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Hu Zhou
Shanghai Institute of Materia Medica, CAS
November 12, 2014
Technology Development for Membrane
Protein Analysis and Its Applications
Outline
2
1. Technology Development for Membrane Protein Analysis
2. Applications in Drug-treated samples and Clinic Samples
Outline
3
1. Technology Development for Membrane Protein Analysis
2. Applications in Drug-treated samples and Clinic Samples
Flowchart of Proteomic Analysis
4
--
- --
-
-
-
-
+
+++
++
+++
++++
++
+++
+
+
+
++
++
++
+++
++++++
Sample
Loading
Elution
Ion Strength
pH
Principle of Strong Cation Exchange
5
Proteomic Reactor
Figeys D, et al J Proteome Res. 2006 Oct;5(10):2754-9.6
Membrane Protein Analysis is an Important and
Challenging Topic in Proteomics
7
supernatant
pellet
glass
homogenizer
(10 strokes)sucrose cushion
(1.12 M)
100,000 × g
90 min, 4
°C
collect
interfacepellet
541,000 ×g
16 min, 4
°C
pellet
541,000 ×g
30 min, 4
°C
pelletmicrosomal
membranes
plasma
membranes
cells (100-mm)
ball-bearing
homogenizer
11,000 × g
20 min, 4
°C
1
2
3b
3a
4
+ 100 mM
Na2CO3, pH 11,
30 min, 22 oC
541,000 ×g
30 min, 4
°C
5
+ 100 mM Na2CO3,
pH 11, 30 min, 22 oC
+ 4 M urea, pH
7.4, 30 min, 4 oC
10
250150
100
75
50
37
2520
15
kDa 1
2
3
4
5
6
7
7
9
10
cell lysateplasma membrane
microsomal membrane
cell lysateplasma membrane
microsomal membrane
calnexin
actin
lamin A/C
+ 4 M urea, pH
7.4, 30 min, 4 oC
Subcellular Fractionation of Membrane Proteins
8Zhou H, et al. Mol Cell Proteomics. 2011 Oct;10(10):O111.008425.
SCX Column(in high
pressurized vessel)
N 2
Pro t ein
-
pH ~ 12
Tr ypsin
-C
Bead in EP tube
Zhou H, et al. Mol Cell Proteomics. 2011 Oct;10(10):O111.008425.
Centrifugal Proteomic Reactor
Cell/tiissue
extraction buffer
absorption to SCX
reduction (20 mM DTT)
collect pellet
collect pellet
alkylation (100 mM IAA)
washing (formic acid)
1
2
4
5
solubilized protein
acidification (formic acid) &
delipidation (methanol)
3
collect pellet
digestion (2 µ g trypsin)6
7
8
successive elution at pH
2.5, 3.0, 3.5, 4.0, 4.5, 5.0,
5.5, 6.0, 8.0, & 12
LC-MS/MS
(LTQ-Orbitrap)
data
9
Centrifugal Proteomic Reactor
Method CPR In-gel Digesion
Sample Plasma
membrane
Microsome
Membrane
Plasma
membrane
Microsome
Membrane
# of proteins 945 955 110 128
# of
membrane
proteins
591
(63%)
447
(47%)
72
(65%)
70
(55%)
Improvements
• User-friendly , only need acentrifuge
• Comparing to traditional in-geldigestion, # of proteins increase7 fold, # of membrane proteinsincrease 6 fold
• The percentage of membraneproteins in plasma membranefraction is >60%
10Zhou H, et al. Mol Cell Proteomics. 2011 Oct;10(10):O111.008425.
Outline
11
1. Technology Development for Membrane Protein Analysis
2. Applications in Drug-treated samples and Clinic Samples
Apply Centrifugal Proteomic Reactor to Analyze Drug-
treated Samples and Clinic Samples
Paired Samples
12
Quantitative proteomic analysis reveals the neuroprotective effects
of huperzine A for amyloid beta treated neuroblastoma N2a cells
Proteomics. 2013 Apr;13(8):1314-24.
13
Quantitative proteomic analysis reveals the neuroprotective effects
of huperzine A for amyloid beta treated neuroblastoma N2a cells
Proteomics. 2013 Apr;13(8):1314-24.
14
Quantitative proteomic analysis reveals the neuroprotective effects
of huperzine A for amyloid beta treated neuroblastoma N2a cells
Proteomics. 2013 Apr;13(8):1314-24.
15
Quantitative Proteomic Analysis of Human Endometrial
Tissues in Receptive and Proliferative Phases
Recent data on the
worldwide prevalence of
infertility indicate that an
estimated 48.5 million
couples worldwide would
need some form of medical
intervention to achieve a
pregnancy
Approximately two-thirdsof implantation failures
result from abnormal
uterine receptivity
Cited from Nat Med. 2012 Dec;18(12):1754-67.16
Quantitative Proteomic Analysis of Human Endometrial
Tissues in Receptive and Proliferative Phases
17Unpublished data
0
1
2
3
4
5
6
7
8
9
-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7Log2 Ratio (N/P)
-lo
g1
0 p
-va
lue
Quantitative Proteomic Analysis of Human Endometrial
Tissues in Receptive and Proliferative Phases
18
1694
888 858 817
459 380 321 264 220 218 186 112 86 360
200400600800
100012001400160018002000
Cellular Component (GO)
Theoretical pI
Quantitative Proteomic Analysis of Human Endometrial
Tissues in Receptive and Proliferative Phases
19
Direct Protein-Protein Interaction Network Analysis
20
Ckb Protein is Up-regulated in Receptive Phase
21
KEGG Pathway Analysis
P<0.05
Ratio<0.5
(N/P)
P<0.05
Ratio>2
(N/P)
22
KEGG Pathway Analysis
23
KEGG Pathway Analysis
P<0.05
Ratio<0.5
(N/P)
P<0.05
Ratio>2
(N/P)
24
KEGG Pathway Analysis
25
Alpha-actinin is Down-regulated in Receptive
Phase
26
Quantitative Proteomic Analysis of Human Endometrial
Tissues in Receptive and Proliferative Phases
Ovarian hormones
Proliferative phase Receptive phase
?
Proteins involved in
cell-cell focal adhesion
Proteins involved in
desmosome
Human Endometrial Tissue
27
Acknowledgements
Dr. Daniel Figeys
The lab members in Dr. Figeys’ lab, University of Ottawa
One Hundred Talent, CAS
Key Laboratory for Receptor Research, CAS
National Natural Science Foundation of China, No. 21375138
Collaborators:
Dr. Haiyan Zhang (SIMM)
Dr. Huaiyu Yang (SIMM)
Dr. Hualiang Jiang (SIMM)
Dr. Chen Xu (Ruijin Hospital)
Thank you!