生命现象的物质基础是核酸和蛋白质，作为基因的载

体，核酸编制了所有的信息，而基因的表达产物蛋白
质，才是各种生物功能的执行者。

• 蛋白质组是功能基因组的新前沿，对基因表达的时空
规律进行揭示，比较了不同生理条件下或不同发育时
期的功能蛋白质组的差别和多样性（ mRNA 剪切的结
果或翻译后加工的结果），才能在分子水平上全面、
具体了解生命活动的过程，为个种生物学特征和生产
应用提供理论基础。
基因 疾病

环境
REAL COMPLEXITY…
IS IN CELLULAR ROTEOMES
• BEYOND THE GENOME…
• Tissue Specific Expression
• Alternate Splicing, (1/3 of all
genes)
• Post-Translational Modifications
– Types and Level:
– Signal Sequence cleavage
– Glycosylation
– Phosphorylation
– Farnylation
– Isoprenylation
– Acetylation
• All combine > 100-1000
fold increase in complexity

Future (challenging) research

后基因组时代的蛋白质组学

1, 蛋白质组的基本概念和历史
2 ，蛋白质组的特征
3 ，蛋白质组与基因组的关系
4 ，蛋白质组的研究技术和研究策略
5 ，蛋白质组研究的范例
1, 蛋白质组的基本概念和历史
Proteome
• 1994 M.Wilkins and K.W.Williams

• Macquarie University in
Sydney

•Total Proteins
Complement of a
PROTEOME : PROTEins expressed by a genOME

Proteomics
Large-scale Study of Proteins
 Why is proteomics necessary

Displaying and studying the products of genes directly is an

attractive way of studying disease and complex problem in biology

The study of gene expression can also be attempted

at the level of mRNA (microarray...)

However ……
 Why is proteomics necessary

You can have a protein in the cell when its mRNA is no longer present;

You can have lots of mRNA without tranlation of the message to protein

No good correlation between mRNA abudence and protein amout in

a cell at a given time.
Why is proteomics necessary

Protein post-translational modification

Protein subcellular location

Proteomics directly contributes to

Annotation of genome
 The Proteome and Technology

The technology required to separate large number of proteins, to

identify them, and to study their modifications are not straightforward

There are many more proteins in a proteome than genes in a genome,

especially for eukaryotes.
various ways a gene is spliced in constructing mRNA
various ways a protein can be post-translationally altered
 蛋白质组学

—— 结构蛋白质组学
—— 功能蛋白质组学
—— 亚细胞蛋白质组学
—— 修饰蛋白质组学
—— 相互作用组学
 蛋白质组研究简况
Pre-stage (1970’s-1994)
- Two-dimensional electrophoesis
- database
- Human Protein Index

Initiation stage (1994-1997):

- Term “proteome” was created by Wilkins, M. and Williams, K. in1994.
- The first meeting on proteome was hold in 1997.

Progression stage (1998-present):

- Projects of proteomics were activated internationally.
- Database of proteome were expanded.
-The milestone of proteomic research.

-Human Proteomics Organization (HUPO)

HUPO (The Human Proteome Organization)

Proteomics is in essence the study of the function,

regulation and expression of proteins in relation to
the normal function of the cell and in the initiation or
progression of a disease state. Proteomics is of
particular importance as it is the level of protein
activity that most disease are manifested.
Consequently proteomics seeks to correlate directly
the involvement of specific proteins and/or protein
complexes in given disease state
2 ，蛋白质组的特征
 蛋白质组的确定？

1 ，蛋白质组的完整性：
—— 基因组内基因（或 ORF ）数量？
—— 基因表达？
2 ，蛋白质组的测定：
—— 修饰蛋白质的判据？
—— 蛋白质的动态变化（降解或转运）？
 蛋白质的“四维”研究

—— 特定的时间：合成与降解

—— 特定的空间：区域性与运动性
 蛋白质组研究技术的复杂性

——20 种化学和物理性质不同的氨基酸
—— 蛋白质量的动态差别（ 106 ）
—— 蛋白质的不稳定性
—— 蛋白质修饰的多样性和复杂性
Dynamic Proteomics ( 动态蛋白质组学 )

• 动 态 表 达

• 动 态 组 成

• 动 态 定 位

• 动 态 关 系
3 ，蛋白质组与基因组的关系
 互相补充

转录水平 翻译水平
基因芯片 蛋白质组研究
 互相帮助

1 ，基因组的注释（ Annotation ）
2, 蛋白质的质谱鉴定
3 ，依赖基因组研究技术的蛋白质组研究技术（双
杂交技术、噬菌体显示技术 ）
蛋白质组研究不同于基因组研究

相对独立

结构基因组 功能基因组

紧密连接

结构蛋白质组

功能蛋白质组
4 ，蛋白质组的研究技术和研究策略
蛋白质组研究的策略：平台与整合

动物模型 / 细胞 人类疾病

功
能
分 蛋白 质组研究
析

生物信息学分析 蛋白质组新技术
蛋白质组生物信息学

Bench works (2D, MS, etc)

Bioinformatics

Databases
 Proteome Research
• Cell Proteins

• Separation and Resolution

• Identification

• Structure and Function of proteins

蛋白质组学的主要研究方法

1 、基于二维电泳 - 质谱技术的蛋白质组研究

2 、基于质谱技术的蛋白质组研究
— 液相电泳 - 质谱技术
— 液相色谱 - 质联用技术
— Shotgun 质谱技术
— ICAT 技术

3 、蛋白质芯片
 Two dimensional electrophoresis

1975 High-resolution IEF-SDS-PAGE O’ Farrell, Klose

Scheele

Separation of over 1000 proteins in E. coli

Drawback: carrier ampholytes used to generate pH gradients is

difficult to achieve good reproducibility.
Limited loading amount.
 Two-dimensional Electrophoresis (2-DE)

1st dimension: isoelectric focusing (IEF)

proteins are separated in a pH gradient ubtil reach a
stationary position where their net charge is zero.
The pH at which a protein has zero net charge is called
isoelectric point (pI)

2nd dimension: SDS-PAGE

Proteins are separated according thire molecular weight (Mr)

Two-dimensional Electrophoresis (2-DE)

!980s
The introduction of immobilised pH gradient (IPG)
eliminlated the problem of gradient instability and poor
sample loading capacity
Two-dimensional Electrophoresis (2-DE)

Immobilised pH gradient (IPG)

pH gradient is co-polymerised with the acrylamide gel matrix

to form completely stable gradients.

The commercial precast IPG stripes is available in various

pH ranges (3-!0,4-7...).
 Two-dimensional Electrophoresis (2-DE)

2-DE is the only methode currently available which is

capable of simultaneously separating thousands of proteins

Two-dimensional Electrophoresis (2-DE)

!980s
The introduction of immobilised pH gradient (IPG)
eliminlated the problem of gradient instability and poor
sample loading capacity
Sample Preparation

Urea, thiourea

Triton X-100, NP-40,

CHAPS, SB 3-10,
ASB 14

DTT, DTE, TBP

 Cell culture

Protein
extraction

2-DE

Image analysis

Scanning

Silver staining
 荧光差 异电泳 （ DIGE ）

Introduction to the Ettan DIGE System

EttanTM Workflow

Typhoon
CyDye IPGphor + Ettan
Scanner
DIGE Fluors Dalttwelve units
Sample Prep (minimal dyes)

DeCyder Software

Ettan
MALDI Pro
MS
Ettan Spotter Ettan Picker
Ettan Digester
 Mass Spectrometry
High Vacuum System

Ion Mass Detector Data

Inlet Source Filter System

Sample MALDI TOF Microchannle PC’

Target ESI Quadruple Electron
Ion Ion Trap Multiplier SunSPAR
HPLC Spray Magnetic Hybrid
GC API Sector
CE LSIMSS FTMS
EI
CI
Generalized schematic of proton energy absorption
by the matrix in MALDI.
TOF-MS instrument with a single acceleration stage

F ＝ Eq (1)
F = ma (2)
a = Eq/m (3)

Principle of PSD analysis in MALDI/MS

 ESI
Electrospray Source
Triple-quadrupole ESI mass spectrometer possesses ion
selection and fragmentation capabilities.
ESI-ion trap
 Designation for peptide CID fragment ions
an bn cn
H+

O O O
NH2-CH C NH CH-C-NH-CH-C-OH
R1 R2 R3

xn yn zn
 MALDI-TOF-MS of peptide mixture

Zhou04_163 #1-31 RT: 0.13-1.94 AV: 7 NL: 7.33E6

T: + c NSI Full ms [ 700.00-2000.00]
1472.0
100
1554.0
95 1896.0
90 1895.0
85

80

75
1816.1
70 1473.0
Relative Abundance

65
1553.0
60 1555.1
1817.1
55 1566.9 1788.0

50

45 1567.9
40 1183.9 1474.0

35 1818.1
1185.0
30
1200.0 1938.0
1781.0
25 1275.9 1568.9
1779.9
1460.0
20 1675.9 1772.1
1201.1 1536.0 1940.0
1729.9 1819.0
15 1611.0
1277.0 1475.9
10 1331.9 1373.1 1425.9 1857.1
1247.5 1970.7
5

0
1200 1300 1400 1500 1600 1700 1800 1900 2000
m /z
 RT: 0.01 - 60.05

100

95
35.27
* NL:
1.48E5
Base Peak
F: + d Z
90 ms

85

80

75

70

65
T4 43.48
Relative Abundance

60

55
T5
50
T2 37.34
45 35.01
5.73 6.66 T6
40

35
38.60
30 48.90

25 8.06 44.87

20
34.45
42.63
15 3.60 4.43 T1
55.07
10 8.49
45.91 49.18 53.17
5 9.35 41.12
10.70
15.08 18.28 22.38 28.33 30.05 33.55 59.22
0
5 10 15 20 25 30 35 40 45 50 55 60
Time (min)

Base peak chromatogram obtained by LC-MS analysis of tryptic digests from spot
7120. 6 peptides (T1~T6) were confidently matched to heat shock 27 kD protein
(HSP27)
 S#: 769 RT: 35.22 AV: 1 NL: 3.22E6
T: + c Full ms [ 400.00 - 2000.00]
892.8
100

95
[M+2H]2+
90

85

80

75

70
Relative Abundance

65

60 953.8
55

50

45

40

35

30

25

20 1163.6

1164.7
[M+H]+
15 595.9 955.7
1783.7
1082.8 1146.1 1615.9
10 672.0 1497.9 1929.9 1962.9
1197.8 1309.0 1387.5
538.1 601.6 765.5 857.4 1056.6 1679.0 1758.5
5 521.6

0
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z

Mass spectrum of one tryptic peptide from spot 7120. The spectrum shows that the
most abundant ion with m/z 892.81 was produced from that peptide
MS for Characterization of
Proteins and peptide

Mass Determination

Peptide mass fingerprinting

Sequence-specific MS/MS

Database identification
 MS for Characterization of
Proteins and peptide

Peptide mass fingerprinting Digestion

(MALDI-TOF)

Matching
& Scoring

Digestion
Database theoretical digestion
 MS for Characterization of
Proteins and peptide

Digestion
Peptide Mass

Sequence-specific MS/MS GHVASTYK

Database identification
Dynamic Proteomics ( 动态蛋白质组学 )

• 动 态 表 达

• 动 态 组 成

• 动 态 定 位

• 动 态 关 系
 动态表达

研究内容：
疾病发生 , 发展 , 预后过程中的蛋白质差异谱构建 ,
跟踪关键蛋白质在疾病不同时期动态表达

研究方法：

多重稳定同位素标记

多重稳定同位素氨基酸参入

合成特定的同位素肽片段，进行蛋白质 “绝对 ”定量

 动态组成

研究内容：
蛋白质翻译后修饰 , 如磷酸化，糖基化 ,
乙酰化等的大规模分析和监测

研究方法：

亲和捕获修饰蛋白质

特异检测修饰蛋白质

修饰位点的标记和检测

修饰的同位素标签定量分析
 动态定位

研究内容：

亚细胞蛋白质组分析及定位，与功能相关的蛋白质
空间动态变化分析

研究方法：

亚细胞分级

高通量鉴定

差异定位

免疫荧光定位
 动态关系

研究内容：
蛋白质功能复合物，蛋白质 - 蛋白质作用网络，
蛋白质与小分子配体相互作用分析

研究方法：

多级亲和标签与质谱分析

小分子亲和标签与质谱分析

蛋白质芯片
Thank
you !