7. HYDROXYUREA
MULTICENTER STUDY OF HUMULTICENTER STUDY OF HU
Patients often improved clinically before a
significant rise in HbF levels
Patients often improved clinically before a
significant rise in HbF levels
HU – induced alterations of additional cellular
mechanisms that are yet to be identified
HU – induced alterations of additional cellular
mechanisms that are yet to be identified
8. HYDROXYUREA
2D - DIGE2D - DIGE Mass spectrometryMass spectrometry
To determine the elemental
composition of an unknown
substance.
To determine the elemental
composition of an unknown
substance.
It utilizes the charged
properties of molecules to
separate them
It utilizes the charged
properties of molecules to
separate them
Ability to separate complex
mixtures of proteins
Ability to separate complex
mixtures of proteins
9. MATERIALS AND METHODS
Homozygous SS patients 10ml blood samples
by
venipuncture
Southwestern Comprehensive Sickle Cell Center
No patient had received any HU or blood transfusion within the preceding 4
months
SUBJECTSSUBJECTS
10. MATERIALS AND METHODS
1. INCUBATION OF SS WHOLE BLOOD WITH HU1. INCUBATION OF SS WHOLE BLOOD WITH HU
Equal volumes of the same
SS blood sample +/- 50 μM
HU
Equal volumes of the same
SS blood sample +/- 50 μM
HU
incubatedincubated
37 ° C, 15
hours with
constant
shaking
37 ° C, 15
hours with
constant
shaking
2. PREPARATION OF ERYTHROCYTE MEMBRANES2. PREPARATION OF ERYTHROCYTE MEMBRANES
RBCs were
sedimented at 1,000 g
for 10 minutes at 4°C
RBCs were
sedimented at 1,000 g
for 10 minutes at 4°C
11. MATERIALS AND METHODS
2. PREPARATION OF ERYTHROCYTE MEMBRANES2. PREPARATION OF ERYTHROCYTE MEMBRANES
RBCs were resuspended
in 10 volumes of PBS and
sedimented at 2,000g
for 10mins.
RBCs were resuspended
in 10 volumes of PBS and
sedimented at 2,000g
for 10mins.
The washed RBCs were lysed in 6
volumes of lysis buffer and were
sedimented at 31,000g for
30mins.
The washed RBCs were lysed in 6
volumes of lysis buffer and were
sedimented at 31,000g for
30mins.
The pellet becomes white or light
pink
12. MATERIALS AND METHODS
3. MINIMAL LABELING OF SS MEMBRANE PROTEINS3. MINIMAL LABELING OF SS MEMBRANE PROTEINS
4. SEPARATION OF PROTEINS IN FIRST DIMENSION4. SEPARATION OF PROTEINS IN FIRST DIMENSION
SS membrane proteins = 100μg each of (-)HU and (+) HU samples
solubilized in lysis buffer were minimally labeled with Cy3 and Cy5
fluorophores
SS membrane proteins = 100μg each of (-)HU and (+) HU samples
solubilized in lysis buffer were minimally labeled with Cy3 and Cy5
fluorophores
100μg each of the control
and drug tested samples
were mixed together
100μg each of the control
and drug tested samples
were mixed together
Rehydration
buffer
Rehydration
buffer
Final protein
mixture
Final protein
mixture
13. MATERIALS AND METHODS
4. SEPARATION OF PROTEINS IN FIRST DIMENSION4. SEPARATION OF PROTEINS IN FIRST DIMENSION
The sample, included
in the rehydration
solution
The sample, included
in the rehydration
solution
IPG
(Immobilized
pH Gradient)
strip holder
IPG
(Immobilized
pH Gradient)
strip holder
Immobiline Dry-
Strip gel was
placed over the
sample
Immobiline Dry-
Strip gel was
placed over the
sample
IPG cover fluidIPG cover fluid
Minimize
evaporation and
urea crystallization
Minimize
evaporation and
urea crystallization
REHYDRATION – 12
hours at 20 °C
14. MATERIALS AND METHODS
5. REDUCTION OF ALKYLATION OF THE IEF SEPARATED PROTEINS5. REDUCTION OF ALKYLATION OF THE IEF SEPARATED PROTEINS
6. SEPARATION OF PROTEINS IN SECOND DIMENSION6. SEPARATION OF PROTEINS IN SECOND DIMENSION
Immobiline strips with
separated proteins were
equilibriated and reduced
Immobiline strips with
separated proteins were
equilibriated and reduced
Equilibriation and
Alkylation at room
temperature
Equilibriation and
Alkylation at room
temperature
Proteins were further
separated on a 10% SDS-
polyacrylamide gel
Proteins were further
separated on a 10% SDS-
polyacrylamide gel
15. MATERIALS AND METHODS
7. GEL IMAGING AND ANALYSIS7. GEL IMAGING AND ANALYSIS
8. STATISTICAL ANALYSIS8. STATISTICAL ANALYSIS
Cy3 and Cy5 fluorophores
were detected using 2920
2D-Master Imager
Cy3 and Cy5 fluorophores
were detected using 2920
2D-Master Imager
DeCyder DIA
software
DeCyder DIA
software
Detects
protein spots
Quantifies the
spot volume for
each imageExpresses
volumes in
ratio
GOALGOAL
To discover protein
spots whose mean
ratios differed
significantly from one
To discover protein
spots whose mean
ratios differed
significantly from one
16. MATERIALS AND METHODS
9. PROTEIN IDENTIFICATION BY TANDEM MASS SPECTROMETRY AND DATABASE SEARCH9. PROTEIN IDENTIFICATION BY TANDEM MASS SPECTROMETRY AND DATABASE SEARCH
10. IMMUNOBLOT ANALYSIS10. IMMUNOBLOT ANALYSIS
Also known as MS/MS or MS2
involves
multiple steps of mass spectrometry
selection
Also known as MS/MS or MS2
involves
multiple steps of mass spectrometry
selection
2D-electrophoresis
Proteins
were
electroblotted
Proteins
were
electroblotted
Nitrocellulose
membrane in transfer
buffer overnight at 4°C
and 25 V
Membranes were
blocked for an hour
Membranes were
incubated overnight
Immunoreactive
spots were
detected
17. RESULTS
2D DIGE + mass spectrometry2D DIGE + mass spectrometry
To identify alterations in the sickle RBC
membrane proteome in response to a
clinically relevant dose of HU
To identify alterations in the sickle RBC
membrane proteome in response to a
clinically relevant dose of HU
18. RESULTS
Each circle represents a protein spot whose position is determined by its log
volume on the X axis and its intensity on the Y axis and the spots lying within the
2.5-fold threshold are shown in green.
Blue – increase in protein content by more than 2.5-fold
Red – decrease in protein content by more than 2.5-fold
Each circle represents a protein spot whose position is determined by its log
volume on the X axis and its intensity on the Y axis and the spots lying within the
2.5-fold threshold are shown in green.
Blue – increase in protein content by more than 2.5-fold
Red – decrease in protein content by more than 2.5-fold
20. RESULTS
IMMUNOBLOT ANALYSIS OF CATALASEIMMUNOBLOT ANALYSIS OF CATALASE
Positions of SS RBC
membrane proteins
that showed a
statistically significant
change in their
composition on
exposure to 50 μM
HU.
Positions of SS RBC
membrane proteins
that showed a
statistically significant
change in their
composition on
exposure to 50 μM
HU.
23. DISCUSSION
FACTORS INVOLVED IN IMPROVING THE PATHOLOGY OF RBC MEMBRANE AFTER
HU TREATMENT
FACTORS INVOLVED IN IMPROVING THE PATHOLOGY OF RBC MEMBRANE AFTER
HU TREATMENT
These indicate the
likelihood of HU
targeting the
membrane and acting
through cellular
mechanisms that are
independent of
elevated HbF and are
yet to be identified
These indicate the
likelihood of HU
targeting the
membrane and acting
through cellular
mechanisms that are
independent of
elevated HbF and are
yet to be identified
With these, the researchers used a proteomics approach to identify HU-induced changes
in the SS RBC membrane
With these, the researchers used a proteomics approach to identify HU-induced changes
in the SS RBC membrane
25. DISCUSSION
2D-DIGE coupled with LC/MS/MS
-this was used since mature RBCs are enucleated cells lacking active protein
synthesis
- To quantitate post-translational protein modifications in SS RBC membranes with
and without physiologic concentrations of HU
-Based on the results and literature, the researchers hypothesized that HU
induces oxidative stress at a concentration of 50 μM.
-Their hypothesis is further strengthened by the fact that equal amounts of the
same SS RBC membrane sample +/- HU were subjected to 2D-DIGE such that the
levels of oxidative stress before and after HU exposure were comparable.
26. DISCUSSION
Thus, HU stimulated increased tyrosine phosphorylation of catalase presents an
attractive model for potential activation of catalase in mature sickle RBCs that do
not contain the necessary machinery for new protein synthesis
This work is the first attempt to identify and characterize in vitro HU-induced
protein modifications in SS RBC membranes using 2D-DIGE protein profiling and
tandem mass spectrometry,
