF State 1: Aspirate
C4
600 ms (sip sensor)
RF State 2: Load/Wash
3000 ms
RF State 3: Elute
4000 ms
RF State 4: Re-equilibrate
500 ms
Pump 1 flow rate
1.5 mL/min
February
26, 2014
Pump 2 flow
rate
1.25 mL/min
Pump 3 flow rate
1.25 mL/min
MS Source Parameters
• SPE purification was optimized for small
molecules with phosphate groups
Drying gas temp.
ESI (-) Agilent 6490
225 °C
Drying gas flow
15 L/min
Sheath gas temp.
300 °C
Sheath gas flow
12 L/min
Nebulizer pressure
20 psi
Nozzle voltage
1000 V
Capillary voltage
2000 V
• The final SPE method had a sample to
sample cycle time of 8.5 seconds
• MS/MS detection was optimized on an
Agilent 6490 QQQ
February 26, 2014
Direct Measurement of PPi-PFK Substrate and Product
PPi
Pi
OH
O
PPi-PFK
PO4H2
H2O4P
HO
Fructose-6-phosphate
(F6P)
activation
F16bP, F26bP
LOD = 7.5 nM
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inhibition
OH
Fructose-1,6-bisphosphate
(F16bP)
Pi
LOD = 3.2 nM
February 26, 2014
Allosteric Activation of PPi-PFK by F16bP and F26bP
T
Less
active
R
More
active
PPi-PFK has a tetrameric structure,
composed of two  and two  subunits,
that shifts into a more active
conformation upon the binding of
allosteric effectors.
Nature (1990) 343:140-45.
The coupled assay creates a steady state level of F16bP , and the allosteric effect of F16bP wasn’t appreciated for 13 years.
The steady state level of F16bP depended on experimental conditions, which varied broadly and resulted in variant PPi-PFK activity.
Consequently, many kinetic parameters for PPi-PFK reported in the literature don’t agree.
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Plant Physiol. (1995) 108:69-73.
February 26, 2014
Kact of F26bP and F16bP for PPi-PFK
Kact = 3.0 M
Kact = 3.4 M
•
To circumvent non-linear enzyme kinetics, F26bP can be added to the assay
•
For our studies, assays were supplemented with saturating concentrations of 13C6-F16bP
• Reactions were carried out with enzyme in the fully activated conformation
• No detriment to assay sensitivity
• provided an internal standard for an additional option for data normalization
Results agree with previous reports: Plant Physiol. (1995) 108:69-73. FEBS Lett (1988) 228:60-64.
Agilent Confidential
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February 26, 2014
Substrate and Enzyme Titrations
on PPi-PFK Reactions
Hill slope = 2.8
½ Vmax = 2 mM F6P
KM = 18 M
Results agree with previous report: Eur J Biochem. (1982) Dec;129(1):191-5.
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February 26, 2014
Inhibition of PPi-PFK by Phosphate
IC50 = 750 M
Noncompetitive with F6P
Mechanistic studies on phosphate
show inhibition that is noncompetitive
with F6P and PPi.
Noncompetitive with PPi
Results agree with previous report:
Plant Physiol. (1989) 89:628-33.
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February 26, 2014
Inhibition of PPi-PFK by a Pyrophosphate Analog
MDPA
IC50 = 2.4 mM
Noncompetitive with F6P
Mechanistic studies on MDPA show
inhibition that is noncompetitive with
F6P and competitive with PPi
MDPA does not affect Vmax
Competitive with PPi
Results agree with previous report:
Biochemistry (1986) 25:4682-4687.
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February 26, 2014
Measurement of Multiple Glycolysis Reactants
Molecules that can be measured using the single analytical method
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February 26, 2014
Concentration Responses for Eight Additional Glycolysis
Reactants Using a Single Analytical Method
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February 26, 2014
February 26, 2014
Concentration Response Experiments
MS/MS Methods and Tabulation of Results
R^2 of fit
LOD (nM)
Q1
(unit)
Q3
(unit)
CE
CAV
G6P
glucose-6-phosphate
0.9939
6.5
259.0
79.0
65
2
F6P
fructose-6-phosphate
0.9998
7.5
259.0
79.0
65
2
F1,6bP
Fructose-1,6-bisphosphate
0.9999
3.2
338.9
79.0
65
2
G3P
glyceraldehyde-3-phosphate
0.9954
5.8
168.9
79.0
35
5
2,3bPG
2,3-bisphosphoglycerate (isomer of 1,3bPG)
0.9983
18.5
264.9
79.0
45
5
3PG
3-phosphoglycerate
0.9995
1.0
184.9
79.0
80
7
2PG
2-phosphoglycerate
0.9991
4.0
184.9
79.0
40
7
PEP
phosphoenolpyruvate
0.9988
1.0
167.0
79.0
65
7
ATP
adenosine triphosphate
0.9983
1.7
506.0
79.0
35
5
ADP
adenosine diphosphate
0.9990
4.7
426.0
79.0
35
5
0.9982
5.4
AVERAGE
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February 26, 2014
Conclusions
•
The RapidFire-MS/MS method presented here is very robust and versatile
•
High-throughput label-free detection of biochemical reactions (8.5 seconds per sample)
•
good sensitivity (single digit nanomolar range)
•
broad linear response range (up to single digit micromolar)
•
effective with the majority of reactants in the glycolysis pathway
•
Studying in vitro glycolysis reactions by SPE-MS/MS is advantageous over other measurement options
•
•
Direct measurement circumvents coupled reactions
–
Avoid data artifacts which can arise through non-intentional reactions/interactions
–
Simplify/expedite the experimental design and execution
–
Maximize results that are specific to target
Enables multiple glycolysis reactants to be measured simultaneously
–
•
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Allowing biochemical data to be normalized (% conversion, product over internal standard, etc)
The ability of SPE-MS/MS to measure multiple reactants within the glycolysis pathway, utilizing a single set
of parameters, demonstrates the potential of this platform for pathway based analyses
February 26, 2014
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February 26, 2014