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

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+

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+

+

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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！