10/27/2005 Chaoqun Wu,Fudan University 1
Proteomic
复旦大学生命科学学院吴超群
10/27/2005 Chaoqun Wu,Fudan University 2
Defining Proteomics
Branch of discovery science focusing on proteins
In 1994 defined as,the complete set of proteins that is
expressed and modified following expression by the
entire genome in the lifetime of a cell”,If we look at an
organism it means that we are looking at the proteome of
3 trillion of cells and ~1000 different cell types with
different protein profiles.
Can be more specific such as the complement of
proteins expressed by a cell at any one time.
Today proteomics is a scientific discipline that will bridge
the gap between our understanding of genome
sequences and cellular behavior,
10/27/2005 Chaoqun Wu,Fudan University 3
Genomics and Proteomics a new
field with a new vocabulary
-Omics,
means area
of research
DNA
RNA
proteins
Metabolites
Protein-protein,Protein-DNA,
Protein-RNA interactions
Genome
Transcriptome
Proteome
Metabolome
Interactome
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The,omics” nomenclature…
Genomics
DNA (Gene)
Functional
Genomics
Transcriptomics RNA
Proteomics
PROTEIN
Metabolomics METABOLITE
Transcription
Translation
Enzymatic
reaction
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Why study protein expression?
Cytosol
Nucleus
Inactive mRNA
RNA
Degradation
control
RNA
Transport
control
Translation control
Post-translational
control
Primary
RNA
transcript
Transcriptional
control
RNA
Processing
control
mRNA
mRNA
DNA
Modified
protein
protein
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The proteome
– Protein complement of a genome
Variable
– In different cell and tissue types in same organism
– In different growth and developmental stages of
organism
Dynamic
– Depends on response of genome to environmental
factors
Disease state
Drug challenge
Growth conditions
Stress
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10/27/2005 Chaoqun Wu,Fudan University 8
Content
I,Introduction
II,Proteomics and Genomics
III,Identification and Quantification
of Proteins
IV,Structural Proteomics
V,Identification of Protein Post-
translational Modifications
VI,Protease degradomics:
VII,Platforms for proteomics
VIII,Mass Spectrometry
IX,Post-MS Analysis
X,Bioinformatics for Proteomics
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Part I.
Introduction
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二十世纪中期以来，随着DNA双螺旋结构的提出和蛋白质空间结构的
X射线解析，开始了分子生物学时代，
对遗传信息载体DNA和生命功能的主要体现者蛋白质的研究，成为生命科学研究的主要内容。
二十世纪中期以来，随着双螺旋结构的提出和蛋白质空间结构的射线解析，开始了分子生物学时代，
对遗传信息载体和生命功能的主要体现者蛋白质的研究，成为生命科学研究的主要内容。
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人类蛋白质组研究对人类蛋白质组研究主要聚焦在特异的组织、
细胞和疾病上。人的各种组织、器官、细胞乃至各种细胞器已被广泛研究。人的各种体液（血液、淋巴、脊髓、乳汁、尿液等）都被用于研究与某些疾病的关系。许多科学家致力于各种肿瘤组织与正常组织之间蛋白质谱差异的研究。这些研究已经找到了一些肿瘤特异性的标志物。例如，在对肾癌的研究中发现有四种蛋白质存在于正常肾组织而在肾癌细胞中消失。其中两种分别是辅酶Q蛋白色素还原酶和线粒体泛醌氧化—还原复合物I，这提示线粒体功能低下可能与肿瘤的发生有关。
人类蛋白质组研究对人类蛋白质组研究主要聚焦在特异的组织、
细胞和疾病上。人的各种组织、器官、细胞乃至各种细胞器已被广泛研究。人的各种体液（血液、淋巴、脊髓、乳汁、尿液等）都被用于研究与某些疾病的关系。许多科学家致力于各种肿瘤组织与正常组织之间蛋白质谱差异的研究。这些研究已经找到了一些肿瘤特异性的标志物。例如，在对肾癌的研究中发现有四种蛋白质存在于正常肾组织而在肾癌细胞中消失。其中两种分别是辅酶蛋白色素还原酶和线粒体泛醌氧化还原复合物，这提示线粒体功能低下可能与肿瘤的发生有关。
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蛋白质组（proteome）由澳大利亚学者Wilkins
和Williams等于1994年提出，指的是由基因组编码的全部蛋白质，即某一物种、个体、器官、组织乃至细胞的全部蛋白质。与以往的蛋白质化学的研究不同，
蛋白质组研究的对象不是单一或少数的蛋白质，它着重的是全面性和整体性，需要获得体系内所有蛋白质组分的物理、化学及生物学参数，如分子量、等电点、
表达量等。它是动态的，有它的时间性、可调节性；
进而能够在细胞和生命有机体的整体水平上阐明生命现象的本质和活动规律。
蛋白质组（）由澳大利亚学者和等于年提出，指的是由基因组编码的全部蛋白质，即某一物种、个体、器官、组织乃至细胞的全部蛋白质。与以往的蛋白质化学的研究不同，
蛋白质组研究的对象不是单一或少数的蛋白质，它着重的是全面性和整体性，需要获得体系内所有蛋白质组分的物理、化学及生物学参数，如分子量、等电点、
表达量等。它是动态的，有它的时间性、可调节性；
进而能够在细胞和生命有机体的整体水平上阐明生命现象的本质和活动规律。
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蛋白质组研究的核心是系统识别一个细胞或组织中表达的每一个蛋白质,以及确定每个蛋白质的突出特征。其分析技术包括分离蛋白质和肽的分离科学、识别和定量分析物的分析科学和数据管理及分析的生物信息学。
?Global Proteomics – Identify and catalog all
possible proteins in an organism.
?Targeted Proteomics – Identify and catalog a
subset of proteins involved in a specific pathway,
disease,or other specific facet you wish to study.
?Structural proteomics – Determine the 3-D
structure of all proteins.
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随着大规模基因组测序计划的完成，人类又面临一个新的、更加艰辛的科学挑战—
蛋白质组学的研究.
蛋白质组学的研究是一项系统性的多方位的科学探索。其研究内容包括：
蛋白质结构、蛋白质分布、
蛋白质功能、蛋白质的丰度变化、
蛋白质修饰、
蛋白质与蛋白质的相互作用、
蛋白质与疾病的关联性。
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Patterson and
Aebersold,Nature
Genetics (supp.),
33,311 (2003)
protein-ligand
interactions
protein
complexes
(machines)
protein families
(activity or structural)
post-translational
modified proteins
Eukaryotic cell.
Examples of protein
properties are shown,
including the interaction of
proteins and protein
modifications.
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What is Proteomics?
Defined as,the analysis of the entire protein
complement in a given cell,tissue,or
organism.”
Proteomics,also assesses activities,
modifications,localization,and interactions
of proteins in complexes.”
Proteomes of organisms share intrinsic
differences across species and growth
conditions.
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The Role of Proteomics
? The existence of an ORF does not imply the
existence of a functional gene.
? Limitations of comparative genomics.
? mRNA levels may not correlate with protein
levels.
? Protein modifications ? post-transcriptional
modifications,isoforms,post-translational
modifications,mutants.
? Issues of proteolysis,sequestration,etc,
relevant only at the protein level.
? Protein complex composition,protein-protein
interactions,structures,
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Proteins classes for Analysis
Membrane
Soluble proteins
Nuclear
Chromosome-associated
Phosphorylated
Glycosylated
Complexes
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Proteomics
A functional understanding of the cell
Definition:
“A proteome is the entire protein
complement expressed by a given
biological system in given
conditions,
Wilkins et al.,BioTechnology 14,61-65 (1996)
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Expression proteomics:
up and down-regulation of protein level,
e.g,normal and tumor cell,rest and drug-
induced cell,mature and inmature cell,
malignant,metastatic cancer cells etc.
Functional proteomics:
characterization of cellular compartments,
multiprotein complexes,protein interaction
net and signaling pathway,etc.
Classification
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Clinical Proteomics
This area of proteomics focuses on accelerating
drug development for diseases through the
systematic identification of potential drug targets.
How could this be accomplished?
Hopefully,we will have more specific information,
instead of raw genes,that will make those complex
differential equations much simpler in the coming
years.
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Applications of Proteomics
Mining,identification of proteins (catalog
the proteins)
Protein-expression profile,identification of
proteins in a particular state of the
organism
Protein-network mapping,protein
interactions in living systems
Mapping of protein modifications,how and
where proteins are modified.
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Proteomics and Systems Biology
“The identification,characterization and
quantification of all proteins involved in a particular
pathway,organelle,cell,tissue,organ or organism
that can be studied in concert to provide accurate
and comprehensive data about that system.”
Systems Biology
http://www.inproteomics.com/prodef.html
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Genomics
Functional
genomics
global Targeted
Transcriptomics
Proteomics
global Targeted
Targetedglobal
Metabolomics
Targetedglobal
System Biology
Interactomics
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“Systems biology is an approach to
studying complex biological systems made
possible through technological
breakthroughs such as the human genome
project,…systems biology simultaneously
studies the complex interaction of many
levels of biological information to
understand how they work together.”
http://www.systems biology.org/
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Epigenetics,Factors that affact
the proteome of a cell
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Levels of genome research in eukaryotes
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mRNA
primary protein
product
protein
degradation
mature?
protein
Active
protein
Transcriptome
primary RNA
transcript
DNA
Genome
Why studying proteomes?
Proteome
Proteomic is a functional approach
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Importance of Proteins:
they serve as catalysts that maintain metabolic
processes in the cell,
they serve as structural elements both within and
outside the cell,
they are signals secreted by one cell or deposited
in the extracellular matrix that are recognized by
other cells,
they are receptors that convey information about
the extracellular milieu to the cell,
they serve as intracellular signaling components
that mediate the effects of receptors,
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Importance of Proteins:
? they are key components of the machinery that
determines which genes are expressed and
whether mRNAs are translated into proteins,
? they are involved in manipulation of DNA and
RNA through processes such as,DNA replication,
DNA recombination,RNA splicing or editing.
http://www-users.med.cornell.edu/~jawagne/proteins_&_purification.html
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Central lesson from
eukaryotic genome projects
Evolutionary complexity is not primarily determined
by increasing the number of genes,but by
increasing variation on the level of the synthesized
proteins.
This is achieved by generating MULTIPLE proteins
from only ONE gene,e.g,by
– different combinations of exons by alternative splicing
– post-translational protein processing (e.g,cleavage of
pro-peptides)
– post-translational protein modifications (e.g,acetylation,
glycosylation)
– modified central dogma,DNA --> RNA --> protein(s)
– it is important to perform analyses on the level of gene
PRODUCTS Key
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But what about the Genome?
What does having the genome of an organism
give us?
– A great diagram,or,blueprint,” of the genes
within an organism,
– Think of the genome as code that needs
compiled into functional units.
– The genome gets,compiled” into the proteome
via the central dogma of biology.
– Proteomic strategies attempt to utilize information
from the genome in an attempt to conceptualize
protein function.
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The proteomic process,
Strong / reliable models
- Organism,organ,tissue,cell,
cellular organal,complex.
Protein separation,
- One or two-dimensional gel
electrophoresis
-
Mass spectrometry
Protein identification,
- Blotting with antibodies
- Amino Acid composition
- Edman degradation
- Mass spectrometry
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The proteomic features,
Key advantage of proteomics
Researchers work on the level of gene products and
deal with genes that are really expressed to give a
detectable PRODUCT and are not just,expressed”
which only says they produce a detectable mRNA but it
is not clear whether there is a gene product or not.
Key limitation of proteomics
Usually,only a fraction of the proteins synthesized can
be detected in a proteomics experiment,whereas the
expression of ALL genes can be monitored in a whole-
genome array experiment.
Key prerequisite of proteomics
A genome sequence for the investigated organism or at
least a collection of many cDNA sequences is required.
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Bottlenecks of Proteomics
Experimental:
Large-scale protein analysis is difficult
because,
Proteins are fragile
They can exist in multiple isoforms
There is no protein equivalent of PCR
for amplification of a small sample
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Bottlenecks of Proteomics
Data Analysis,
-Data contains a lot of noise that is difficult to
separate from actual signal,This results in
wastage of computing resources on
searching for unlikely spectra.
-Database searches for matching spectra
only give scores,leaving manual intervention
necessary for eliminating false positives
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Bottlenecks of Proteomics
Biomedical limitations
-In practice,it is very difficult to trace the
complete progression of a disease.
-Hence,using proteomics for monitoring
the biochemistry of a disease is like
using a photo camera to record a
football match.
10/27/2005 Chaoqun Wu,Fudan University 38
Part II.
Proteomics and
Genomics
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基因和蛋白质基因翻译蛋白质
27，000条>10万种
1,基因编码蛋白质，
2,一个基因可翻译几种蛋白质，
3,基因的功能通过翻译成蛋白质来实现，
4,基因的异常引起翻译成的蛋白质异常，
而影响蛋白质的功能。
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26,383
条人类基因的功能分类核苷酸酶受体转录调控因子原癌基因细胞骨架细胞黏附分子激酶选择调节分子转移酶氧化还原酶水解酶
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The Synergy of
Proteomics and Genomics
Proteomics provides data on the
outcome of gene expression,
Genomics provides the comprehensive
sequence and expression data required
to advance protein research.
Functional genomics includs Proteomics
and Genomics
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Functional Genomics
? Understanding Function of Genes and Other Parts of
Genome
? Study of Biological Systems Based on Global
Knowledge of Genomes,Transcriptomes & Proteomes
Genome,All the Genetic Material in the
Chromosomes
Transcriptome,Entire Set of Gene Transcripts
Proteome,Entire Sets of Proteins
Genome (DNA) Transcriptome (RNA) Proteome (Protein)
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Proteomics is the study of proteins,
whereas genomics is the study of
DNA and the processes which lead
to the creation of proteins,When
used in combination,these two
approaches to the study of gene
expression enable researchers to
analyze regulation at many levels.
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Proteomics,Tools From
Genome to Proteome
It sounds like mission impossible?
– Follow the changes taking place inside a cell by
identifying the thousands of different proteins the
cell produces and watching how they change
and flow over time.
– To a growing band of researchers,however,
such a mission is becoming more realistic,
thanks to the increasing volume of sequence
data and to improved analytical techniques for
proteins like MALDI-MS,etc..
10/27/2005 Chaoqun Wu,Fudan University 45
Predicting Life Processes,
Reverse Engineering Living Systems
Transcription
Translation
Proteins
Biochemical Circuitry
Phenotypes (Traits)
Metabolomics
(storage)
DNA
Gene Expression
Proteomics
Environment
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Example 1 - Signal Transduction
When a cell receives a signal,such as a
growth factor,its immediate response is at the
protein level,Cell surface protein receptors
are activated and modified,In addition,
transmission of information from the activated
receptor to the nucleus often involves physical
movement of proteins,These activities can be
detected and analyzed using proteomic
technologies,
growth factor,its immediate response is at the
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Example 2 - A signalling network central
to tumour suppression.
Most disease processes manifest themselves
at the level of protein activity,but until recently,
high throughput analysis of proteins was not
possible,
The development of several technologies now
makes it feasible to perform mass screening
of proteins and is creating a revolution in
proteomics.
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Part III.
Identification and
Quantification of
Proteins
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General
flow for
proteomics
analysis
SEPARATION
ID
ENTIF
I
CATION
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Identification of gel separated
proteins prior to MS
MS analysis
Excision of the spots of interest
Reduction & Alkylation
In gel digestion
Extraction of the peptides
Purification & concentration of
the peptides on a
nano reverse phase column?
1) Peptide mapping
2) Peptide sequencing
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2D gels are covalently
bound to glass,
allowing long-term
storage of gels while
minimizing gel-to-gel
size variation.
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Reproducibility
Reproducibility is one of the key developments in proteomics
was the development of gels which deliver reproducible
results,In a reproducibility experiment:
The x-axis is the Isoelectric point (pI) which is
analagous to pH,while the y-axis is molecular weight
(Mw) or size,
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MS analyses
MS identifies analytes by:
?Producing gas phase ions
?Separating these ions according to m/z ratio
?Detecting the ions
Sensitive:
Detect attomole concentrations
10/27/2005 Chaoqun Wu,Fudan University 57
?Total extract
?Serum dopletion
?Fractionation
?High resolution
SDS-PAGE
?1D + 2D
?M-HPLC
?Post-seperation
fluorescence
?Differentially
expressed
proteins
?Robotic process
from gel to
peptide pool
?MALDI-TOF and
tandem MS
?Fractionation
Computational
analysis
(data treatment)
Current Proteome Analysis
Biological
sample
Sample
preparation
Protein
extract
Peptide
sequence
Separation Detection Selection
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Current
protocol
Almost no limitation
when the entire
genome is sequenced.
Limited to ~1000 proteins
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Differential 2D analysis
Comparison between different protein samples run on 2-D gels,Proteins
from either a normal liver tissue sample ora liver tumor sample were
run on two 2-D gels and stained,images of the gelswere captured
using the FLA-3000 scanner,Images from the two gels were then
pseudo-colored either pink or green,overlaid and matched spot-to-spot
using Z3 software(Compugen),Matched spot intensities that differ by
More than 20% are represented as green spots (for proteins expressed
higher in the liver tumor sample) or pink spots (for proteins expressed
higher in the normal liver tissue sample).Matched spots that differ by
less than 20.0% are black.
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LC 2D analysis
?Proteins Fractionated using 2D Chromatography
?Proteins resolved by both chromatofocusing and
reversed phase chromatography
?Proteins unique to diseased state - Isolated
*
*
*
*
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2D-DIGE (双向荧光差异凝胶电泳)
Differential in-gel electrophoresis (2D-DIGE)
2D-DIGE analysis,employing Cy3 or Cy5 dyes to
label two different protein populations,eliminates
inter-gel variation and facilitates quantitative
evaluation of differentially expressed proteins,
Populations of unique and differentially expressed
proteins from wild type and rin mutant tomato fruit
at different developmental stages were
trypsinized and analyzed by mass spectrometry,
The resulting data were used to query databases
in order to identify cognate genes/proteins,
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Mass Spectrometry and
Quantitative Measurements
A B
m/z
Rel,Abund.
Q
H
E
Mass spectrometry is
inherently not a
quantitative technique,
The intensity of a peptide
ion signal does not
accurately reflect the
amount of peptide in the
sample.
equimolar mixture
of 2 peptides
516.725 516.828
m/z
(M+2H)
2+
,[
12
C]-ion
[Val
5
]-Angiotensin II
1031.5188 (monoisotopic)
Lys-des-Arg
9
-Bradykinin
1031.5552 (monoisotopic)
= 0.036
equimolar mixture
of 2 peptides
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Mass Spectrometry and
Quantitative Measurements
equimolar mixture
of 2 peptides
Q
H
E
A B
Rel,Abund.
Two peptides of identical chemical structure that differ in mass
because they differ in isotopic composition are expected to
generate identical specific signals in a mass spectrometer.
Q
H
E
13
C
13
C
13
C
A B
2
D
2
D
m/z
Methods coupling mass spectrometry and stable isotope tagging
have been developed for quantitative proteomics.
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ICAT,Isotope-Coded Affinity Tag
(同位素亲和标签同位素亲和标签
)
Alkylating (烷基化物烷基化物
)group covalently attaches the reagent to
reduces Cys(半胱氨酸半胱氨酸
)-residues.
A polyether mass-encoded linker contains 8 hydrogens (氢氢,
d0) or 8 deuteriums (氘氘
,d8) that represents the isotope
dilution.
A biotin affinity tag is used to selectively isolate tagged
peptides (by avidin purification).
10/27/2005 Chaoqun Wu,Fudan University 66
ICAT,Isotope-Coded Affinity Tag
MS/MS
identifies
the protein
The Cys-residues in sample 1 is labeled with d0-ICAT and sample 2 is
labeled with d8-ICAT.
The combined samples are digested,and the biotinylated ICAT-labeled
peptides are enriched by avidin affinity chromatography and analyzed by
LC-MS/MS.
Each Cys-peptide appears as a pair of signals differing by the mass
differential encoded in the tag,The ratio of the signal intensities indicates
the abundance ratio of the protein from which the peptide originates.
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Stable Isotope Amino Acid or
15
N-
in vivo Labeling
Metabolic stable isotope
coding of proteomes
An equivalent number of
cells from 2 distinct cultures
are grown on media
supplemented with either
normal amino acids or
14
N-
minimal media,or stable
isotope amino acids
(
2
D/
13
C/
15
N) or
15
N-enriched
media.
These mass tags are
incorporated into proteins
during translation.
10/27/2005 Chaoqun Wu,Fudan University 68
Enzymatic Stable Isotope
Coding of Proteomes
Enzymatic digestion in the presence of
18
O-water incorporates
18
O at the
carboxy-terminus of peptides
Proteins from 2 different samples are
enzymatically digested in normal
water or H
2
18
O.
R
3
R
4
NH
2
-CH-CO-NH-CH-COOH...NH-CH-CO-NH-CH-CO-
18
OH
R
1
R
2
...NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH
R
1
R
2
R
3
R
4
Trypsin /H
2
18
O
(Arg,Lys)
C-terminal peptide
10/27/2005 Chaoqun Wu,Fudan University 69
Identification of Low
Abundance Proteins
The identification of low
abundance proteins in the
presence of high abundance
proteins is problematic (e.g.,
“needle in a haystack”)
Pre-fractionation of complex
protein mixtures can alleviate
some difficulties
– gel electrophoresis,
– chromatography,etc
Removal of known high
abundance proteins allows less
abundant species to be visualized
and detected
10/27/2005 Chaoqun Wu,Fudan University 70
Identification of Low Abundance
Proteins
GenWay Biotech
10/27/2005 Chaoqun Wu,Fudan University 71
Protein-Protein Interactions
affinity support
(1) Affinity Purification
10/27/2005 Chaoqun Wu,Fudan University 72
(2) Phage Display
10/27/2005 Chaoqun Wu,Fudan University 73
(3) Yeast 2-hybrid
10/27/2005 Chaoqun Wu,Fudan University 74
(4) Protein Chips
10/27/2005 Chaoqun Wu,Fudan University 75
Part IV.
Identification of Protein
Post-translational
Modifications
10/27/2005 Chaoqun Wu,Fudan University 76
Proteomics and post-translational
modifications
Patterson and
Aebersold,Nature
Genetics (supp.),
33,311 (2003)
protein-ligand
interactions
protein-ligand
interactions
protein
complexes
(machines)
protein
complexes
(machines)
protein families
(activity or structural)
protein families
(activity or structural)
post-translational
modified proteins
post-translational
modified proteins
Eukaryotic cell.
Examples of protein
properties are shown,
including the interaction
of proteins and protein
modifications.
10/27/2005 Chaoqun Wu,Fudan University 77
Post translational modifications
in the ER
Removal of signal peptide
Addition of carbohydrates – glycosylation
Addition of GPI anchors
Folding
– Disulfide bond formation
Formation of multimeric proteins
Only properly modified proteins are transported
from ER to the final destination
10/27/2005 Chaoqun Wu,Fudan University 78
Proteomic analysis of PTMs
醌酰肉豆蔻酰棕榈酰
10/27/2005 Chaoqun Wu,Fudan University 79
( Be continued )
羟(基)脯氨酸脱酰胺焦谷氨酸糖基磷脂酰肌醇锚
Mann and Jensen,Nature Biotech,21,255 (2003)
10/27/2005 Chaoqun Wu,Fudan University 80
Proteomic Analysis of Post-
translational Modifications
Post-translational modifications (PTMs)
– Covalent processing events that change the
properties of a protein
proteolytic cleavage
addition of a modifying group to one or more amino
acids
– Determine its activity state,localization,turnover,
interactions with other proteins
– Mass spectrometry and other biophysical methods
can be used to determine and localize potential PTMs
However,PTMs are still challenging aspects of
proteomics with current methodologies
10/27/2005 Chaoqun Wu,Fudan University 81
Complexity of the Proteome
Protein processing and modification comprise an
important third dimension of information,beyond those of
DNA sequence and protein sequence,
Complexity of the human proteome is far beyond the
more than 30,000 human genes.
The thousands of component proteins of a cell and their
post-translational modifications may change with the cell
cycle,environmental conditions,developmental stage,
and metabolic state.
Proteomic approaches that advance beyond identifying
proteins to elucidating their post-translational
modifications are needed.
10/27/2005 Chaoqun Wu,Fudan University 82
Mass spectrometry,powerful tool for proteomics
Mass spectrometry measures the mass of proteins or peptides
from analysis of the masstocharge (m/z) ratio.
Mass spectrometers first ionize the sample,and then the ions
are introduced into the mass analyser,which separates and
detects the sample ions according to their mass,
Fragmentation of the protein sample results in a collection of
ions that have different masses,and the spectrometer
measures the relative abundance of each ion,according to
their m/z ratios,to obtain a spectrum of masses,hence the
term mass spectrometry.Proteomic analyses typically use
matrix-assisted laser desorptionionization (MALDI) or
electrospray ionization (ESI) sources followed by introduction
into time-of-flight (TOF) or quadrupole (Q) mass analysers,
which are described in more detail below.
10/27/2005 Chaoqun Wu,Fudan University 83
Use MS to
determine PTM
of isolated
protein
Enzymatic or
chemical
degradation of
modified protein
HPLC
separation of
peptides
MALDI and/or
ESI used to
identify PTM
MS/MS used to
determine
location of
PTM(s)
10/27/2005 Chaoqun Wu,Fudan University 84
Ionization sources
Matrix-assisted laser desorption-ionization (MALDI),Protein or
peptide samples on solid matrices are ionized by a pulsed laser,
entrained in the resulting ionized gas plume,and introduced
into the mass analyser.
Electrospray ionization (ESI),Protein or peptide samples are
passed through a fine needle to which a voltage is applied,
which results in a fine spray of sample-containing droplets,
Samples are delivered to the mass analyser after the breakup
and evaporation of the droplets,which releases the protein or
peptide samples to the gas phase,A low-flow rate liquid
chromatography system can be coupled to the needle to allow
for protein fractionation of the samples before mass analysis.
10/27/2005 Chaoqun Wu,Fudan University 85
Mass analysers
Time-of-flight (TOF),Time-of-flight analysers separate ions
on the basis of their flight times over a known distance,The
lower the mass of the ion,the greater the velocity and hence
the shorter the flight time.Travel time from the ion source to
the detector is transformed into the m/z ratio,from which the
mass of the ionized sample can be calculated with extreme
accuracy.
Quadrupole (Q) mass filter,A quadrupole mass filter
consists of four parallel rods through which direct current and
radio frequency electric fields are applied to sort the
introduced ions,For each combination of voltages and
frequencies,only ions with a specific m/z ratio pass
undeflected through the quadrupole mass filter,Precise
stepping of these settings therefore allows the quadrupole to
be used as a mass analyser to scan for ions over a large m/z
range.
10/27/2005 Chaoqun Wu,Fudan University 86
Glycoprotein Gel Stain
CandyCane glycoprotein molecular weight standards
containing alternating glycosylated and
nonglycosylated proteins were electrophoresed
through a 13% polyacrylamide gel,After separation,
the gel was stained with SYPRO Ruby protein gel
stain to detect all eight marker proteins (left),
Subsequently,the gel was stained by the standard
periodic acid–Schiff base (PAS) method in the Pro-Q
Fuchsia Glycoprotein Gel Stain Kit to detect the
glycoproteins alpha
2
-macroglobulin,glucose oxidase,
alpha
1
-glycoprotein and avidin.
Pro-Q? Glycoprotein
Stain (DDAO phosphate)
Molecular Formula,
C
15
H
18
Cl
2
N
3
O
5
P (MW
422.20)
Detection of glycoproteins and total protein on an SDS-
polyacrylamide gel using the Pro-Q Fuchsia Glycoprotein Gel
Stain Kit.
10/27/2005 Chaoqun Wu,Fudan University 87
Nitro-Tyrosine Modification
Oxidative modification of amino acid side chains include
methionine oxidation to the corresponding sulfone,S-
nitrosation or S-nitrosoglutationylation of cysteine residues,
and tyrosine modification to yield o,o’-dityrosine,3-
nitrotyrosine and 3-chlorotyrosine.
Nitric oxide (NO) synthases provide the biological precursor
for nitrating agents that perform this modification in vivo,
NO can form nitrating agents in a number of ways including
reacting with superoxide to make peroxynitrite (HOONO)
and through enzymatic oxidation of nitrite to generate NO
·
2
Tyrosine nitration is a well-established protein modification
that occurs in disease states associated with oxidative
stress and increased nitric oxide synthase activity.
The combination of 2D-PAGE,western blotting,and mass
spectrometry has been the more typical strategy to identify
3-nitrotyrosine-modified proteins,
10/27/2005 Chaoqun Wu,Fudan University 88
Nitro-Tyrosine Modification
“Proteomic method identifies proteins nitrated in vivo during inflammatory
challenge,” K,S,Aulak,M,Miyagi,L,Yan,K,A,West,D,Massillon,J,W,
Crabb,and D,J,Stuehr,Proc,Natl,Acad,Sci,USA 2001; 98,12056-12061.
Anti-nitrotyrosine immunopositive proteins in lung of rats induced with LPS.
10/27/2005 Chaoqun Wu,Fudan University 89
Phosphorylation
Analysis of the entire complement of phosphorylated proteins in
cells:,phosphoproteome”
Qualitative and quantitative information regarding protein
phosphorylation important
Important in many cellular processes
– signal transduction,gene regulation,cell cycle,apoptosis
Most common sites of phosphorylation,Ser,Thr,Tyr
MS can be used to detect and map
locations for phosphorylation
– MW increase from addition of
phosphate group
– treatment with phosphatase allows
determination of number of
phosphate groups
– digestion and tandem MS allows for
determination of phosphorylation
sites
10/27/2005 Chaoqun Wu,Fudan University 90
MS/MS and Phosphorylation
Detection of phosphopeptides in complex mixtures can
be facilitated by neutral loss and precurson ion
scanning using tandem mass spectrometers
Allow selective visualization of peptides containing
phosphorylated residues
Most commonly performed with triple quadrupole mass
spectrometers
precursor ion
transmission
collision
cell
(chamber)
mass
analysis of
product ions
10/27/2005 Chaoqun Wu,Fudan University 91
MS/MS and Phosphorylation
Precursor ion scan
– Q1 is set to allow all the components of the mixture to enter
the collision cell and undergo CAD
– Q3 is fixed at a specific mass value,so that only analytes
which fragment to give a fragment ion of this specific mass
will be detected
– Phospho-peptide fragments by CAD to give an ion at m/z 79
(PO
3
)
– Set Q3 to m/z 79,only species which fragment to give a
fragment ion of 79 reach the detector and hence indicating
phosphorylation
Q1 Q2
collision cell
Q3
detector
10/27/2005 Chaoqun Wu,Fudan University 92
MS/MS and Phosphorylation
Neutral loss scan
– Q1 and Q3 are scanned synchronously but with a specific
m/z offset
– The entire mixture is allowed to enter the collision cell,but
only those species which fragment to yield a fragment
with the same mass as the offset will be observed at the
detector
– pSer and pThr peptides readily lose phosphoric acid
during CAD (98 Da)
– For 2+ ion set offset at 49
Any species which loses 49 from a doubly charged ion
would be observed at the detector and be indicative of
phosphorylation
10/27/2005 Chaoqun Wu,Fudan University 93
Enrichment strategies to analyze
phosphoproteins/peptides
Phosphospecific antibodies
– Anti-pY quite successful
– Anti-pS and anti-pT not as successful,but may be used (M,
Gr?nborg,T,Z,Kristiansen,A,Stensballe,J,S,Andersen,O,
Ohara,M,Mann,O,N,Jensen,and A,Pandey,“Approach for
Identification of Serine/Threonine-phosphorylated Proteins by
Enrichment with Phospho-specific Antibodies.” Mol,Cell,
Proteomics 2002,1:517–527.
Immobilized metal affinity chromatography (IMAC)
– Negatively charged phosphate groups bind to postively
charged metal ions (e.g.,Fe
3+
,Ga
3+
) immobilized to a
chromatographic support
– Limitation,non-specific binding to acidic side chains (D,E)
Derivatize all peptides by methyl esterification to reduce non-
specific binding by carboxylate groups.
Ficarro et al.,Nature Biotech,(2002),20,301.
10/27/2005 Chaoqun Wu,Fudan University 94
Direct MS of phosphopeptides
bound to IMAC beads
Raska et al.,Anal,Chem,
2002,74,3429
IMAC beads placed directly on
MALDI target
Matrix solution spotted onto
target
MALDI-MS of peptides bound
to IMAC bead
MALDI-MS/MS (*) to identify
phosphorylation site(s)
10/27/2005 Chaoqun Wu,Fudan University 95
MALDI-MS
spectrum obtained
from peptide bound
to IMAC beads
applied directly to
MALDI target
MALDI-MS/MS (Q-
TOF) to locate
phosphorylation site
Sample enrichment
with minimal sample
handling
contains
phosphorylated
residue
10/27/2005 Chaoqun Wu,Fudan University 96
Enrichment strategies to analyze
phosphoproteins/peptides
Chemical derivatization
– Introduce affinity tag to enrich for
phosphorylated molecules
e.g.,biotin binding to immobilized
avidin/streptavidin
10/27/2005 Chaoqun Wu,Fudan University 97
Enrichment strategies to analyze
phosphoproteins/peptides
Oda et al.,Nature Biotech,2001,19,379 for analysis of
pS and pT
Remove Cys-reactivity by oxidation with performic acid
Base hydrolysis induce?-elimination of phosphate from
pS/pT
Addition of ethanedithiol allows coupling to biotin
Avidin affinity chromatography to purify phosphoproteins
10/27/2005 Chaoqun Wu,Fudan University 98
Enrichment strategies to analyze
phosphoproteins/peptides
Zhou et al.,Nature Biotech,2001,19,375
Reduce and alkylate Cys-residues to eliminate their reactivity
Protect amino groups with t-butyl-dicarbonate (tBoc)
Phosphoramidate adducts at
phosphorylated residues are
formed by carbodiimide
condensation with cystamine
Free sulfhydryls are covalently
captured onto glass beads
coupled to iodoacetic acid
Elute with trifluoroacetic acid
10/27/2005 Chaoqun Wu,Fudan University 99
Chemical derivatization to
enrich for phosphoproteins
Developed because other
methods based on
affinity/adsorption (e.g.,IMAC)
displayed some non-specific
binding
Chemical derivatization
methods may be overly
complex to be used routinely
Sensitivity may not be sufficient
for some experiments (low pmol)
10/27/2005 Chaoqun Wu,Fudan University 100
Phosphoprotein Stain
PeppermintStick phosphoprotein
molecular weight standards
separated on a 13% SDS
polyacrylamide gel,The markers
contain (from largest to smallest)
beta-galactosidase,bovine serum
albumin (BSA),ovalbumin,beta-
casein,avidin and lysozyme,
Ovalbumin and beta-casein are
phosphorylated,The gel was
stained with Pro-Q Diamond
phosphoprotein gel stain (blue)
followed by SYPRO Ruby protein
gel stain (red),The digital images
were pseudocolored.
Phospho
10/27/2005 Chaoqun Wu,Fudan University 101
Phosphoprotein Stain
Visualization of total protein and
phosphoproteins in a 2-D gel
Proteins from a Jurkat T-cell
lymphoma line cell lysate were
separated by 2-D gel electrophoresis
and stained with Pro-Q Diamond
phosphoprotein gel stain (blue)
followed by SYPRO Ruby protein gel
stain (red),After each dye staining,
the gel was imaged and the resulting
composite image was digitally
pseudocolored and overlaid.
T.H,Steinberg et al.,Global quantitative
phosphoprotein analysis using Multiplexed Proteomics
technology,Proteomics 2003,3,1128-1144
10/27/2005 Chaoqun Wu,Fudan University 102
Global Analysis of Protein Phosphorylation
RAW 264.7 exposed to DEP
Sypro Ruby
IEF
9.53.54.5 5.1 5.5 6.0 7.0 8.4
Pro-Q Diamond
9.53.54.5 5.1 5.5 6.0 7.0 8.4
TNFα convertase
MAGUK p55
PDI
Protein phosphatase 2A
JNK-1
p38 MAPK alpha
ERK-1
ERK-2
ErbB-2
TNF
HSP 27
5
3
4
12
6 7
20
30
37
98
55
8
9
10
11
12
13
14
98
55
37
30
20
Xiao,Loo,and Nel - UCLA
10/27/2005 Chaoqun Wu,Fudan University 103
Proteomics in Practice,A Laboratory Manual of
Proteome Analysis
Reiner Westermeier,Tom Naven
Wiley-VCH,2002
PART II,COURSE MANUAL
Step 1,Sample Preparation
Step 2,Isoelectric Focusing
Step 3,SDS Polyacrylamide Gel Electrophoresis
Step 4,Staining of the Gels
Step 5,Scanning of Gels and Image Analysis
Step 6,2D DIGE
Step 7,Spot Excision
Step 8,Sample Destaining
Step 9,In-gel Digestion
Step 10,Microscale Purification
Step 11,Chemical Derivatisation of the Peptide Digest
Step 12,MS Analysis
Step 13,Calibration of the MALDI-ToF MS
Step 14,Preparing for a Database Search
Step 15,PMF Database Search Unsuccessful
PART I,PROTEOMICS
TECHNOLOGY
Introduction
Expression Proteomics
Two-dimensional Electrophoresis
Spot Handling
Mass Spectrometry
Protein Identification by Database
Searching
Methods of Proteomics
10/27/2005 Chaoqun Wu,Fudan University 104
Proteins and Proteomics:
A Laboratory Manual
Richard J,Simpson
Cold Spring Harbor Laboratory (2002)
Chapter 1,Introduction to Proteomics
Chapter 2,One–dimensional Polyacrylamide Gel Electrophoresis
Chapter 3,Preparing Cellular and Subcellular Extracts
Chapter 4,Preparative Two–dimensional Gel Electrophoresis with
Immobilized pH Gradients
Chapter 5,Reversed–phase High–performance Liquid Chromatography
Chapter 6,Amino– and Carboxy– terminal Sequence Analysis
Chapter 7,Peptide Mapping and Sequence Analysis of Gel–resolved Proteins
Chapter 8,The Use of Mass Spectrometry in Proteomics
Chapter 9,Proteomic Methods for Phosphorylation Site Mapping
Chapter 10,Characterization of Protein Complexes
Chapter 11,Making Sense of Proteomics,Using Bioinformatics to Discover a
Protein’s Structure,Functions,and Interactions
10/27/2005 Chaoqun Wu,Fudan University 105
Part V.
Protease
Degradomics
10/27/2005 Chaoqun Wu,Fudan University 106
Degradomics — the application of genomic
and proteomic approaches to identify the
protease and protease-substrate repertoires,
or ‘degradomes’,on an organism-wide scale —
promises to uncover new roles for proteases
in vivo,
10/27/2005 Chaoqun Wu,Fudan University 107
Proteolysis
Many cellular processes,including
embryogenesis,gene expression,cell
cycle,programmed cell death,intracellular
protein targeting and endocrine/neural
functions are regulated by limited
proteolysis of precursor proteins,These
functions are carried out by proteolytic
enzyme families that are strategically
localized within cells or on cell surfaces
10/27/2005 Chaoqun Wu,Fudan University 108
Proteolytic enzyme families
Current numbers of proteases in humans and model species
Catalytic class of protease
Species Total* Aspartic Cysteine Metallo Serine Threonine
Homo sapiens 461 18 121 159 140 23
C elegans 353 26 93 151 62 21
Drosophila 513 38 59 157 225 34
Mus musculus 383 11 93 120 137 22
Rattus 227 10 41 77 80 19
*The total numbers are accurate as of the date of release of the most recent
MEROPS database v 5.7 (17 December 2001),but will
continue to grow as new proteases are discovered and characterized in the existing
databases and in new sequence deposits.
天(门)冬氨酸,半胱氨酸,金属蛋白,丝氨酸,色氨酸
10/27/2005 Chaoqun Wu,Fudan University 109
Genomics and proteomics,the parents of degradomics
Degradome and degradomics are two terms that have been adapted from the
fields of genomics and proteomics,and these terms are defined below together
with other 'omics' terms that appear in this Review.
Genome,The entire collection of genes in the complete DNA sequence of an
organism.
Transcriptome,The complete set of mRNAs that are transcribed from the
genome.
Proteome,The expressed set of proteins that are encoded by the genome.
Genomics,Investigations and techniques for identifying the genome.
Proteomics,Investigations and techniques for identifying the proteome.
Degradomics,All genomic and proteomic investigations and techniques
regarding the genetic,structural and functional identification and
characterization of proteases,and their substrates and inhibitors,that are
present in an organism.
Degradomes,The complete set of proteases that are expressed at a specific
time by a cell,tissue or organism,The degradome of a protease is its substrate
repertoire.
10/27/2005 Chaoqun Wu,Fudan University 110
Relationship of degradomics to the fields of proteomics and
genomics,and of the degradome to the proteome and genome.
10/27/2005 Chaoqun Wu,Fudan University 111
Protease
An enzyme that cleaves proteins by the
catalysis of peptide-bond hydrolysis,On the
basis of their catalytic mechanism,proteases
belong to one of five classes (aspartic,
cysteine,metallo,serine,or threonine).
Proteolytic processing
Proteolysis that is distinct from degradation in
that it represents highly specific and limited
substrate cleavage,which results in a specific
change of protein function.
10/27/2005 Chaoqun Wu,Fudan University 112
Genomics and proteomics,
the parents of degradomics
Degradomics,All genomic and proteomic
investigations and techniques regarding the
genetic,structural and functional identification and
characterization of proteases,and
their substrates and inhibitors,that are present in
an organism.
Degradomes,The complete set of proteases that
are expressed at a specific time by a cell,tissue or
organism,The degradome of a protease is its
substrate repertoire.
10/27/2005 Chaoqun Wu,Fudan University 113
Proteases initiate,modulate and terminate
many important cellular functions by highly
specific and limited substrate cleavage,This
mechanism,called proteolytic processing,
allows the precise cellular control of several
biological processes,including DNA
replication,cell-cycle progression,cell
proliferation,wound healing,immunity,
angiogenesis and apoptosis.
10/27/2005 Chaoqun Wu,Fudan University 114
10/27/2005 Chaoqun Wu,Fudan University 115
Degradomics approches,activity profiling
This knowledge will facilitate the identification of new
pharmaceutical targets to treat disease,
10/27/2005 Chaoqun Wu,Fudan University 116
The recent identification of many bioactive
molecules — including cytokines,cell-
adhesion molecules and receptors — and
intracellular targets (such as transcription
factors and kinases) as new protease
substrates that are precisely processed by
proteolytic activity is redefining,
10/27/2005 Chaoqun Wu,Fudan University 117
The hierarchical importance of proteases in a system
is influenced by specific activity of the protease,
redundancy,expression levels,temporal–spatial
distribution,zymogen activation,protease turnover
and inhibition properties.
Organism-wide degradomics approaches are
therefore required to identify the members of the
~500 protease human degradome that are expressed
by a cell or tissue in disease,and to determine the
complete natural substrate repertoire — the so-called
"substrate degradome" — for each protease.
10/27/2005 Chaoqun Wu,Fudan University 118
H
+
Lysosome
ATP ADP + P
i
Vacuolar ATPase
low
internal
pH
Lumen
contains
hydrolytic
enzymes,
Lysosomes contain
a large variety of
hydrolytic enzymes
that degrade proteins
& other substances
taken in by
endocytosis.
plasma membrane may
be processed first in an
endosomal compartment
and then delivered into the lumen of a lysosome by fusion of
a transport vesicle.
Solute transporters embedded in the lysosomal membrane
catalyze exit of products of lysosomal digestion (e.g.,amino
acids,sugars,cholesterol) to the cytosol,
10/27/2005 Chaoqun Wu,Fudan University 119
“The Nobel Prize in Chemistry 2004 for the discovery
of ubiquitin-mediated protein degradation"
b,1926b,1937
(in Karcag,Hungary)
b,1947
University of California
Irvine,CA,USA
Technion – Israel
Institute of Technology
Haifa,Israel
Technion – Israel
Institute of
Technology
Haifa,Israel
USA Israel Israel
1/3 of the prize 1/3 of the prize 1/3 of the prize
Irwin RoseAvram HershkoAaron Ciechanover
10/27/2005 Chaoqun Wu,Fudan University 120
Ubiquitin
76 Amino Acid polypeptide
Highly conserved in evolution
3 Amino acid differences between
yeast and human homologues
C-Terminal Gly residue is activated
via an ATP to form a thiol ester(硫硫羟酸酯羟酸酯
)
H
3
N
+
CCOO
CH
2
CH
2
CH
2
CH
2
NH
3
H
+
lysine
isopeptide bond(异异肽键肽键) between C-
terminal Gly of Ub
and εε-amino group
of Lys on a target
protein
Most cell proteins are
degraded via the ubiquitin/
proteasome pathway.
10/27/2005 Chaoqun Wu,Fudan University 121
Ubiquitin–Mediated Proteolysis in
Cellular Processes
Regulation of:
Cell cycle
Differentiation & development
Extracellular effectors
Cell surface receptors & ion channels
DNA repair
Immune and inflammatory responses
Biogenesis organelles
10/27/2005 Chaoqun Wu,Fudan University 122
Ubiquitination
Ub
E1
Ub
E2
Ub
E3
Ub
26s proteosome degradation
Target
Ub
Ub
Ub
In general,multiple ubiquitin units are arranged in
polyubiquitin chains linked via Lys
48
of ubiquitin,
targeting the protein for degradation
10/27/2005 Chaoqun Wu,Fudan University 123
Degradation of proteins is a specific
energy dependent process via
polyubiquitination and 26S proteasome
degradation,
Know the function and general
biochemistry of E
1
,E
2
and E
3
enzymes.
Functional description of the proteasome.
Functional differences between mono and
polyubiquitination.
Functional differences between
sumoylation and ubiquitination.
10/27/2005 Chaoqun Wu,Fudan University 124
ATP Driven Activation is a
Common Theme in Biochemistry
Ub-E1(poly-Ub/protein degradation)
aa-tRNA (proteins)
Acetyl-CoA (FA)
UDP-glucose (glycogen)
Dolichol-GlcNAc (N-Linked glycoproteins)
10/27/2005 Chaoqun Wu,Fudan University 125
Proteins Targeted by Ubiquitin
Cell cycle regulators
Tumor suppressors & growth modulators
Transcriptional activators & inhibitors
Cell surface receptors
Mutant and damaged proteins
10/27/2005 Chaoqun Wu,Fudan University 126
Degradation of a Protein Via the
Ubiquitin-Proteasome Involves Two
Successive Steps
Covalent attachment of multiple ubiquitin
molecules to a protein substrate.
Degradation of the tagged protein by the
26s proteasome.
(ubiquitin is recycled)
10/27/2005 Chaoqun Wu,Fudan University 127
Ubiquitin Conjugation:
A 3-Step Mechanism
Ub forms isopeptide bond
between C-terminal Gly of Ub
and ε
ε
-amino group of Lys
on a target protein
Ubiquitin ligase E
3
Ub is transferred to a Cys of
E
2
forming a new thiol ester
Ubiquitin conjugating
enzymes E
2
High energy thiol ester is
formed between C-terminal
Gly of ubiqutin and a Cys in
the E
1
active site (ATP/AMP)
ATP-dependent
ubiquitin (Ub)
activating enzyme E
1
10/27/2005 Chaoqun Wu,Fudan University 128
E
1
,E
2
,& E
3
E,ubiquitin-activating enzyme，泛素激活酶
With each step increasing level of regulatory
specificity:
E1,only one (?)
E2,about 30 (homologous family)
E3,100 and structurally unrelated
(ultimate biological specificity)
10/27/2005 Chaoqun Wu,Fudan University 129
Proteasome Complexes
11S (PA28) Activator of 20S Proteasome
(human)
20S Proteasome (human)
20S Proteasome (S,cerevisiae)
26S Proteasome (human)
About 30000 proteasomes in human.
10/27/2005 Chaoqun Wu,Fudan University 130
The ubiquitin/
proteasome
pathway
The substrate
proteins are
polyubiquitinated
through the ubiquitin
pathway following
degradation by the
26S proteasome.
JARQ 39 (1),1 – 4 (2005)
10/27/2005 Chaoqun Wu,Fudan University 131
Cell cycle controls depending on ubiquitins
The ubiquitin-
proteasome system
controls not only the
master regulatory
Cdk activities,but
also the execution of
many cell-cycle
events downstream
from Cdk regulatory
circuit,
For example,
Cyc (cyclin) A,
CycB,CycD,and
B-type CDK are
degraded by 26S
proteasome in a
cell cycle-
dependent
manner.
10/27/2005 Chaoqun Wu,Fudan University 132
Degradation of cell-cycle regulatory proteins
Various cell-cycle regulatory proteins are degraded through
the ubiquitin/proteasome pathway at specific cell-cycle stages
in plant cells.
10/27/2005 Chaoqun Wu,Fudan University 133
Cell division is propelled by the oscillation of
cyclin-dependent kinase (Cdk) activities,which in
turn are regulated by the periodic synthesis and
degradation of their regulatory subunits,cyclins,
In addition to cyclins,other key regulators,such
as p27,p53,anaphase inhibitor,Cdc20,polo-like
kinase,aurora kinase,Nek2 kinase and kinesin-
related motor proteins,are all regulated by
ubiquitin-dependent proteolytic pathways in the
cell cycle,
10/27/2005 Chaoqun Wu,Fudan University 134
Phosphorylation of
I
Κ
Βα leads to
ubiquitination and
degradation of the
inhibitor and
activation of the
transcription factor.
NFκB pathway
10/27/2005 Chaoqun Wu,Fudan University 135
ATP consuming process
10/27/2005 Chaoqun Wu,Fudan University 136
Modification of Protein Substrates by Ub/UBLs
E,ubiquitin-activating enzyme，泛素激活酶
10/27/2005 Chaoqun Wu,Fudan University 137
Ubiquitin (Ub) and ubiquitin-like molecules (UBLs)
are expressed as precursors,either in form of
concatemers(连接) or C-terminal extensions,A
subset of Ub/UBL-specific proteases cleaves
precursor polypeptides to obtain the free Ub/UBL
monomers (recognition and proteolysis of a
peptide bond),Conjugation of Ub and UBLs to
target polypeptides is achieved by several
enzymatic steps via a cascade involving E1,E2,
and E3 enzymes,Conjugated protein substrates
are then subjected to deconjugation by Ub/UBL-
specific proteases that specifically recognize and
cleave isopeptide bonds,Individual proteases may
recognize a specific subset of conjugated
substrates.
10/27/2005 Chaoqun Wu,Fudan University 138
Mono-Ub,Another Function
Histone regulation
Endocytosis
Budding of retroviruses from plasma
membrane
10/27/2005 Chaoqun Wu,Fudan University 139
Ubiquitin like Molecule
like Molecule (UBLs)
Small Ubiquitin Related Modifier (SUMO)
SUMO does not have the Lys-48 found in
ubiquitin
SUMO does not make multi-chain forms
SUMO-1,2,3 are the mammalian forms
SUMO-1,101 amino acids,C-terminal
Gly,18% identical to ubiquitin.
10/27/2005 Chaoqun Wu,Fudan University 140
SUMO
10/27/2005 Chaoqun Wu,Fudan University 141
SUMO Enzymes
SUMO-activating enzyme,heterodimer
SUMO-conjugating enzyme,Ubc9,an E
2
enzyme that can’t be ubiquitinated
SUMO-ligase,E3 enzyme that is specific
for sumoylation
E3-like proteins increase affinity between
SUMO-Ubc9 and target protein
10/27/2005 Chaoqun Wu,Fudan University 142
Sumoylation and Desumoylation Cycle
with E1-,E2-,E3-like Enzymes
10/27/2005 Chaoqun Wu,Fudan University 143
Biological Roles of SUMO
Modification
Antagonistic role against ubiquitin,IκBα is
sumoylated preventing ubiquitination and
degradation (NF-κB pathway).
Protein translocation,Ran-GAP1/RanBp2
Modulation of transcriptional activity,activates
transcriptional activity of p53.
Subnuclear structure formation,protein targeting
to nuclear bodies,PML,tumor suppressor,
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Proteins Modified BY SUMO
Polarized cell growth and
cytokinesis
Septins
Mutations causing premature
aging
Werner’s syndrome protein
Regulates p53Mdm2
Tumor suppressorp53
Inflammatory responseIκBα
Affected by translocations
causing leukemias
PML
Nuclear transportRanGAP1
FunctionProtein modified by SUMO
Proteomic
复旦大学生命科学学院吴超群
10/27/2005 Chaoqun Wu,Fudan University 2
Defining Proteomics
Branch of discovery science focusing on proteins
In 1994 defined as,the complete set of proteins that is
expressed and modified following expression by the
entire genome in the lifetime of a cell”,If we look at an
organism it means that we are looking at the proteome of
3 trillion of cells and ~1000 different cell types with
different protein profiles.
Can be more specific such as the complement of
proteins expressed by a cell at any one time.
Today proteomics is a scientific discipline that will bridge
the gap between our understanding of genome
sequences and cellular behavior,
10/27/2005 Chaoqun Wu,Fudan University 3
Genomics and Proteomics a new
field with a new vocabulary
-Omics,
means area
of research
DNA
RNA
proteins
Metabolites
Protein-protein,Protein-DNA,
Protein-RNA interactions
Genome
Transcriptome
Proteome
Metabolome
Interactome
10/27/2005 Chaoqun Wu,Fudan University 4
The,omics” nomenclature…
Genomics
DNA (Gene)
Functional
Genomics
Transcriptomics RNA
Proteomics
PROTEIN
Metabolomics METABOLITE
Transcription
Translation
Enzymatic
reaction
10/27/2005 Chaoqun Wu,Fudan University 5
Why study protein expression?
Cytosol
Nucleus
Inactive mRNA
RNA
Degradation
control
RNA
Transport
control
Translation control
Post-translational
control
Primary
RNA
transcript
Transcriptional
control
RNA
Processing
control
mRNA
mRNA
DNA
Modified
protein
protein
10/27/2005 Chaoqun Wu,Fudan University 6
The proteome
– Protein complement of a genome
Variable
– In different cell and tissue types in same organism
– In different growth and developmental stages of
organism
Dynamic
– Depends on response of genome to environmental
factors
Disease state
Drug challenge
Growth conditions
Stress
10/27/2005 Chaoqun Wu,Fudan University 7
10/27/2005 Chaoqun Wu,Fudan University 8
Content
I,Introduction
II,Proteomics and Genomics
III,Identification and Quantification
of Proteins
IV,Structural Proteomics
V,Identification of Protein Post-
translational Modifications
VI,Protease degradomics:
VII,Platforms for proteomics
VIII,Mass Spectrometry
IX,Post-MS Analysis
X,Bioinformatics for Proteomics
10/27/2005 Chaoqun Wu,Fudan University 9
Part I.
Introduction
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二十世纪中期以来，随着DNA双螺旋结构的提出和蛋白质空间结构的
X射线解析，开始了分子生物学时代，
对遗传信息载体DNA和生命功能的主要体现者蛋白质的研究，成为生命科学研究的主要内容。
二十世纪中期以来，随着双螺旋结构的提出和蛋白质空间结构的射线解析，开始了分子生物学时代，
对遗传信息载体和生命功能的主要体现者蛋白质的研究，成为生命科学研究的主要内容。
10/27/2005 Chaoqun Wu,Fudan University 11
人类蛋白质组研究对人类蛋白质组研究主要聚焦在特异的组织、
细胞和疾病上。人的各种组织、器官、细胞乃至各种细胞器已被广泛研究。人的各种体液（血液、淋巴、脊髓、乳汁、尿液等）都被用于研究与某些疾病的关系。许多科学家致力于各种肿瘤组织与正常组织之间蛋白质谱差异的研究。这些研究已经找到了一些肿瘤特异性的标志物。例如，在对肾癌的研究中发现有四种蛋白质存在于正常肾组织而在肾癌细胞中消失。其中两种分别是辅酶Q蛋白色素还原酶和线粒体泛醌氧化—还原复合物I，这提示线粒体功能低下可能与肿瘤的发生有关。
人类蛋白质组研究对人类蛋白质组研究主要聚焦在特异的组织、
细胞和疾病上。人的各种组织、器官、细胞乃至各种细胞器已被广泛研究。人的各种体液（血液、淋巴、脊髓、乳汁、尿液等）都被用于研究与某些疾病的关系。许多科学家致力于各种肿瘤组织与正常组织之间蛋白质谱差异的研究。这些研究已经找到了一些肿瘤特异性的标志物。例如，在对肾癌的研究中发现有四种蛋白质存在于正常肾组织而在肾癌细胞中消失。其中两种分别是辅酶蛋白色素还原酶和线粒体泛醌氧化还原复合物，这提示线粒体功能低下可能与肿瘤的发生有关。
10/27/2005 Chaoqun Wu,Fudan University 12
蛋白质组（proteome）由澳大利亚学者Wilkins
和Williams等于1994年提出，指的是由基因组编码的全部蛋白质，即某一物种、个体、器官、组织乃至细胞的全部蛋白质。与以往的蛋白质化学的研究不同，
蛋白质组研究的对象不是单一或少数的蛋白质，它着重的是全面性和整体性，需要获得体系内所有蛋白质组分的物理、化学及生物学参数，如分子量、等电点、
表达量等。它是动态的，有它的时间性、可调节性；
进而能够在细胞和生命有机体的整体水平上阐明生命现象的本质和活动规律。
蛋白质组（）由澳大利亚学者和等于年提出，指的是由基因组编码的全部蛋白质，即某一物种、个体、器官、组织乃至细胞的全部蛋白质。与以往的蛋白质化学的研究不同，
蛋白质组研究的对象不是单一或少数的蛋白质，它着重的是全面性和整体性，需要获得体系内所有蛋白质组分的物理、化学及生物学参数，如分子量、等电点、
表达量等。它是动态的，有它的时间性、可调节性；
进而能够在细胞和生命有机体的整体水平上阐明生命现象的本质和活动规律。
10/27/2005 Chaoqun Wu,Fudan University 13
蛋白质组研究的核心是系统识别一个细胞或组织中表达的每一个蛋白质,以及确定每个蛋白质的突出特征。其分析技术包括分离蛋白质和肽的分离科学、识别和定量分析物的分析科学和数据管理及分析的生物信息学。
?Global Proteomics – Identify and catalog all
possible proteins in an organism.
?Targeted Proteomics – Identify and catalog a
subset of proteins involved in a specific pathway,
disease,or other specific facet you wish to study.
?Structural proteomics – Determine the 3-D
structure of all proteins.
10/27/2005 Chaoqun Wu,Fudan University 14
随着大规模基因组测序计划的完成，人类又面临一个新的、更加艰辛的科学挑战—
蛋白质组学的研究.
蛋白质组学的研究是一项系统性的多方位的科学探索。其研究内容包括：
蛋白质结构、蛋白质分布、
蛋白质功能、蛋白质的丰度变化、
蛋白质修饰、
蛋白质与蛋白质的相互作用、
蛋白质与疾病的关联性。
10/27/2005 Chaoqun Wu,Fudan University 15
Patterson and
Aebersold,Nature
Genetics (supp.),
33,311 (2003)
protein-ligand
interactions
protein
complexes
(machines)
protein families
(activity or structural)
post-translational
modified proteins
Eukaryotic cell.
Examples of protein
properties are shown,
including the interaction of
proteins and protein
modifications.
10/27/2005 Chaoqun Wu,Fudan University 16
What is Proteomics?
Defined as,the analysis of the entire protein
complement in a given cell,tissue,or
organism.”
Proteomics,also assesses activities,
modifications,localization,and interactions
of proteins in complexes.”
Proteomes of organisms share intrinsic
differences across species and growth
conditions.
10/27/2005 Chaoqun Wu,Fudan University 17
The Role of Proteomics
? The existence of an ORF does not imply the
existence of a functional gene.
? Limitations of comparative genomics.
? mRNA levels may not correlate with protein
levels.
? Protein modifications ? post-transcriptional
modifications,isoforms,post-translational
modifications,mutants.
? Issues of proteolysis,sequestration,etc,
relevant only at the protein level.
? Protein complex composition,protein-protein
interactions,structures,
10/27/2005 Chaoqun Wu,Fudan University 18
Proteins classes for Analysis
Membrane
Soluble proteins
Nuclear
Chromosome-associated
Phosphorylated
Glycosylated
Complexes
10/27/2005 Chaoqun Wu,Fudan University 19
Proteomics
A functional understanding of the cell
Definition:
“A proteome is the entire protein
complement expressed by a given
biological system in given
conditions,
Wilkins et al.,BioTechnology 14,61-65 (1996)
10/27/2005 Chaoqun Wu,Fudan University 20
Expression proteomics:
up and down-regulation of protein level,
e.g,normal and tumor cell,rest and drug-
induced cell,mature and inmature cell,
malignant,metastatic cancer cells etc.
Functional proteomics:
characterization of cellular compartments,
multiprotein complexes,protein interaction
net and signaling pathway,etc.
Classification
10/27/2005 Chaoqun Wu,Fudan University 21
Clinical Proteomics
This area of proteomics focuses on accelerating
drug development for diseases through the
systematic identification of potential drug targets.
How could this be accomplished?
Hopefully,we will have more specific information,
instead of raw genes,that will make those complex
differential equations much simpler in the coming
years.
10/27/2005 Chaoqun Wu,Fudan University 22
Applications of Proteomics
Mining,identification of proteins (catalog
the proteins)
Protein-expression profile,identification of
proteins in a particular state of the
organism
Protein-network mapping,protein
interactions in living systems
Mapping of protein modifications,how and
where proteins are modified.
10/27/2005 Chaoqun Wu,Fudan University 23
Proteomics and Systems Biology
“The identification,characterization and
quantification of all proteins involved in a particular
pathway,organelle,cell,tissue,organ or organism
that can be studied in concert to provide accurate
and comprehensive data about that system.”
Systems Biology
http://www.inproteomics.com/prodef.html
10/27/2005 Chaoqun Wu,Fudan University 24
Genomics
Functional
genomics
global Targeted
Transcriptomics
Proteomics
global Targeted
Targetedglobal
Metabolomics
Targetedglobal
System Biology
Interactomics
10/27/2005 Chaoqun Wu,Fudan University 25
“Systems biology is an approach to
studying complex biological systems made
possible through technological
breakthroughs such as the human genome
project,…systems biology simultaneously
studies the complex interaction of many
levels of biological information to
understand how they work together.”
http://www.systems biology.org/
10/27/2005 Chaoqun Wu,Fudan University 26
Epigenetics,Factors that affact
the proteome of a cell
10/27/2005 Chaoqun Wu,Fudan University 27
Levels of genome research in eukaryotes
10/27/2005 Chaoqun Wu,Fudan University 28
mRNA
primary protein
product
protein
degradation
mature?
protein
Active
protein
Transcriptome
primary RNA
transcript
DNA
Genome
Why studying proteomes?
Proteome
Proteomic is a functional approach
10/27/2005 Chaoqun Wu,Fudan University 29
Importance of Proteins:
they serve as catalysts that maintain metabolic
processes in the cell,
they serve as structural elements both within and
outside the cell,
they are signals secreted by one cell or deposited
in the extracellular matrix that are recognized by
other cells,
they are receptors that convey information about
the extracellular milieu to the cell,
they serve as intracellular signaling components
that mediate the effects of receptors,
10/27/2005 Chaoqun Wu,Fudan University 30
Importance of Proteins:
? they are key components of the machinery that
determines which genes are expressed and
whether mRNAs are translated into proteins,
? they are involved in manipulation of DNA and
RNA through processes such as,DNA replication,
DNA recombination,RNA splicing or editing.
http://www-users.med.cornell.edu/~jawagne/proteins_&_purification.html
10/27/2005 Chaoqun Wu,Fudan University 31
Central lesson from
eukaryotic genome projects
Evolutionary complexity is not primarily determined
by increasing the number of genes,but by
increasing variation on the level of the synthesized
proteins.
This is achieved by generating MULTIPLE proteins
from only ONE gene,e.g,by
– different combinations of exons by alternative splicing
– post-translational protein processing (e.g,cleavage of
pro-peptides)
– post-translational protein modifications (e.g,acetylation,
glycosylation)
– modified central dogma,DNA --> RNA --> protein(s)
– it is important to perform analyses on the level of gene
PRODUCTS Key
10/27/2005 Chaoqun Wu,Fudan University 32
But what about the Genome?
What does having the genome of an organism
give us?
– A great diagram,or,blueprint,” of the genes
within an organism,
– Think of the genome as code that needs
compiled into functional units.
– The genome gets,compiled” into the proteome
via the central dogma of biology.
– Proteomic strategies attempt to utilize information
from the genome in an attempt to conceptualize
protein function.
10/27/2005 Chaoqun Wu,Fudan University 33
The proteomic process,
Strong / reliable models
- Organism,organ,tissue,cell,
cellular organal,complex.
Protein separation,
- One or two-dimensional gel
electrophoresis
-
Mass spectrometry
Protein identification,
- Blotting with antibodies
- Amino Acid composition
- Edman degradation
- Mass spectrometry
10/27/2005 Chaoqun Wu,Fudan University 34
The proteomic features,
Key advantage of proteomics
Researchers work on the level of gene products and
deal with genes that are really expressed to give a
detectable PRODUCT and are not just,expressed”
which only says they produce a detectable mRNA but it
is not clear whether there is a gene product or not.
Key limitation of proteomics
Usually,only a fraction of the proteins synthesized can
be detected in a proteomics experiment,whereas the
expression of ALL genes can be monitored in a whole-
genome array experiment.
Key prerequisite of proteomics
A genome sequence for the investigated organism or at
least a collection of many cDNA sequences is required.
10/27/2005 Chaoqun Wu,Fudan University 35
Bottlenecks of Proteomics
Experimental:
Large-scale protein analysis is difficult
because,
Proteins are fragile
They can exist in multiple isoforms
There is no protein equivalent of PCR
for amplification of a small sample
10/27/2005 Chaoqun Wu,Fudan University 36
Bottlenecks of Proteomics
Data Analysis,
-Data contains a lot of noise that is difficult to
separate from actual signal,This results in
wastage of computing resources on
searching for unlikely spectra.
-Database searches for matching spectra
only give scores,leaving manual intervention
necessary for eliminating false positives
10/27/2005 Chaoqun Wu,Fudan University 37
Bottlenecks of Proteomics
Biomedical limitations
-In practice,it is very difficult to trace the
complete progression of a disease.
-Hence,using proteomics for monitoring
the biochemistry of a disease is like
using a photo camera to record a
football match.
10/27/2005 Chaoqun Wu,Fudan University 38
Part II.
Proteomics and
Genomics
10/27/2005 Chaoqun Wu,Fudan University 39
基因和蛋白质基因翻译蛋白质
27，000条>10万种
1,基因编码蛋白质，
2,一个基因可翻译几种蛋白质，
3,基因的功能通过翻译成蛋白质来实现，
4,基因的异常引起翻译成的蛋白质异常，
而影响蛋白质的功能。
10/27/2005 Chaoqun Wu,Fudan University 40
26,383
条人类基因的功能分类核苷酸酶受体转录调控因子原癌基因细胞骨架细胞黏附分子激酶选择调节分子转移酶氧化还原酶水解酶
10/27/2005 Chaoqun Wu,Fudan University 41
The Synergy of
Proteomics and Genomics
Proteomics provides data on the
outcome of gene expression,
Genomics provides the comprehensive
sequence and expression data required
to advance protein research.
Functional genomics includs Proteomics
and Genomics
10/27/2005 Chaoqun Wu,Fudan University 42
Functional Genomics
? Understanding Function of Genes and Other Parts of
Genome
? Study of Biological Systems Based on Global
Knowledge of Genomes,Transcriptomes & Proteomes
Genome,All the Genetic Material in the
Chromosomes
Transcriptome,Entire Set of Gene Transcripts
Proteome,Entire Sets of Proteins
Genome (DNA) Transcriptome (RNA) Proteome (Protein)
10/27/2005 Chaoqun Wu,Fudan University 43
Proteomics is the study of proteins,
whereas genomics is the study of
DNA and the processes which lead
to the creation of proteins,When
used in combination,these two
approaches to the study of gene
expression enable researchers to
analyze regulation at many levels.
10/27/2005 Chaoqun Wu,Fudan University 44
Proteomics,Tools From
Genome to Proteome
It sounds like mission impossible?
– Follow the changes taking place inside a cell by
identifying the thousands of different proteins the
cell produces and watching how they change
and flow over time.
– To a growing band of researchers,however,
such a mission is becoming more realistic,
thanks to the increasing volume of sequence
data and to improved analytical techniques for
proteins like MALDI-MS,etc..
10/27/2005 Chaoqun Wu,Fudan University 45
Predicting Life Processes,
Reverse Engineering Living Systems
Transcription
Translation
Proteins
Biochemical Circuitry
Phenotypes (Traits)
Metabolomics
(storage)
DNA
Gene Expression
Proteomics
Environment
10/27/2005 Chaoqun Wu,Fudan University 46
Example 1 - Signal Transduction
When a cell receives a signal,such as a
growth factor,its immediate response is at the
protein level,Cell surface protein receptors
are activated and modified,In addition,
transmission of information from the activated
receptor to the nucleus often involves physical
movement of proteins,These activities can be
detected and analyzed using proteomic
technologies,
growth factor,its immediate response is at the
10/27/2005 Chaoqun Wu,Fudan University 47
10/27/2005 Chaoqun Wu,Fudan University 48
Example 2 - A signalling network central
to tumour suppression.
Most disease processes manifest themselves
at the level of protein activity,but until recently,
high throughput analysis of proteins was not
possible,
The development of several technologies now
makes it feasible to perform mass screening
of proteins and is creating a revolution in
proteomics.
10/27/2005 Chaoqun Wu,Fudan University 49
10/27/2005 Chaoqun Wu,Fudan University 50
Part III.
Identification and
Quantification of
Proteins
10/27/2005 Chaoqun Wu,Fudan University 51
General
flow for
proteomics
analysis
SEPARATION
ID
ENTIF
I
CATION
10/27/2005 Chaoqun Wu,Fudan University 52
10/27/2005 Chaoqun Wu,Fudan University 53
Identification of gel separated
proteins prior to MS
MS analysis
Excision of the spots of interest
Reduction & Alkylation
In gel digestion
Extraction of the peptides
Purification & concentration of
the peptides on a
nano reverse phase column?
1) Peptide mapping
2) Peptide sequencing
10/27/2005 Chaoqun Wu,Fudan University 54
2D gels are covalently
bound to glass,
allowing long-term
storage of gels while
minimizing gel-to-gel
size variation.
10/27/2005 Chaoqun Wu,Fudan University 55
Reproducibility
Reproducibility is one of the key developments in proteomics
was the development of gels which deliver reproducible
results,In a reproducibility experiment:
The x-axis is the Isoelectric point (pI) which is
analagous to pH,while the y-axis is molecular weight
(Mw) or size,
10/27/2005 Chaoqun Wu,Fudan University 56
MS analyses
MS identifies analytes by:
?Producing gas phase ions
?Separating these ions according to m/z ratio
?Detecting the ions
Sensitive:
Detect attomole concentrations
10/27/2005 Chaoqun Wu,Fudan University 57
?Total extract
?Serum dopletion
?Fractionation
?High resolution
SDS-PAGE
?1D + 2D
?M-HPLC
?Post-seperation
fluorescence
?Differentially
expressed
proteins
?Robotic process
from gel to
peptide pool
?MALDI-TOF and
tandem MS
?Fractionation
Computational
analysis
(data treatment)
Current Proteome Analysis
Biological
sample
Sample
preparation
Protein
extract
Peptide
sequence
Separation Detection Selection
10/27/2005 Chaoqun Wu,Fudan University 58
Current
protocol
Almost no limitation
when the entire
genome is sequenced.
Limited to ~1000 proteins
10/27/2005 Chaoqun Wu,Fudan University 59
Differential 2D analysis
Comparison between different protein samples run on 2-D gels,Proteins
from either a normal liver tissue sample ora liver tumor sample were
run on two 2-D gels and stained,images of the gelswere captured
using the FLA-3000 scanner,Images from the two gels were then
pseudo-colored either pink or green,overlaid and matched spot-to-spot
using Z3 software(Compugen),Matched spot intensities that differ by
More than 20% are represented as green spots (for proteins expressed
higher in the liver tumor sample) or pink spots (for proteins expressed
higher in the normal liver tissue sample).Matched spots that differ by
less than 20.0% are black.
10/27/2005 Chaoqun Wu,Fudan University 60
LC 2D analysis
?Proteins Fractionated using 2D Chromatography
?Proteins resolved by both chromatofocusing and
reversed phase chromatography
?Proteins unique to diseased state - Isolated
*
*
*
*
10/27/2005 Chaoqun Wu,Fudan University 61
2D-DIGE (双向荧光差异凝胶电泳)
Differential in-gel electrophoresis (2D-DIGE)
2D-DIGE analysis,employing Cy3 or Cy5 dyes to
label two different protein populations,eliminates
inter-gel variation and facilitates quantitative
evaluation of differentially expressed proteins,
Populations of unique and differentially expressed
proteins from wild type and rin mutant tomato fruit
at different developmental stages were
trypsinized and analyzed by mass spectrometry,
The resulting data were used to query databases
in order to identify cognate genes/proteins,
10/27/2005 Chaoqun Wu,Fudan University 62
10/27/2005 Chaoqun Wu,Fudan University 63
Mass Spectrometry and
Quantitative Measurements
A B
m/z
Rel,Abund.
Q
H
E
Mass spectrometry is
inherently not a
quantitative technique,
The intensity of a peptide
ion signal does not
accurately reflect the
amount of peptide in the
sample.
equimolar mixture
of 2 peptides
516.725 516.828
m/z
(M+2H)
2+
,[
12
C]-ion
[Val
5
]-Angiotensin II
1031.5188 (monoisotopic)
Lys-des-Arg
9
-Bradykinin
1031.5552 (monoisotopic)
= 0.036
equimolar mixture
of 2 peptides
10/27/2005 Chaoqun Wu,Fudan University 64
Mass Spectrometry and
Quantitative Measurements
equimolar mixture
of 2 peptides
Q
H
E
A B
Rel,Abund.
Two peptides of identical chemical structure that differ in mass
because they differ in isotopic composition are expected to
generate identical specific signals in a mass spectrometer.
Q
H
E
13
C
13
C
13
C
A B
2
D
2
D
m/z
Methods coupling mass spectrometry and stable isotope tagging
have been developed for quantitative proteomics.
10/27/2005 Chaoqun Wu,Fudan University 65
ICAT,Isotope-Coded Affinity Tag
(同位素亲和标签同位素亲和标签
)
Alkylating (烷基化物烷基化物
)group covalently attaches the reagent to
reduces Cys(半胱氨酸半胱氨酸
)-residues.
A polyether mass-encoded linker contains 8 hydrogens (氢氢,
d0) or 8 deuteriums (氘氘
,d8) that represents the isotope
dilution.
A biotin affinity tag is used to selectively isolate tagged
peptides (by avidin purification).
10/27/2005 Chaoqun Wu,Fudan University 66
ICAT,Isotope-Coded Affinity Tag
MS/MS
identifies
the protein
The Cys-residues in sample 1 is labeled with d0-ICAT and sample 2 is
labeled with d8-ICAT.
The combined samples are digested,and the biotinylated ICAT-labeled
peptides are enriched by avidin affinity chromatography and analyzed by
LC-MS/MS.
Each Cys-peptide appears as a pair of signals differing by the mass
differential encoded in the tag,The ratio of the signal intensities indicates
the abundance ratio of the protein from which the peptide originates.
10/27/2005 Chaoqun Wu,Fudan University 67
Stable Isotope Amino Acid or
15
N-
in vivo Labeling
Metabolic stable isotope
coding of proteomes
An equivalent number of
cells from 2 distinct cultures
are grown on media
supplemented with either
normal amino acids or
14
N-
minimal media,or stable
isotope amino acids
(
2
D/
13
C/
15
N) or
15
N-enriched
media.
These mass tags are
incorporated into proteins
during translation.
10/27/2005 Chaoqun Wu,Fudan University 68
Enzymatic Stable Isotope
Coding of Proteomes
Enzymatic digestion in the presence of
18
O-water incorporates
18
O at the
carboxy-terminus of peptides
Proteins from 2 different samples are
enzymatically digested in normal
water or H
2
18
O.
R
3
R
4
NH
2
-CH-CO-NH-CH-COOH...NH-CH-CO-NH-CH-CO-
18
OH
R
1
R
2
...NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH
R
1
R
2
R
3
R
4
Trypsin /H
2
18
O
(Arg,Lys)
C-terminal peptide
10/27/2005 Chaoqun Wu,Fudan University 69
Identification of Low
Abundance Proteins
The identification of low
abundance proteins in the
presence of high abundance
proteins is problematic (e.g.,
“needle in a haystack”)
Pre-fractionation of complex
protein mixtures can alleviate
some difficulties
– gel electrophoresis,
– chromatography,etc
Removal of known high
abundance proteins allows less
abundant species to be visualized
and detected
10/27/2005 Chaoqun Wu,Fudan University 70
Identification of Low Abundance
Proteins
GenWay Biotech
10/27/2005 Chaoqun Wu,Fudan University 71
Protein-Protein Interactions
affinity support
(1) Affinity Purification
10/27/2005 Chaoqun Wu,Fudan University 72
(2) Phage Display
10/27/2005 Chaoqun Wu,Fudan University 73
(3) Yeast 2-hybrid
10/27/2005 Chaoqun Wu,Fudan University 74
(4) Protein Chips
10/27/2005 Chaoqun Wu,Fudan University 75
Part IV.
Identification of Protein
Post-translational
Modifications
10/27/2005 Chaoqun Wu,Fudan University 76
Proteomics and post-translational
modifications
Patterson and
Aebersold,Nature
Genetics (supp.),
33,311 (2003)
protein-ligand
interactions
protein-ligand
interactions
protein
complexes
(machines)
protein
complexes
(machines)
protein families
(activity or structural)
protein families
(activity or structural)
post-translational
modified proteins
post-translational
modified proteins
Eukaryotic cell.
Examples of protein
properties are shown,
including the interaction
of proteins and protein
modifications.
10/27/2005 Chaoqun Wu,Fudan University 77
Post translational modifications
in the ER
Removal of signal peptide
Addition of carbohydrates – glycosylation
Addition of GPI anchors
Folding
– Disulfide bond formation
Formation of multimeric proteins
Only properly modified proteins are transported
from ER to the final destination
10/27/2005 Chaoqun Wu,Fudan University 78
Proteomic analysis of PTMs
醌酰肉豆蔻酰棕榈酰
10/27/2005 Chaoqun Wu,Fudan University 79
( Be continued )
羟(基)脯氨酸脱酰胺焦谷氨酸糖基磷脂酰肌醇锚
Mann and Jensen,Nature Biotech,21,255 (2003)
10/27/2005 Chaoqun Wu,Fudan University 80
Proteomic Analysis of Post-
translational Modifications
Post-translational modifications (PTMs)
– Covalent processing events that change the
properties of a protein
proteolytic cleavage
addition of a modifying group to one or more amino
acids
– Determine its activity state,localization,turnover,
interactions with other proteins
– Mass spectrometry and other biophysical methods
can be used to determine and localize potential PTMs
However,PTMs are still challenging aspects of
proteomics with current methodologies
10/27/2005 Chaoqun Wu,Fudan University 81
Complexity of the Proteome
Protein processing and modification comprise an
important third dimension of information,beyond those of
DNA sequence and protein sequence,
Complexity of the human proteome is far beyond the
more than 30,000 human genes.
The thousands of component proteins of a cell and their
post-translational modifications may change with the cell
cycle,environmental conditions,developmental stage,
and metabolic state.
Proteomic approaches that advance beyond identifying
proteins to elucidating their post-translational
modifications are needed.
10/27/2005 Chaoqun Wu,Fudan University 82
Mass spectrometry,powerful tool for proteomics
Mass spectrometry measures the mass of proteins or peptides
from analysis of the masstocharge (m/z) ratio.
Mass spectrometers first ionize the sample,and then the ions
are introduced into the mass analyser,which separates and
detects the sample ions according to their mass,
Fragmentation of the protein sample results in a collection of
ions that have different masses,and the spectrometer
measures the relative abundance of each ion,according to
their m/z ratios,to obtain a spectrum of masses,hence the
term mass spectrometry.Proteomic analyses typically use
matrix-assisted laser desorptionionization (MALDI) or
electrospray ionization (ESI) sources followed by introduction
into time-of-flight (TOF) or quadrupole (Q) mass analysers,
which are described in more detail below.
10/27/2005 Chaoqun Wu,Fudan University 83
Use MS to
determine PTM
of isolated
protein
Enzymatic or
chemical
degradation of
modified protein
HPLC
separation of
peptides
MALDI and/or
ESI used to
identify PTM
MS/MS used to
determine
location of
PTM(s)
10/27/2005 Chaoqun Wu,Fudan University 84
Ionization sources
Matrix-assisted laser desorption-ionization (MALDI),Protein or
peptide samples on solid matrices are ionized by a pulsed laser,
entrained in the resulting ionized gas plume,and introduced
into the mass analyser.
Electrospray ionization (ESI),Protein or peptide samples are
passed through a fine needle to which a voltage is applied,
which results in a fine spray of sample-containing droplets,
Samples are delivered to the mass analyser after the breakup
and evaporation of the droplets,which releases the protein or
peptide samples to the gas phase,A low-flow rate liquid
chromatography system can be coupled to the needle to allow
for protein fractionation of the samples before mass analysis.
10/27/2005 Chaoqun Wu,Fudan University 85
Mass analysers
Time-of-flight (TOF),Time-of-flight analysers separate ions
on the basis of their flight times over a known distance,The
lower the mass of the ion,the greater the velocity and hence
the shorter the flight time.Travel time from the ion source to
the detector is transformed into the m/z ratio,from which the
mass of the ionized sample can be calculated with extreme
accuracy.
Quadrupole (Q) mass filter,A quadrupole mass filter
consists of four parallel rods through which direct current and
radio frequency electric fields are applied to sort the
introduced ions,For each combination of voltages and
frequencies,only ions with a specific m/z ratio pass
undeflected through the quadrupole mass filter,Precise
stepping of these settings therefore allows the quadrupole to
be used as a mass analyser to scan for ions over a large m/z
range.
10/27/2005 Chaoqun Wu,Fudan University 86
Glycoprotein Gel Stain
CandyCane glycoprotein molecular weight standards
containing alternating glycosylated and
nonglycosylated proteins were electrophoresed
through a 13% polyacrylamide gel,After separation,
the gel was stained with SYPRO Ruby protein gel
stain to detect all eight marker proteins (left),
Subsequently,the gel was stained by the standard
periodic acid–Schiff base (PAS) method in the Pro-Q
Fuchsia Glycoprotein Gel Stain Kit to detect the
glycoproteins alpha
2
-macroglobulin,glucose oxidase,
alpha
1
-glycoprotein and avidin.
Pro-Q? Glycoprotein
Stain (DDAO phosphate)
Molecular Formula,
C
15
H
18
Cl
2
N
3
O
5
P (MW
422.20)
Detection of glycoproteins and total protein on an SDS-
polyacrylamide gel using the Pro-Q Fuchsia Glycoprotein Gel
Stain Kit.
10/27/2005 Chaoqun Wu,Fudan University 87
Nitro-Tyrosine Modification
Oxidative modification of amino acid side chains include
methionine oxidation to the corresponding sulfone,S-
nitrosation or S-nitrosoglutationylation of cysteine residues,
and tyrosine modification to yield o,o’-dityrosine,3-
nitrotyrosine and 3-chlorotyrosine.
Nitric oxide (NO) synthases provide the biological precursor
for nitrating agents that perform this modification in vivo,
NO can form nitrating agents in a number of ways including
reacting with superoxide to make peroxynitrite (HOONO)
and through enzymatic oxidation of nitrite to generate NO
·
2
Tyrosine nitration is a well-established protein modification
that occurs in disease states associated with oxidative
stress and increased nitric oxide synthase activity.
The combination of 2D-PAGE,western blotting,and mass
spectrometry has been the more typical strategy to identify
3-nitrotyrosine-modified proteins,
10/27/2005 Chaoqun Wu,Fudan University 88
Nitro-Tyrosine Modification
“Proteomic method identifies proteins nitrated in vivo during inflammatory
challenge,” K,S,Aulak,M,Miyagi,L,Yan,K,A,West,D,Massillon,J,W,
Crabb,and D,J,Stuehr,Proc,Natl,Acad,Sci,USA 2001; 98,12056-12061.
Anti-nitrotyrosine immunopositive proteins in lung of rats induced with LPS.
10/27/2005 Chaoqun Wu,Fudan University 89
Phosphorylation
Analysis of the entire complement of phosphorylated proteins in
cells:,phosphoproteome”
Qualitative and quantitative information regarding protein
phosphorylation important
Important in many cellular processes
– signal transduction,gene regulation,cell cycle,apoptosis
Most common sites of phosphorylation,Ser,Thr,Tyr
MS can be used to detect and map
locations for phosphorylation
– MW increase from addition of
phosphate group
– treatment with phosphatase allows
determination of number of
phosphate groups
– digestion and tandem MS allows for
determination of phosphorylation
sites
10/27/2005 Chaoqun Wu,Fudan University 90
MS/MS and Phosphorylation
Detection of phosphopeptides in complex mixtures can
be facilitated by neutral loss and precurson ion
scanning using tandem mass spectrometers
Allow selective visualization of peptides containing
phosphorylated residues
Most commonly performed with triple quadrupole mass
spectrometers
precursor ion
transmission
collision
cell
(chamber)
mass
analysis of
product ions
10/27/2005 Chaoqun Wu,Fudan University 91
MS/MS and Phosphorylation
Precursor ion scan
– Q1 is set to allow all the components of the mixture to enter
the collision cell and undergo CAD
– Q3 is fixed at a specific mass value,so that only analytes
which fragment to give a fragment ion of this specific mass
will be detected
– Phospho-peptide fragments by CAD to give an ion at m/z 79
(PO
3
)
– Set Q3 to m/z 79,only species which fragment to give a
fragment ion of 79 reach the detector and hence indicating
phosphorylation
Q1 Q2
collision cell
Q3
detector
10/27/2005 Chaoqun Wu,Fudan University 92
MS/MS and Phosphorylation
Neutral loss scan
– Q1 and Q3 are scanned synchronously but with a specific
m/z offset
– The entire mixture is allowed to enter the collision cell,but
only those species which fragment to yield a fragment
with the same mass as the offset will be observed at the
detector
– pSer and pThr peptides readily lose phosphoric acid
during CAD (98 Da)
– For 2+ ion set offset at 49
Any species which loses 49 from a doubly charged ion
would be observed at the detector and be indicative of
phosphorylation
10/27/2005 Chaoqun Wu,Fudan University 93
Enrichment strategies to analyze
phosphoproteins/peptides
Phosphospecific antibodies
– Anti-pY quite successful
– Anti-pS and anti-pT not as successful,but may be used (M,
Gr?nborg,T,Z,Kristiansen,A,Stensballe,J,S,Andersen,O,
Ohara,M,Mann,O,N,Jensen,and A,Pandey,“Approach for
Identification of Serine/Threonine-phosphorylated Proteins by
Enrichment with Phospho-specific Antibodies.” Mol,Cell,
Proteomics 2002,1:517–527.
Immobilized metal affinity chromatography (IMAC)
– Negatively charged phosphate groups bind to postively
charged metal ions (e.g.,Fe
3+
,Ga
3+
) immobilized to a
chromatographic support
– Limitation,non-specific binding to acidic side chains (D,E)
Derivatize all peptides by methyl esterification to reduce non-
specific binding by carboxylate groups.
Ficarro et al.,Nature Biotech,(2002),20,301.
10/27/2005 Chaoqun Wu,Fudan University 94
Direct MS of phosphopeptides
bound to IMAC beads
Raska et al.,Anal,Chem,
2002,74,3429
IMAC beads placed directly on
MALDI target
Matrix solution spotted onto
target
MALDI-MS of peptides bound
to IMAC bead
MALDI-MS/MS (*) to identify
phosphorylation site(s)
10/27/2005 Chaoqun Wu,Fudan University 95
MALDI-MS
spectrum obtained
from peptide bound
to IMAC beads
applied directly to
MALDI target
MALDI-MS/MS (Q-
TOF) to locate
phosphorylation site
Sample enrichment
with minimal sample
handling
contains
phosphorylated
residue
10/27/2005 Chaoqun Wu,Fudan University 96
Enrichment strategies to analyze
phosphoproteins/peptides
Chemical derivatization
– Introduce affinity tag to enrich for
phosphorylated molecules
e.g.,biotin binding to immobilized
avidin/streptavidin
10/27/2005 Chaoqun Wu,Fudan University 97
Enrichment strategies to analyze
phosphoproteins/peptides
Oda et al.,Nature Biotech,2001,19,379 for analysis of
pS and pT
Remove Cys-reactivity by oxidation with performic acid
Base hydrolysis induce?-elimination of phosphate from
pS/pT
Addition of ethanedithiol allows coupling to biotin
Avidin affinity chromatography to purify phosphoproteins
10/27/2005 Chaoqun Wu,Fudan University 98
Enrichment strategies to analyze
phosphoproteins/peptides
Zhou et al.,Nature Biotech,2001,19,375
Reduce and alkylate Cys-residues to eliminate their reactivity
Protect amino groups with t-butyl-dicarbonate (tBoc)
Phosphoramidate adducts at
phosphorylated residues are
formed by carbodiimide
condensation with cystamine
Free sulfhydryls are covalently
captured onto glass beads
coupled to iodoacetic acid
Elute with trifluoroacetic acid
10/27/2005 Chaoqun Wu,Fudan University 99
Chemical derivatization to
enrich for phosphoproteins
Developed because other
methods based on
affinity/adsorption (e.g.,IMAC)
displayed some non-specific
binding
Chemical derivatization
methods may be overly
complex to be used routinely
Sensitivity may not be sufficient
for some experiments (low pmol)
10/27/2005 Chaoqun Wu,Fudan University 100
Phosphoprotein Stain
PeppermintStick phosphoprotein
molecular weight standards
separated on a 13% SDS
polyacrylamide gel,The markers
contain (from largest to smallest)
beta-galactosidase,bovine serum
albumin (BSA),ovalbumin,beta-
casein,avidin and lysozyme,
Ovalbumin and beta-casein are
phosphorylated,The gel was
stained with Pro-Q Diamond
phosphoprotein gel stain (blue)
followed by SYPRO Ruby protein
gel stain (red),The digital images
were pseudocolored.
Phospho
10/27/2005 Chaoqun Wu,Fudan University 101
Phosphoprotein Stain
Visualization of total protein and
phosphoproteins in a 2-D gel
Proteins from a Jurkat T-cell
lymphoma line cell lysate were
separated by 2-D gel electrophoresis
and stained with Pro-Q Diamond
phosphoprotein gel stain (blue)
followed by SYPRO Ruby protein gel
stain (red),After each dye staining,
the gel was imaged and the resulting
composite image was digitally
pseudocolored and overlaid.
T.H,Steinberg et al.,Global quantitative
phosphoprotein analysis using Multiplexed Proteomics
technology,Proteomics 2003,3,1128-1144
10/27/2005 Chaoqun Wu,Fudan University 102
Global Analysis of Protein Phosphorylation
RAW 264.7 exposed to DEP
Sypro Ruby
IEF
9.53.54.5 5.1 5.5 6.0 7.0 8.4
Pro-Q Diamond
9.53.54.5 5.1 5.5 6.0 7.0 8.4
TNFα convertase
MAGUK p55
PDI
Protein phosphatase 2A
JNK-1
p38 MAPK alpha
ERK-1
ERK-2
ErbB-2
TNF
HSP 27
5
3
4
12
6 7
20
30
37
98
55
8
9
10
11
12
13
14
98
55
37
30
20
Xiao,Loo,and Nel - UCLA
10/27/2005 Chaoqun Wu,Fudan University 103
Proteomics in Practice,A Laboratory Manual of
Proteome Analysis
Reiner Westermeier,Tom Naven
Wiley-VCH,2002
PART II,COURSE MANUAL
Step 1,Sample Preparation
Step 2,Isoelectric Focusing
Step 3,SDS Polyacrylamide Gel Electrophoresis
Step 4,Staining of the Gels
Step 5,Scanning of Gels and Image Analysis
Step 6,2D DIGE
Step 7,Spot Excision
Step 8,Sample Destaining
Step 9,In-gel Digestion
Step 10,Microscale Purification
Step 11,Chemical Derivatisation of the Peptide Digest
Step 12,MS Analysis
Step 13,Calibration of the MALDI-ToF MS
Step 14,Preparing for a Database Search
Step 15,PMF Database Search Unsuccessful
PART I,PROTEOMICS
TECHNOLOGY
Introduction
Expression Proteomics
Two-dimensional Electrophoresis
Spot Handling
Mass Spectrometry
Protein Identification by Database
Searching
Methods of Proteomics
10/27/2005 Chaoqun Wu,Fudan University 104
Proteins and Proteomics:
A Laboratory Manual
Richard J,Simpson
Cold Spring Harbor Laboratory (2002)
Chapter 1,Introduction to Proteomics
Chapter 2,One–dimensional Polyacrylamide Gel Electrophoresis
Chapter 3,Preparing Cellular and Subcellular Extracts
Chapter 4,Preparative Two–dimensional Gel Electrophoresis with
Immobilized pH Gradients
Chapter 5,Reversed–phase High–performance Liquid Chromatography
Chapter 6,Amino– and Carboxy– terminal Sequence Analysis
Chapter 7,Peptide Mapping and Sequence Analysis of Gel–resolved Proteins
Chapter 8,The Use of Mass Spectrometry in Proteomics
Chapter 9,Proteomic Methods for Phosphorylation Site Mapping
Chapter 10,Characterization of Protein Complexes
Chapter 11,Making Sense of Proteomics,Using Bioinformatics to Discover a
Protein’s Structure,Functions,and Interactions
10/27/2005 Chaoqun Wu,Fudan University 105
Part V.
Protease
Degradomics
10/27/2005 Chaoqun Wu,Fudan University 106
Degradomics — the application of genomic
and proteomic approaches to identify the
protease and protease-substrate repertoires,
or ‘degradomes’,on an organism-wide scale —
promises to uncover new roles for proteases
in vivo,
10/27/2005 Chaoqun Wu,Fudan University 107
Proteolysis
Many cellular processes,including
embryogenesis,gene expression,cell
cycle,programmed cell death,intracellular
protein targeting and endocrine/neural
functions are regulated by limited
proteolysis of precursor proteins,These
functions are carried out by proteolytic
enzyme families that are strategically
localized within cells or on cell surfaces
10/27/2005 Chaoqun Wu,Fudan University 108
Proteolytic enzyme families
Current numbers of proteases in humans and model species
Catalytic class of protease
Species Total* Aspartic Cysteine Metallo Serine Threonine
Homo sapiens 461 18 121 159 140 23
C elegans 353 26 93 151 62 21
Drosophila 513 38 59 157 225 34
Mus musculus 383 11 93 120 137 22
Rattus 227 10 41 77 80 19
*The total numbers are accurate as of the date of release of the most recent
MEROPS database v 5.7 (17 December 2001),but will
continue to grow as new proteases are discovered and characterized in the existing
databases and in new sequence deposits.
天(门)冬氨酸,半胱氨酸,金属蛋白,丝氨酸,色氨酸
10/27/2005 Chaoqun Wu,Fudan University 109
Genomics and proteomics,the parents of degradomics
Degradome and degradomics are two terms that have been adapted from the
fields of genomics and proteomics,and these terms are defined below together
with other 'omics' terms that appear in this Review.
Genome,The entire collection of genes in the complete DNA sequence of an
organism.
Transcriptome,The complete set of mRNAs that are transcribed from the
genome.
Proteome,The expressed set of proteins that are encoded by the genome.
Genomics,Investigations and techniques for identifying the genome.
Proteomics,Investigations and techniques for identifying the proteome.
Degradomics,All genomic and proteomic investigations and techniques
regarding the genetic,structural and functional identification and
characterization of proteases,and their substrates and inhibitors,that are
present in an organism.
Degradomes,The complete set of proteases that are expressed at a specific
time by a cell,tissue or organism,The degradome of a protease is its substrate
repertoire.
10/27/2005 Chaoqun Wu,Fudan University 110
Relationship of degradomics to the fields of proteomics and
genomics,and of the degradome to the proteome and genome.
10/27/2005 Chaoqun Wu,Fudan University 111
Protease
An enzyme that cleaves proteins by the
catalysis of peptide-bond hydrolysis,On the
basis of their catalytic mechanism,proteases
belong to one of five classes (aspartic,
cysteine,metallo,serine,or threonine).
Proteolytic processing
Proteolysis that is distinct from degradation in
that it represents highly specific and limited
substrate cleavage,which results in a specific
change of protein function.
10/27/2005 Chaoqun Wu,Fudan University 112
Genomics and proteomics,
the parents of degradomics
Degradomics,All genomic and proteomic
investigations and techniques regarding the
genetic,structural and functional identification and
characterization of proteases,and
their substrates and inhibitors,that are present in
an organism.
Degradomes,The complete set of proteases that
are expressed at a specific time by a cell,tissue or
organism,The degradome of a protease is its
substrate repertoire.
10/27/2005 Chaoqun Wu,Fudan University 113
Proteases initiate,modulate and terminate
many important cellular functions by highly
specific and limited substrate cleavage,This
mechanism,called proteolytic processing,
allows the precise cellular control of several
biological processes,including DNA
replication,cell-cycle progression,cell
proliferation,wound healing,immunity,
angiogenesis and apoptosis.
10/27/2005 Chaoqun Wu,Fudan University 114
10/27/2005 Chaoqun Wu,Fudan University 115
Degradomics approches,activity profiling
This knowledge will facilitate the identification of new
pharmaceutical targets to treat disease,
10/27/2005 Chaoqun Wu,Fudan University 116
The recent identification of many bioactive
molecules — including cytokines,cell-
adhesion molecules and receptors — and
intracellular targets (such as transcription
factors and kinases) as new protease
substrates that are precisely processed by
proteolytic activity is redefining,
10/27/2005 Chaoqun Wu,Fudan University 117
The hierarchical importance of proteases in a system
is influenced by specific activity of the protease,
redundancy,expression levels,temporal–spatial
distribution,zymogen activation,protease turnover
and inhibition properties.
Organism-wide degradomics approaches are
therefore required to identify the members of the
~500 protease human degradome that are expressed
by a cell or tissue in disease,and to determine the
complete natural substrate repertoire — the so-called
"substrate degradome" — for each protease.
10/27/2005 Chaoqun Wu,Fudan University 118
H
+
Lysosome
ATP ADP + P
i
Vacuolar ATPase
low
internal
pH
Lumen
contains
hydrolytic
enzymes,
Lysosomes contain
a large variety of
hydrolytic enzymes
that degrade proteins
& other substances
taken in by
endocytosis.
plasma membrane may
be processed first in an
endosomal compartment
and then delivered into the lumen of a lysosome by fusion of
a transport vesicle.
Solute transporters embedded in the lysosomal membrane
catalyze exit of products of lysosomal digestion (e.g.,amino
acids,sugars,cholesterol) to the cytosol,
10/27/2005 Chaoqun Wu,Fudan University 119
“The Nobel Prize in Chemistry 2004 for the discovery
of ubiquitin-mediated protein degradation"
b,1926b,1937
(in Karcag,Hungary)
b,1947
University of California
Irvine,CA,USA
Technion – Israel
Institute of Technology
Haifa,Israel
Technion – Israel
Institute of
Technology
Haifa,Israel
USA Israel Israel
1/3 of the prize 1/3 of the prize 1/3 of the prize
Irwin RoseAvram HershkoAaron Ciechanover
10/27/2005 Chaoqun Wu,Fudan University 120
Ubiquitin
76 Amino Acid polypeptide
Highly conserved in evolution
3 Amino acid differences between
yeast and human homologues
C-Terminal Gly residue is activated
via an ATP to form a thiol ester(硫硫羟酸酯羟酸酯
)
H
3
N
+
CCOO
CH
2
CH
2
CH
2
CH
2
NH
3
H
+
lysine
isopeptide bond(异异肽键肽键) between C-
terminal Gly of Ub
and εε-amino group
of Lys on a target
protein
Most cell proteins are
degraded via the ubiquitin/
proteasome pathway.
10/27/2005 Chaoqun Wu,Fudan University 121
Ubiquitin–Mediated Proteolysis in
Cellular Processes
Regulation of:
Cell cycle
Differentiation & development
Extracellular effectors
Cell surface receptors & ion channels
DNA repair
Immune and inflammatory responses
Biogenesis organelles
10/27/2005 Chaoqun Wu,Fudan University 122
Ubiquitination
Ub
E1
Ub
E2
Ub
E3
Ub
26s proteosome degradation
Target
Ub
Ub
Ub
In general,multiple ubiquitin units are arranged in
polyubiquitin chains linked via Lys
48
of ubiquitin,
targeting the protein for degradation
10/27/2005 Chaoqun Wu,Fudan University 123
Degradation of proteins is a specific
energy dependent process via
polyubiquitination and 26S proteasome
degradation,
Know the function and general
biochemistry of E
1
,E
2
and E
3
enzymes.
Functional description of the proteasome.
Functional differences between mono and
polyubiquitination.
Functional differences between
sumoylation and ubiquitination.
10/27/2005 Chaoqun Wu,Fudan University 124
ATP Driven Activation is a
Common Theme in Biochemistry
Ub-E1(poly-Ub/protein degradation)
aa-tRNA (proteins)
Acetyl-CoA (FA)
UDP-glucose (glycogen)
Dolichol-GlcNAc (N-Linked glycoproteins)
10/27/2005 Chaoqun Wu,Fudan University 125
Proteins Targeted by Ubiquitin
Cell cycle regulators
Tumor suppressors & growth modulators
Transcriptional activators & inhibitors
Cell surface receptors
Mutant and damaged proteins
10/27/2005 Chaoqun Wu,Fudan University 126
Degradation of a Protein Via the
Ubiquitin-Proteasome Involves Two
Successive Steps
Covalent attachment of multiple ubiquitin
molecules to a protein substrate.
Degradation of the tagged protein by the
26s proteasome.
(ubiquitin is recycled)
10/27/2005 Chaoqun Wu,Fudan University 127
Ubiquitin Conjugation:
A 3-Step Mechanism
Ub forms isopeptide bond
between C-terminal Gly of Ub
and ε
ε
-amino group of Lys
on a target protein
Ubiquitin ligase E
3
Ub is transferred to a Cys of
E
2
forming a new thiol ester
Ubiquitin conjugating
enzymes E
2
High energy thiol ester is
formed between C-terminal
Gly of ubiqutin and a Cys in
the E
1
active site (ATP/AMP)
ATP-dependent
ubiquitin (Ub)
activating enzyme E
1
10/27/2005 Chaoqun Wu,Fudan University 128
E
1
,E
2
,& E
3
E,ubiquitin-activating enzyme，泛素激活酶
With each step increasing level of regulatory
specificity:
E1,only one (?)
E2,about 30 (homologous family)
E3,100 and structurally unrelated
(ultimate biological specificity)
10/27/2005 Chaoqun Wu,Fudan University 129
Proteasome Complexes
11S (PA28) Activator of 20S Proteasome
(human)
20S Proteasome (human)
20S Proteasome (S,cerevisiae)
26S Proteasome (human)
About 30000 proteasomes in human.
10/27/2005 Chaoqun Wu,Fudan University 130
The ubiquitin/
proteasome
pathway
The substrate
proteins are
polyubiquitinated
through the ubiquitin
pathway following
degradation by the
26S proteasome.
JARQ 39 (1),1 – 4 (2005)
10/27/2005 Chaoqun Wu,Fudan University 131
Cell cycle controls depending on ubiquitins
The ubiquitin-
proteasome system
controls not only the
master regulatory
Cdk activities,but
also the execution of
many cell-cycle
events downstream
from Cdk regulatory
circuit,
For example,
Cyc (cyclin) A,
CycB,CycD,and
B-type CDK are
degraded by 26S
proteasome in a
cell cycle-
dependent
manner.
10/27/2005 Chaoqun Wu,Fudan University 132
Degradation of cell-cycle regulatory proteins
Various cell-cycle regulatory proteins are degraded through
the ubiquitin/proteasome pathway at specific cell-cycle stages
in plant cells.
10/27/2005 Chaoqun Wu,Fudan University 133
Cell division is propelled by the oscillation of
cyclin-dependent kinase (Cdk) activities,which in
turn are regulated by the periodic synthesis and
degradation of their regulatory subunits,cyclins,
In addition to cyclins,other key regulators,such
as p27,p53,anaphase inhibitor,Cdc20,polo-like
kinase,aurora kinase,Nek2 kinase and kinesin-
related motor proteins,are all regulated by
ubiquitin-dependent proteolytic pathways in the
cell cycle,
10/27/2005 Chaoqun Wu,Fudan University 134
Phosphorylation of
I
Κ
Βα leads to
ubiquitination and
degradation of the
inhibitor and
activation of the
transcription factor.
NFκB pathway
10/27/2005 Chaoqun Wu,Fudan University 135
ATP consuming process
10/27/2005 Chaoqun Wu,Fudan University 136
Modification of Protein Substrates by Ub/UBLs
E,ubiquitin-activating enzyme，泛素激活酶
10/27/2005 Chaoqun Wu,Fudan University 137
Ubiquitin (Ub) and ubiquitin-like molecules (UBLs)
are expressed as precursors,either in form of
concatemers(连接) or C-terminal extensions,A
subset of Ub/UBL-specific proteases cleaves
precursor polypeptides to obtain the free Ub/UBL
monomers (recognition and proteolysis of a
peptide bond),Conjugation of Ub and UBLs to
target polypeptides is achieved by several
enzymatic steps via a cascade involving E1,E2,
and E3 enzymes,Conjugated protein substrates
are then subjected to deconjugation by Ub/UBL-
specific proteases that specifically recognize and
cleave isopeptide bonds,Individual proteases may
recognize a specific subset of conjugated
substrates.
10/27/2005 Chaoqun Wu,Fudan University 138
Mono-Ub,Another Function
Histone regulation
Endocytosis
Budding of retroviruses from plasma
membrane
10/27/2005 Chaoqun Wu,Fudan University 139
Ubiquitin like Molecule
like Molecule (UBLs)
Small Ubiquitin Related Modifier (SUMO)
SUMO does not have the Lys-48 found in
ubiquitin
SUMO does not make multi-chain forms
SUMO-1,2,3 are the mammalian forms
SUMO-1,101 amino acids,C-terminal
Gly,18% identical to ubiquitin.
10/27/2005 Chaoqun Wu,Fudan University 140
SUMO
10/27/2005 Chaoqun Wu,Fudan University 141
SUMO Enzymes
SUMO-activating enzyme,heterodimer
SUMO-conjugating enzyme,Ubc9,an E
2
enzyme that can’t be ubiquitinated
SUMO-ligase,E3 enzyme that is specific
for sumoylation
E3-like proteins increase affinity between
SUMO-Ubc9 and target protein
10/27/2005 Chaoqun Wu,Fudan University 142
Sumoylation and Desumoylation Cycle
with E1-,E2-,E3-like Enzymes
10/27/2005 Chaoqun Wu,Fudan University 143
Biological Roles of SUMO
Modification
Antagonistic role against ubiquitin,IκBα is
sumoylated preventing ubiquitination and
degradation (NF-κB pathway).
Protein translocation,Ran-GAP1/RanBp2
Modulation of transcriptional activity,activates
transcriptional activity of p53.
Subnuclear structure formation,protein targeting
to nuclear bodies,PML,tumor suppressor,
10/27/2005 Chaoqun Wu,Fudan University 144
Proteins Modified BY SUMO
Polarized cell growth and
cytokinesis
Septins
Mutations causing premature
aging
Werner’s syndrome protein
Regulates p53Mdm2
Tumor suppressorp53
Inflammatory responseIκBα
Affected by translocations
causing leukemias
PML
Nuclear transportRanGAP1
FunctionProtein modified by SUMO
