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Part VI.
Structural
Proteomics
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Structural proteomics
Structural proteomics has the goal of
obtaining useful,three-dimensional
models of all proteins by a combina-
tion of experimental structure
determination and comparative model
building.
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The experimental (left) and computational (right)
hierarchies will increasingly become codependent as the
research community models greater biological complexity.
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Global structural genomics efforts will be major players in
Completing the protein family and fold landscape,
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It is feasible to determine at least 10,000
protein structures within the next five years,
No new discoveries or methods are needed
for this task.
The average cost per structure is expected
to decrease significantly from $200,000 per
structure to perhaps less than $20,000 per
structure,
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Large-scale protein structure modeling.
A small sample of the 1,100 comparative models calculated
for the proteins in the yeast genome is displayed over an
image of a yeast cell
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Experimental structure determination
蛋白质的生物活性检测选择性蛋白质分子的突变蛋白质的体外翻译
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1,Target selection (Grouping)
The targets for structure determination will be
1,Individual domains rather than multi-domain
proteins (Chris Sander,Millenium Information),
2,Easier to determine by X-ray crystallography or
NMR spectroscopy than the more flexible multi-
domain proteins.
3,Done by pairwise comparison of all protein
sequences,followed by clustering into groups of
domains,
4,The representatives of the groups without
structurally defined members,
5,Members that share at least 30% sequence
identity (30-seq families),rather than those that
correspond to superfamiles or fold families.
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A variety of different criteria likely will be used
for this task; for example:
null size of the family,
null biological knowledge about the family,
null distribution of the members among various
organisms,
null the pharmaceutical relevance,
null the likelihood of a successful structure
determination,
and so forth,
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2,Cloning,expression and purification
Obtaining protein samples for crystallization
trials and NMR measurements likely will be the
bottleneck in the structural genomics process,
Although the current methods are sufficiently
powerful to begin the project,entirely new
technologies may arise,such as an in vitro system
that already has allowed researchers to produce
proteins up to concentrations of 6 mg / ml
(Shigeyuki Yokoyama,Tokyo University).
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3,Crystallization and X-ray crystallography
Crystals have a higher chance of being formed
when the protein species in solution is
Conformationally homogeneous,This can be
evaluated by dynamic light scattering,as well
as by limited proteolysis combined with mass
spectroscopy,For example,candidates that
are monodisperse under standard aqueous
conditions have a probability of at least 75% to
yield crystals suitable for X-ray studies,in
comparison to 10% for poly-disperse samples,
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Atomic resolution structure
determinations of proteins by X-
ray crystallography require:
protein construct design,
expression and purification,
crystallization trials,
phase determination,
model building.
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Mass Spectrometry can be used to
1,verify that the desired protein construct has been
correctly expressed,
2,to define compact domains in the target protein,
3,to assess the components contained within the
protein crystals,
4,to screen for successful incorporation of seleno-
methionine and other heavy metal reagents used for
phasing,
5,to address issues of modeling,topology,and side-
chain proximity.
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VERIFICATION
1) Mass spectrometry’s unsurpassed accuracy and
speed for measuring mass allows one to quickly
test whether a given protein has been faithfully
expressed.
2) The measurement allows for detection of PCR
errors,mistranslation errors,unwanted
modifications (e.g.,from attachment of beta-
mercaptoethanol to thiols or oxidation of
methionyl residues),and major protein impurities
and byproducts(e.g.,from degradation).
3) detecting desired modifications such as
phosphorylation.
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DOMAIN ELUCIDATION
Numerous computer-aided approaches to help
define folding domains are available,These include
the use of homologous sequence alignment tools,
secondary structure prediction software,and various
“threading” algorithms,Progress in genomic
sequencing has greatly facilitated homology
searching,and there are increasingly available more
reliable secondary structure prediction programs.
In cases of sequences that exhibit low homology
alignments or poorly predicted secondary structural
elements.
Limited proteolysis combined with MS
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PROTEIN CRYSTAL ANALYSIS
Determining the Presence of the
Expected Protein Components
Examining Alteration of Proteins
During Crystallization
Assessing Heavy Metal Incorporation
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PHASING
Extraction of electron density and structural
information from X-ray diffraction data of
protein crystals requires knowledge of the
magnitudes and phase angles of the
diffracted X-rays,
The diffraction data itself contains only the
magnitudes; the associated phases must
be obtained by other means.
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Available,phasing” methods are isomorphous
replacement and molecular replacement
techniques.
In isomorphic replacement,a heavy atom is
introduced into the protein with minimal
perturbation of the native protein fold,
Phasing information is obtained from X-ray
diffraction of a crystal of heavy-atom
derivatized protein.
Heavy atoms used for phasing range in size
from selenium to uranium.
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MODEL BUILDING AND REFINEMENT
After the electron density has been determined
from an X-ray diffraction study of a protein,a
model of the protein structure is built,One
question that frequently arises during model
building is whether a stretch of sequence that
is not observed in the electron density map
is either disordered or altogether missing in
the protein crystal (perhaps as a result of
degradation over time)
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intramolecular protein cross-linking in
combination with mass spectrometry
provided restraint information that is
potentially useful for protein model
building
Limited proteolysis,in conjunction with
mass spectrometry,
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A Case Study
The KcsA potassium channel,
from Streptomyces lividans,
consists of four identical
integral membrane protein
subunits.
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Initial protein constructs were obtained
by purifying a full-length C-terminal
His-Tag protein (170 residues; 18.8
kDa),followed by trypsin treatment to
remove the His-Tag,
Although the trypsin treatment yielded
sufficient quantities of protein,this
material did not produce diffraction
grade crystals.
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Domain elucidation
1,Detergents (e.g.,Mega-9,LDAO) were
found to be suitable for protein purification
and crystallization and did not interfere
with MALDI-MS analysis
2,Proteolysis experiments with trypsin,
chymotrypsin,and subtilisin,then mapped
by MALDI-MS.
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PROTEOLYSIS RESULTS:
Each protease quickly removed about 45 residues
from the C-terminal of the His-Tag KcsA,leaving a
122-residue (by trypsin) or 125-residue (by
chymotrypsin or subtilisin) core domain,There was
little indication that the 45-residue portion that was
proteolytically trimmed from the C-terminal of full-
length KcsA had any inherent structure,
Beyond one day of digestion,the C-terminally-
truncated protein began to show an additional
cleavage site 19 residues (by trypsin) or 25 residues
(by subtilisin) from the N-terminal,Chymotrypsin did
not cleave anywhere in the N-terminal.
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Another problem:
MS revealed that in addition to the expected
chymotryptic Cleavage product (KcsA 1-125),
Cleavage product (KcsA 1-125),an
unanticipated second population of the protein
appeared (KcsA 20-125),not easily
distinguished from the expected product by
SDS-PAGE,and its presence likely would have
adversely affected the crystallization trials.
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Because of trypsin,a common
contaminant found in chymotrypsin
preparations,To avoid the trypsin
activity,TLCK-treated chymotrypsin
was used to ensure a homogenous
population of proteolytically resistant
KcsA,
This chymotrypsin construct provided
high quality diffracting crystals of the
potassium channel.
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Phasing of the KcsA
needs cysteinyl residues
where to place the cysteine within the
protein,different mercurating reagents.
They all need MS to see if it’s right
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Finally,mass spectrometry
was used to assist in the
model building process
that yielded the beautiful
and informative structure
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Ribbon
representa
tion of the
KcsA
potassium
channel
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Detail of the electron
density map of
Deacetoxycephalosporin
C synthase from
Streptomyces clavuligerus.
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4,Nuclear magnetic resonance
(NMR) spectroscopy
Recent and prospective advances in NMR
spectroscopy that will allow structure
determination of larger proteins as well as
increase the accuracy and speed of the analysis,
These advances include,
nullPartial deuteration of protein samples,
nullNew magnets (up to 1 GHz),
nullSuperconducting probes,
nullSoftware for resonance assignments and
structure determination,
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5,Comparative model building
null Templete selection
null Comparative modeling
null Function prediction
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Templete
selection
Protein fold assignment.
A genome-encoded amino acid
sequence (center) is tested for
compatibility with a library of
known 3D protein folds.
An actual library would contain
of the order of 1000 folds; the
one shown here illustrates the
most common protein folding
motifs,
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High-accuracy comparative models are based on
more than 50% sequence identity to their templates,
Comparative modeling
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Before Modeling
1,Identification of modelling templates,
2,Comparative protein modeling requires
at least one sequence of known 3D-
structure with significant similarity to the
target sequence,
3,Aligning the target sequence with the
template Sequence,
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Step By Step Work
Secondary Structure and Backbone
Conformation
Super-secondary structure
Side Chain Conformation
Tertiary Protein Structure and folds
Quaternary Structure
Comparative protein modelling
To evaluate the quality of a model
Secondary Structure and Backbone
Super-secondary structure
Side Chain Conformation
Tertiary Protein Structure and folds
Quaternary Structure
Comparative protein modelling
To evaluate the quality of a model
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Secondary Structure and Backbone
Conformation
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Detail from a three-
dimensional
structure of space-
grown insulin
crystals,
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Framework construction
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Completing the backbone
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Super-secondary structure
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Side Chain Conformation
Here are some conformations
that can be adopted by
Arginines:
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Adding side chains
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Tertiary Protein Structure and folds
The lone helix The helix-turn-helix motif
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The four-helix bundle
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Quaternary Structure
Tertiary domains
Aggregating
together
to form a unit,
The photoreaction
centre is a good
example,
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6,Function prediction
The similarities and differences between two plant and archeal
members of a family of glycosidases that includes a protein
implicated in ageing,
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7,Datebase and software
Databases
Several different databases will be useful in
facilitating the structural genomics project,These
databases will be used for,
1,Target selection,
2,Coordination of the experimental efforts,
3,Archiving of the new structures,
4,Dissemination of the results,
5,Performing new research with the old and new
data,
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Using the structures
Development of the databases and software
will be necessary to integrate raw structural
information and structure-derived results with
other information,such as:
null Genetic and biochemical analysis,
null Gene expression patterns,
null Mutational analysis,
null Binding data from protein chips,
null Interaction data from two-hybrid systems,
null Metabolic pathway information,
and so forth,
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蛋白质水平的分析蛋白质水平的分析蛋白分子的等电点分析蛋白分子的亲水性分析蛋白分子的抗原性分析进化树分析氨基酸糖基化位点预测同源性分析同源性分析功能域分析功能域分析结构域分析结构域分析膜蛋白分析膜蛋白分析分泌蛋白分析分泌蛋白分析立体结构分析立体结构分析蛋白分子建模蛋白分子建模
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结构分析结构分析蛋白质的结构对于蛋白质发挥功能非常重蛋白质的结构对于蛋白质发挥功能非常重要，现在已经有许多蛋白质的三级结构被要，现在已经有许多蛋白质的三级结构被测定，可以通过同源模建的方法分析目标测定，可以通过同源模建的方法分析目标蛋白的三级结构。
蛋白的三级结构。
工具：
工具：
EXPASY的蛋白分析工具或的蛋白分析工具或
NCBI的的
STRUCTURE。
。
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A computational method has been developed for the assignment
of a protein sequence to a folding class in the Structural Classifica-
tion of Proteins (SCOP),Current data places the number between
1,000 and 5,000 distinct protein folds.
This method uses global descriptors of a primary protein seque-
nce in terms of the physical,chemical,and structural properties of
the constituent amino acids.
The method is applicable for the fold assignment of any protein
sequence with or without significant sequence homology to known
proteins,A WWW page for predicting protein folds is available at
URL http://cbcg.lbl.gov/.
Structural Classification of Proteins
(SCOP)
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EXPASY
This is the ExPASy (Expert Protein Analysis System)
proteomics server of the Swiss Institute of
Bioinformatics (SIB),This server is dedicated to the
analysis of protein sequences and structures as well
as 2-D PAGE (Disclaimer
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8,Applications of Strutural Genomics
nullFunction Studies
nullDrug Development
nullMetabolic Pathway
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iNOS,Cys
194
,Glu
371
.
Oxidase-domain eNOS
nNOS
Example
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iNOS,
Cys
194
-heme binding,
Glu
371-
Arginin binding.
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Example:
Mechanism-based
Design of a protein
kinase inhibitor
nature structural biology?
volume 8 number 1?
january 2001 37
Keykavous Parang et al..
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Protein kinases play a critical role in cell signaling
Pathways by catalyzing the transfer of the
γ-phosphoryl group from ATP to the hydroxyl groups
of protein side chains,The protein kinase superfamily
is one of the largest as ～2% of eukaryotic genes encode
them,Because of their importance in contributing to a
variety of pathophysiologic states,including cancer,
inflammatory conditions,autoimmune disorders,and
cardiac diseases,intense efforts have been made to
develop specific protein kinase inhibitors as biological
tools and as therapeutic agents,
γ
～
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Example:
Crystal structure of the binary
complex between cIRK and the
bisubstrate inhibitor,
a,Overall view of the binary
complex in which cIRK is shown in
surface representation and the
bisubstrate inhibitor in stick
representation,
b,Stereo view of the Fo - Fc electron
density for the bisubstrate inhibitor
computed after simulated annealing
(1,000 K),
c,Stereo view of the interactions
between the inhibitor and key
catalytic residues,
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Accuracy and application of
protein structure models.
The different ranges of applic-
ability of comparative protein
Structure modeling,threading,
and de novo structure prediction,
The corresponding accuracy of
protein structure models; and
Their sample applications,
The accuracy of the models
decrease significantly in going from
(A) to (E),but the overall structure
is still roughly correct,
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Part VII.
Platforms
for proteomics
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Pillar Proteomic Technologies
Amino Acid Composition
Array-based Proteomics
2D PAGE ( LC 2D)
Mass Spectrometry
Structural Proteomics
Informatics (and the challenges facing the
Human Proteome Project)
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Platforms for proteomics and functional
genomics,Methodology is shown in the outer
columns,resultant data sets in the middle
columns,and model systems in the centre,
Nature 422,193 - 197 (13 March 2003)
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A,2D gel analysis
2D-gel of
erythroleukemia
cell
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双向凝胶电泳
（two-dimensional gel electrophoresis,2-DE）
基本原理是蛋白质首先根据其等电点在pH
梯度胶中等电聚焦，然后按照它们的分子量大小进行SDS-PAGE第二次电泳分离。
第一向等电聚焦条件和方式可根据待分离的蛋白质分子特性的不同而变化。例如pH梯度
(immobilized pH gradients)中，pH梯度范围可按所需设置。
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2D PAGE
2-D gel electrophoresis is a multi-step
procedure that can be used to separate
hundreds to thousands of proteins with
extremely high resolution,
It works by separation of proteins by their
pI's in one dimension using an immobilized
pH gradient (first dimension,isoelectric
focusing) and then by their MW's in the
second dimension.
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2D PAGE
2-D gel electrophoresis process consists of
these steps,
Sample preparation
First dimension,isoelectric focusing
Second dimension,gel electrophoresis
Staining
Imaging analysis via software
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Advances in 2D gel electrophoresis are
revolutionizing proteomics by providing a
means to separate different proteins for further
analysis.
Protein analysis uses a diseased or treated
sample and a control sample,2D gel
electrophoresis is performed for each sample to
separate proteins based on their molecular
weight and charge,
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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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Two-Dimensional Gel
Electrophoresis
Protein separation
Identification of
the proteins
corresponding to
the selected
spots
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Examples of 2D gels
molecular weight (kDa)
pH
2D separation of
human lymphoma
proteins
Many sources of proteins possible
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2D map of WI-38 human lung fibroblasts
IEFNEpHGE
I.P,I.P.
S
S
S
D
S
M
.
W
.
(
k
D
)
M
.
W
.
(
k
D
)
D
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B,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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Drawbacks of 2D PAGE
Technique precision lacks reliable
reproduction.
Spots often overlap,making identifications
difficult.
More of,an art” than,a science.”
Slow and tedious.
Process contains may,open” phases
where contamination is possible.
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Advantages vs,Disadvantages
Good resolution of
proteins
Detection of post-
translational
modifications
Not for
hydrophobic
proteins
Limited by pH
range
Not easy for low
abundant proteins
Analysis and
quantification are
difficult
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2-D凝胶电泳的挑战和问题
1.低丰度蛋白质的检测、灵敏度不够
2.不同实验间蛋白质所在位置的漂移
3.不能全面代表膜蛋白或疏水蛋白的真实情况
5.某些蛋白质在等电聚焦缓冲液中低溶解度(Insolubility)
6.特大分子量的蛋白质的检测难
7.特小分子量的蛋白质的检测难
8.某些蛋白质带电荷的微小差异(Charge microheterogeneity)
9.容易受污染(Susceptibility to contamination)
10.处在变性的条件(Requirement for denaturing conditions)
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C,2-D analysis by liquid
chromatography(LC)
Gel filtration
Ion-exchange
Hydroxyapatite columns
Hydrophobic
Immobilized reactive dyes
Affinity
Chromatofocusing
HPLC
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Typical proteomics experiment using gel
electrophoresis
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2D - LC/LC
(trypsin)
Peptides all bind
to cation
exchange column
Study protein
complexes
without gel
electrophoresis
Successive elution
with increasing salt
gradients separates
peptides by charge
Peptides are
separated by
hydrophobicity on
reverse phase
column
Complex mixture is
simplified prior to
MS/MS by 2D LC
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Reverse Phase column
Polypeptides enter the column in the mobile phase…
…the hydrophobic,foot” of the polypeptides adsorb to the
hydrophobic (non polar) surface of the reverse-phase
material (stationary phase) where they remain until…
…the organic modifier concentration rises to critical
concentration and desorbs the polypeptides
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2D - LC/MS
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But 2D-PAGE is not a best choice for proteome analysis
because of individual extraction,digestion,and analysis of
each spot from 2D-PAGE is a tedious and time-
consuming process.
The largest 2D-PAGE-based proteome study to date
identified 502 unique proteins for the Hemophilus
influenzae proteome.
Fundamental limitation of 2D gel electrophoresis is its
failure to display the entire protein complement of sample
on the gel surface.
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Large proteins and hydrophobic proteins such as
membrane proteins have difficulty entering the gel.
Acidic proteins(PI<4) or basic proteins(PI>10) are poorly
resolved,Most importantly,the levels of low-copy-number
proteins present in low abundance are below the detection
limit of silver staining techniques.
This limitation is probably most critical because many of the
regulatory proteins that might be involved in the disease
process and are candidates for many drugs are present in
the cell in low concentrations.
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Liquid Chromatography
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Liquid Chromatography
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nullProteins Fractionated using 2D Chromatography
nullProteins resolved by both chromatofocusing and
reversed phase chromatography
nullProteins unique to diseased state - Isolated
*
*
*
*
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ICAT（Isotope Coded Affinity Tages,同位素标记亲和标签）
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Differential LC/MSMS
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Advantage of 2 dimensional liquid
chromatography
When reversed phase is preceded by ion exchange
1,The combined effect of the two chemistries
procedures better purification than either
chemistry alone.
- Ion exchange and reversed phase are
orthogonal separation method,The effects
of the two methods are synergistic; final
resolution is greater than with either
method alone
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2,The ion-exchange chromatogram
can aid locating the desired product
- Sometimes reversed-phase analysis of a
crude synthetic peptide does not clearly
indicate a main peak,When ion exchange run
first,the last major peak on the ion-exchange
chromatogram usually contains the peptide of
interest.
10/27/2005 Chaoqun Wu,Fudan University 107
3,Hydrophbic reaction products are
removed by the ion-exchange step
- This reduces the loading capacity required
for the reversed-phase separation,In addtion,
fouling of the reversed-phase column that
may require cleaning with IPA or stronger
solvents is eliminated.
10/27/2005 Chaoqun Wu,Fudan University 108
4,Strongly acidic cleavage reagent
are removed prior to reversed-phase
- HF and TFA reagents used in cleavage
reactions can attack a silca-based reversed-
phase column,An initial step of ion-exchange
on a chemically resistant polymer-based
column removes these reagents,thereby
protecting the reversed-pahse column.
10/27/2005 Chaoqun Wu,Fudan University 109
Advantages vs,Disadvantages
Determination
of MW and aa,
Sequence
Detection of
posttranslational
modifications
High-throughput
capability
High capital costs
Requires sequence
databases for
analysis
10/27/2005 Chaoqun Wu,Fudan University 110
MudPIT (multidimensional protein
identification technology)
多维液相色谱与串联质谱仪在线联用
Consist of
1,Multidimensional liquid chromatography
2,Tandem mass spectrometry
3,Database searching by the SEQUEST
algorithm
10/27/2005 Chaoqun Wu,Fudan University 111
Yates’ group suggest non-gel-based
chromatography systems to resolve and identify
thousands of proteins from biological sample.
The acidified complex peptide mixture is applied to
a strong cation exchange(SCX) chromatography
column,and a discrete fraction of the absorbed
peptides are displaced onto a reversed-phase(RP)
chromatography column using a salt step gradient.
Peptides are retained on the RP column,but
contaminating salts and buffers are washed away
and diverted to waste.
The peptides are then eluted from the RP column
into the mass spectrometer using a gradient of
increasing acetonitrile.
10/27/2005 Chaoqun Wu,Fudan University 112
Multidimensional protein identification technology (MudPIT)
10/27/2005 Chaoqun Wu,Fudan University 113
Peptides all bind to cation
exchange column
Peptides are separated by
hydrophobicity on reverse
phase column
Successive elutions with
increasing salt gradients
separates peptides by
charge
Complex mixture is
simplified prior to
MS/MS by 2D LC
“MudPIT”
(trypsin)
Study protein
complexes
without gel
electrophoresis
10/27/2005 Chaoqun Wu,Fudan University 114
IEX-HPLC,Ion-exchange high-performance liquid chromatography
离子交换高效液相色谱法
RP-HPLC,Reverse-phase high-performance liquid chromatography
反相高效液相色谱法
MudPIT
Trypsin
+ proteins
p53
IEX-HPLC
RP-HPLC
10/27/2005 Chaoqun Wu,Fudan University 115
Additions to protein solvents
Class examples purpose
Buffer stability
Salts KCl,NaCl,stability
Detergents SDC,stability,
solubility
surfactant Triton X-100 stability,
solubility
Glycerol,sucrose stability
Sodium azide
bacteriostatic
Metal chelator ETDA,EGTA stability
Sulfhydryl agents DTT stability
ligands metals,ATP
stability
Protease inhibitors PMSF stability
10/27/2005 Chaoqun Wu,Fudan University 116
D,Array-based Proteomics
Offer a high-throughput technique for
proteome analysis.
These small plates are able to hold many
different samples at a time.
Current research is ongoing in an attempt
to interface array methodologies with
Mass Spectrometry at ORNL.
10/27/2005 Chaoqun Wu,Fudan University 117
Array-based Proteomics
10/27/2005 Chaoqun Wu,Fudan University 118
Protein Chips Offer Powerful
Method for Probing Protein
Function
10/27/2005 Chaoqun Wu,Fudan University 119
Functional protein microarrays are
critical for the next phase of proteomics
research.
"Profiling proteins will be invaluable,
for example,in distinguishing the proteins
of normal cells from early-stage cancer
cells,and from malignant,metastatic
cancer cells that are the real killers," said
HHMI investigator Stuart L,Schreiber.
10/27/2005 Chaoqun Wu,Fudan University 120
Like DNA chips,protein chips will be able
to analyze thousands of samples
simultaneously,leading the way towards
a complete map of the entire complement
of human proteins,
But,unlike DNA,proteins are not so easy
to attach to chips,Where DNA is robust,
and able to withstand harsh experimental
conditions,proteins are fragile and will
denature if they aren't treated gently,
10/27/2005 Chaoqun Wu,Fudan University 121
Functional protein microarrays are consi-
dered critical to progress in proteomics
research,And,not surprisingly,academic
and industrial researchers alike are expen-
ding considerable time and energy to come
up with an operable system,
Trends in Biotechnology Vol,20,No,4,April 2002
10/27/2005 Chaoqun Wu,Fudan University 122
Protein biochip
analytical system
will enable high-
throughput protein
discovery,protein
profiling,protein
structure and
activity analyses,
as well as protein-
protein and
protein-small
molecule
interaction studies.
10/27/2005 Chaoqun Wu,Fudan University 123
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10/27/2005 Chaoqun Wu,Fudan University 126
Proteins are extremely sensitive to the
physical and chemical properties of the
particular substrate,It is difficult to come
up witha so-called generic solid phase
material,
Proteins must remain in a liquid environ-
ment to retain their activity,
Proteins have one-,two- and three-dimen-
sional configurations as they transform
from a straight chain of amino acids into a
functional unit,
10/27/2005 Chaoqun Wu,Fudan University 127
The arrayer picks up about a microliter of sample from
four wells of a 96- or 384-well plate and deposits about
1 nanoliter of each sample at defined locations on a series
of glass microscope slides,The arrayer uses a pin and
ring system,
the samples are picked up in
small rings that each hold
about 1 microliter and a solid
pin (150 um diameter) then
punches repeatedly through
the ring to deposit the proteins
on the slides,To prevent
evaporation of the nanodroplets,
we include 40% glycerol in the
protein samples,Nanoliter
droplets of 40% glycerol remain
hydrated,even when left
exposedto the atmosphere
overnight.
1,Arraying the proteins
10/27/2005 Chaoqun Wu,Fudan University 128
Arraying Proteins on Glass Slides
The proteins were spotted on BSA-NHS
slides following a 3 hr incubation in a
humid chamber at room temperature,the
slides were inverted and dropped onto a
solution of PBS,pH 8.0 containing 500 mM
glycine,After 1 min,the slides were turned
right side up and immersed in the glycine
solution for 1 hr at room temperature with
gentle agitation,
10/27/2005 Chaoqun Wu,Fudan University 129
Chemically Derivatized Glass Slides
Protein chip slides,displaying activated
amino and carboxyl groups on the
surface of an immobilized layer of
bovine serum albumin (BSA),were
fabricated,
10/27/2005 Chaoqun Wu,Fudan University 130
2,Protein Array typing
(1) Hydrophobic Protein Arrays
Hydrophobic arrays bind proteins through
hydrophobic surface interaction with amino
acids such as alanine,valine,leucine,
isoleucine,phenylalanine,tryptophan and
tyrosine,
Binding occurs in aqueous,high salt
conditions and binding is reduced by
decreasing salt and increasing the
concentration of organics.
10/27/2005 Chaoqun Wu,Fudan University 131
(2) Hydrophilic Protein Arrays
Hydrophilic arrays bind proteins through
electrostatic and dipole-dipole interactions
as well as hydrogen binding,Proteins with
hydrophilic and charged surface animo acids
such as serine,threonine and lysine bind well.
Binding occurs in aqueous buffers with a water
wash prior to analysis.
10/27/2005 Chaoqun Wu,Fudan University 132
(3) Anion Exchange Protein Arrays
Anionic arrays bind proteins through
electrostatic interaction of negatively charged
amino acids such as aspartic acid and glutamic
acid,
Binding occurs at high pH with low salt and
binding decreases as pH decreases and salt
concentration increases.
10/27/2005 Chaoqun Wu,Fudan University 133
(4) Cation Exchange Protein Arrays
Cationic arrays bind proteins through
electrostatic interaction of positively charged
amino acids such as lysine,arginine and
histidine,
Binding occurs at low pH with low salt;
binding decreases as pH and salt
concentration increase.
10/27/2005 Chaoqun Wu,Fudan University 134
(5) Immobilized Metal affinity Protein Arrays
Immobilized metal affinity capture (IMAC) arrays
bind proteins and peptides which have affinity
for metals; proteins with exposed histidine,
tryptophan and/or cysteine typically bind to
metals immobilized on these chip surfaces,
Binding occurs under pH 6-8 and high salt and
decreases as the concentration of imidizole and
glycine increase.
10/27/2005 Chaoqun Wu,Fudan University 135
(6) Preactivated Protein Arrays
ProteinChip contains a carbonyldiimidazole
surface which covalently reacts with amine
groups,DNA and proteins,including antibodies,
can be immobilized on the slids surface.
10/27/2005 Chaoqun Wu,Fudan University 136
(7) 2-D gel is also a Protein Arrays
10/27/2005 Chaoqun Wu,Fudan University 137
(8) Gel- based protein chips
The gel is aqueous,porous (creating a
large surface area) and dimensional
(eliminating solid-liquid interfaces),
The gel substrate is truly three-dimensional
and porous," Burke explained,
It is speculated that the gel provides a
hydrophilic environment that lends itself
to protein-protein interactions,and that
these proteins behave more naturally.
10/27/2005 Chaoqun Wu,Fudan University 138
(9) Silicon- silane based protein chip
Coat the chip surface with a film of silicon
dioxide,Then the molecules of interest
are attached covalently via a silane
coupling reagent to offer ready-to-use
chips as well as pre-activated chips,
10/27/2005 Chaoqun Wu,Fudan University 139
(10) Protein-antibody based chips
The protein-antibody pairs selected will
be used to develop a new generation of
microarrays for detecting proteins,For
instance,imagine creating biochips for
membrane proteins,
……
10/27/2005 Chaoqun Wu,Fudan University 140
3,Classes of capture molecues
for protein microarray
An attachment tag is added to the top layer,
and a protein capture agent (such as an
antibody fragment,a peptide or even a novel
scaffold of antibody-like molecules) gets
bound to its free end,These protein capture
agents can be tailored specifically to snag
the proteins of interest during the actual
experiment.
10/27/2005 Chaoqun Wu,Fudan University 141
Classes of capture molecues
10/27/2005 Chaoqun Wu,Fudan University 142
Classes of capture
Molecues for protein
microarray
10/27/2005 Chaoqun Wu,Fudan University 143
(1) Protein-Protein Interactions
Protein microarrays can be used to screen rapidly for protein-protein
interactions,As a demonstration of this application,selected three
pairs of proteins that are known to interact,
protein G and IgG;
p50 (of the NF-kappa-B complex) and I-kappa-B-alpha;
FRB domain of FRAP and FKBP12,
While the first two interactions occur without any special
requirements,the third interaction in dependent on the presence of
the small molecule rapamycin,When array the first protein of each
pair in quadruplicate on five identical aldehyde slides and probed
each slide with a different fluorescently labeled protein,The results
are shown below
10/27/2005 Chaoqun Wu,Fudan University 144
Detecting protein-protein interactions on
glass slides,
(A) Slide probed with BODIPY-FL-IgG.
(B) Slide probed with Cy3-I-kappa-B-
alpha.
(C) Slide probed with Cy5-FKBP12 and
100 nM rapamycin,
(D) Slide probed with Cy5-FKBP12 and
no rapamycin.
(E) Slide probed with a mixture of
BODIPY-FL-IgG,Cy3-I-kappa-B-alpha,
Cy5-FKBP12,and 100 nM rapamycin,
In all panels,BODIPY-FL,Cy3,
and Cy5 fluorescence were false-
colored blue,green,and red,
respectively.
10/27/2005 Chaoqun Wu,Fudan University 145
A single slide holding 10,800 spots,Protein G was spotted 10,799
times,with a single spot of FRB in row 27,column 109,The slide
was then probed with BODIPY-FL-IgG,Cy5-FKBP12,and 100 nM
rapamycin,BODIPY-FL and Cy5 fluorescence were false-colored
blue and red,respectively.
10/27/2005 Chaoqun Wu,Fudan University 146
(2) Kinase-Substrate Interactions
Protein microarrays can also be used to detect enzyme-substrate
interactions,as long as the enzyme modifies its substrate in a way
that can subsequently be detected,Since kinases modify their
substrates by adding a phosphate group,it is possible to screen for
kinase-substrate interactions by detecting the phosphorylation
event,
10/27/2005 Chaoqun Wu,Fudan University 147
(3) Characterized kinase-substrate
nullPKA and Kemptide (a peptide substrate for PKA);
null casein kinase II (CKII) and protein phosphatase inhibitor 2 (I-2);
null p42 MAP kinase (Erk2) and Elk1.
The protein substrates of each pair were spotted in
quadruplicate on three BSA-NHS slides and each slide
was incubated with a different kinase in the presence of
gamma-33P-ATP,To detect phosphorylation,the slides
were dipped in a photographic emulsion,incubated in the
dark for 4-10 days,and then developed by hand,This
resulted in deposition of silver grains directly on the
slide surface,which were then visualized with an
automated,scanning light microscope,
10/27/2005 Chaoqun Wu,Fudan University 148
Detecting the substrates of
protein kinases on glass
slides,
(A)Slide incubated with the
catalytic subunit of PKA.
(B) Slide incubated with CKII.
(C) Slide incubated with p42
MAP kinase (Erk2).
10/27/2005 Chaoqun Wu,Fudan University 149
(4) Small Molecule-Protein Interactions
Protein microarrays can be used to identify the protein
targets of biologically active small molecules.
10/27/2005 Chaoqun Wu,Fudan University 150
Three proteins for which small molecule ligands were
available,
Anti-DIG — a mouse monoclonal antibody that binds the
steroid digoxigenin;
Streptavidin — a bacterial protein that binds the common
vitamin biotin;
FKBP12 — a human immunophilin that binds the synthetic
pipecolyl alpha-ketoamide AP1497,
Small molecule ligands,
1,a derivative of
digoxigenin;
2,biotin;
3a,AP1497;
3b,AP1767;
3c,AP1780,
The compounds were
coupled to BSA through
their carboxyl groups
(via a flexible linker).
10/27/2005 Chaoqun Wu,Fudan University 151
Detecting small molecule-protein
interactions on glass slides.
(A) Slide probed with Alexa488-
BSA-DIG.
(B) Slide probed with Cy5-BSA-
biotin.
(C) Slide probed with Cy3-BSA-
AP1497.
(D) Slide probed with a mixture of
Alexa488-BSA-DIG,Cy5-BSA-
biotin,and Cy3-BSA-AP1497,
In all panels,Alexa488,Cy3,and
Cy5 fluorescence were false-colored
blue,green,and red,respectively.
10/27/2005 Chaoqun Wu,Fudan University 152
(5) Preparing Protein Microarrays by Soft-
Landing of Mass-Selected Ions
The use of mass spectrometry as a separation
method is expected to provide high selectivity
because components having different molecular
formulae,including isotopomers,can be separated
and individually soft-landed,
This should include separate glycoforms of
proteins,individual polysulfonated forms,and other
post translationally modified variants [ the mass
difference due to acetylation of an immunoglobulin
G antibody can be resolved with a mass resolution
of 3000 m/z].
Science,Vol,301,1351-1354,September 5,2003
10/27/2005 Chaoqun Wu,Fudan University 153
4,Data analysis
Proteins are to be labeled with Cy3 and
Cy5,respectively,using monofunctional
reactive dye.
To visualize fluorescence,the slides are to
be scanned using an fluorescence slide
scanner
10/27/2005 Chaoqun Wu,Fudan University 154
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10/27/2005 Chaoqun Wu,Fudan University 156
10/27/2005 Chaoqun Wu,Fudan University 157
The three steps involved in data
interpretation are,
1,data normalization,
2,data filtering
3,pattern identification.
10/27/2005 Chaoqun Wu,Fudan University 158
10/27/2005 Chaoqun Wu,Fudan University 159
Analysis using software
2D gels are transformed into high
resolution digital protein maps using
staining,imaging,and bioinformatics
software.
10/27/2005 Chaoqun Wu,Fudan University 160
Proprietary technologies and software allow common features
to be matched from gel to gel,enabling protein expression profiling
across different samples in a biological system,
10/27/2005 Chaoqun Wu,Fudan University 161
Bioinformatics Analysis
10/27/2005 Chaoqun Wu,Fudan University 162
5.蛋白质芯片具有巨大的应用前景
null蛋白质功能分析研究
null检测与疾病相关的蛋白质,进而用于疾病诊断
null药物筛选,
null检测蛋白质突变
null其他
10/27/2005 Chaoqun Wu,Fudan University 163
(1) In general,protein chips miniaturize many
types of protein measurements.
including,
nullprotein profiling,
nullprotein discovery,
nullprotein activities,
nullprotein structure,
nullidentification of protein-protein
nullprotein-small molecule interactions
10/27/2005 Chaoqun Wu,Fudan University 164
(2) Protein Biomarker Applications
Robust,highly reproducible data on biomarkers will
advance
many fields of medicine and drug discovery (see picture),
Toxicological biomarkers signal adverse responses to
medication,enabling clinicians to measure drug safety
and prescribe drugs to patients who will tolerate them,
Disease biomarkers enable
researchers to develop
pre-symptomatic diagnostic
tests,If the disease biomarker
has a direct role in a cellular
disease pathway,it also
constitutes a new drug target
10/27/2005 Chaoqun Wu,Fudan University 165
Potential diagnostic markers for
detection of prostate,bladder,
colorectal and ovarian cancers
were discovered in hours or
days,directly from biological
fluids or tissue samples,In a
recent study,researchers at the
National Cancer Institute and the
FDA used Ciphergenas
technology to generate highly
reproducible protein,fingerprints”
correlated with specific phases
of prostate,breast and colorectal
cancer progression,
10/27/2005 Chaoqun Wu,Fudan University 166
E,Two-Hybrid Assay
Modern
Genetic
Analysis.
Interaction
between two
proteins
10/27/2005 Chaoqun Wu,Fudan University 167
F,Structural Proteomics
Pioneering work is undergoing by Baumeister et
al,which can significantly reduce the amount of
painstaking labor in the crystallization of proteins.
Current techniques are not considered,high
throughput” within the structural realm.
Novel solutions combine current technologies,
such as NMR and XRC.
10/27/2005 Chaoqun Wu,Fudan University 168
G,Informatics
Significant improvements are needed in:
– Data presentation standards and formatting
– Software infrastructure
ISB - have created many powerful software packages that
interpret data from different techniques.
EBI and HUPO have come together to promote uniform data
storage and analysis:
– http://psidev.sourceforge.net
The proteomics community has,over the course of the past ten
years,become slightly,less proprietary.” Ron Beavis of U,
Manitoba has developed x! tandem,an open-source search
algorithm as an alternative to SEQUEST.
Development of novel software for both analysis and strategies [for
biologists?] to manage the data are two fronts that we can see as
opportunities for folks with a CS background.
Part VI.
Structural
Proteomics
10/27/2005 Chaoqun Wu,Fudan University 2
Structural proteomics
Structural proteomics has the goal of
obtaining useful,three-dimensional
models of all proteins by a combina-
tion of experimental structure
determination and comparative model
building.
10/27/2005 Chaoqun Wu,Fudan University 3
The experimental (left) and computational (right)
hierarchies will increasingly become codependent as the
research community models greater biological complexity.
10/27/2005 Chaoqun Wu,Fudan University 4
Global structural genomics efforts will be major players in
Completing the protein family and fold landscape,
10/27/2005 Chaoqun Wu,Fudan University 5
10/27/2005 Chaoqun Wu,Fudan University 6
It is feasible to determine at least 10,000
protein structures within the next five years,
No new discoveries or methods are needed
for this task.
The average cost per structure is expected
to decrease significantly from $200,000 per
structure to perhaps less than $20,000 per
structure,
10/27/2005 Chaoqun Wu,Fudan University 7
Large-scale protein structure modeling.
A small sample of the 1,100 comparative models calculated
for the proteins in the yeast genome is displayed over an
image of a yeast cell
10/27/2005 Chaoqun Wu,Fudan University 8
Experimental structure determination
蛋白质的生物活性检测选择性蛋白质分子的突变蛋白质的体外翻译
10/27/2005 Chaoqun Wu,Fudan University 9
1,Target selection (Grouping)
The targets for structure determination will be
1,Individual domains rather than multi-domain
proteins (Chris Sander,Millenium Information),
2,Easier to determine by X-ray crystallography or
NMR spectroscopy than the more flexible multi-
domain proteins.
3,Done by pairwise comparison of all protein
sequences,followed by clustering into groups of
domains,
4,The representatives of the groups without
structurally defined members,
5,Members that share at least 30% sequence
identity (30-seq families),rather than those that
correspond to superfamiles or fold families.
10/27/2005 Chaoqun Wu,Fudan University 10
A variety of different criteria likely will be used
for this task; for example:
null size of the family,
null biological knowledge about the family,
null distribution of the members among various
organisms,
null the pharmaceutical relevance,
null the likelihood of a successful structure
determination,
and so forth,
10/27/2005 Chaoqun Wu,Fudan University 11
2,Cloning,expression and purification
Obtaining protein samples for crystallization
trials and NMR measurements likely will be the
bottleneck in the structural genomics process,
Although the current methods are sufficiently
powerful to begin the project,entirely new
technologies may arise,such as an in vitro system
that already has allowed researchers to produce
proteins up to concentrations of 6 mg / ml
(Shigeyuki Yokoyama,Tokyo University).
10/27/2005 Chaoqun Wu,Fudan University 12
3,Crystallization and X-ray crystallography
Crystals have a higher chance of being formed
when the protein species in solution is
Conformationally homogeneous,This can be
evaluated by dynamic light scattering,as well
as by limited proteolysis combined with mass
spectroscopy,For example,candidates that
are monodisperse under standard aqueous
conditions have a probability of at least 75% to
yield crystals suitable for X-ray studies,in
comparison to 10% for poly-disperse samples,
10/27/2005 Chaoqun Wu,Fudan University 13
10/27/2005 Chaoqun Wu,Fudan University 14
Atomic resolution structure
determinations of proteins by X-
ray crystallography require:
protein construct design,
expression and purification,
crystallization trials,
phase determination,
model building.
10/27/2005 Chaoqun Wu,Fudan University 15
Mass Spectrometry can be used to
1,verify that the desired protein construct has been
correctly expressed,
2,to define compact domains in the target protein,
3,to assess the components contained within the
protein crystals,
4,to screen for successful incorporation of seleno-
methionine and other heavy metal reagents used for
phasing,
5,to address issues of modeling,topology,and side-
chain proximity.
10/27/2005 Chaoqun Wu,Fudan University 16
VERIFICATION
1) Mass spectrometry’s unsurpassed accuracy and
speed for measuring mass allows one to quickly
test whether a given protein has been faithfully
expressed.
2) The measurement allows for detection of PCR
errors,mistranslation errors,unwanted
modifications (e.g.,from attachment of beta-
mercaptoethanol to thiols or oxidation of
methionyl residues),and major protein impurities
and byproducts(e.g.,from degradation).
3) detecting desired modifications such as
phosphorylation.
10/27/2005 Chaoqun Wu,Fudan University 17
DOMAIN ELUCIDATION
Numerous computer-aided approaches to help
define folding domains are available,These include
the use of homologous sequence alignment tools,
secondary structure prediction software,and various
“threading” algorithms,Progress in genomic
sequencing has greatly facilitated homology
searching,and there are increasingly available more
reliable secondary structure prediction programs.
In cases of sequences that exhibit low homology
alignments or poorly predicted secondary structural
elements.
Limited proteolysis combined with MS
10/27/2005 Chaoqun Wu,Fudan University 18
10/27/2005 Chaoqun Wu,Fudan University 19
PROTEIN CRYSTAL ANALYSIS
Determining the Presence of the
Expected Protein Components
Examining Alteration of Proteins
During Crystallization
Assessing Heavy Metal Incorporation
10/27/2005 Chaoqun Wu,Fudan University 20
10/27/2005 Chaoqun Wu,Fudan University 21
10/27/2005 Chaoqun Wu,Fudan University 22
PHASING
Extraction of electron density and structural
information from X-ray diffraction data of
protein crystals requires knowledge of the
magnitudes and phase angles of the
diffracted X-rays,
The diffraction data itself contains only the
magnitudes; the associated phases must
be obtained by other means.
10/27/2005 Chaoqun Wu,Fudan University 23
Available,phasing” methods are isomorphous
replacement and molecular replacement
techniques.
In isomorphic replacement,a heavy atom is
introduced into the protein with minimal
perturbation of the native protein fold,
Phasing information is obtained from X-ray
diffraction of a crystal of heavy-atom
derivatized protein.
Heavy atoms used for phasing range in size
from selenium to uranium.
10/27/2005 Chaoqun Wu,Fudan University 24
MODEL BUILDING AND REFINEMENT
After the electron density has been determined
from an X-ray diffraction study of a protein,a
model of the protein structure is built,One
question that frequently arises during model
building is whether a stretch of sequence that
is not observed in the electron density map
is either disordered or altogether missing in
the protein crystal (perhaps as a result of
degradation over time)
10/27/2005 Chaoqun Wu,Fudan University 25
intramolecular protein cross-linking in
combination with mass spectrometry
provided restraint information that is
potentially useful for protein model
building
Limited proteolysis,in conjunction with
mass spectrometry,
10/27/2005 Chaoqun Wu,Fudan University 26
A Case Study
The KcsA potassium channel,
from Streptomyces lividans,
consists of four identical
integral membrane protein
subunits.
10/27/2005 Chaoqun Wu,Fudan University 27
Initial protein constructs were obtained
by purifying a full-length C-terminal
His-Tag protein (170 residues; 18.8
kDa),followed by trypsin treatment to
remove the His-Tag,
Although the trypsin treatment yielded
sufficient quantities of protein,this
material did not produce diffraction
grade crystals.
10/27/2005 Chaoqun Wu,Fudan University 28
Domain elucidation
1,Detergents (e.g.,Mega-9,LDAO) were
found to be suitable for protein purification
and crystallization and did not interfere
with MALDI-MS analysis
2,Proteolysis experiments with trypsin,
chymotrypsin,and subtilisin,then mapped
by MALDI-MS.
10/27/2005 Chaoqun Wu,Fudan University 29
PROTEOLYSIS RESULTS:
Each protease quickly removed about 45 residues
from the C-terminal of the His-Tag KcsA,leaving a
122-residue (by trypsin) or 125-residue (by
chymotrypsin or subtilisin) core domain,There was
little indication that the 45-residue portion that was
proteolytically trimmed from the C-terminal of full-
length KcsA had any inherent structure,
Beyond one day of digestion,the C-terminally-
truncated protein began to show an additional
cleavage site 19 residues (by trypsin) or 25 residues
(by subtilisin) from the N-terminal,Chymotrypsin did
not cleave anywhere in the N-terminal.
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Another problem:
MS revealed that in addition to the expected
chymotryptic Cleavage product (KcsA 1-125),
Cleavage product (KcsA 1-125),an
unanticipated second population of the protein
appeared (KcsA 20-125),not easily
distinguished from the expected product by
SDS-PAGE,and its presence likely would have
adversely affected the crystallization trials.
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Because of trypsin,a common
contaminant found in chymotrypsin
preparations,To avoid the trypsin
activity,TLCK-treated chymotrypsin
was used to ensure a homogenous
population of proteolytically resistant
KcsA,
This chymotrypsin construct provided
high quality diffracting crystals of the
potassium channel.
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Phasing of the KcsA
needs cysteinyl residues
where to place the cysteine within the
protein,different mercurating reagents.
They all need MS to see if it’s right
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Finally,mass spectrometry
was used to assist in the
model building process
that yielded the beautiful
and informative structure
10/27/2005 Chaoqun Wu,Fudan University 34
Ribbon
representa
tion of the
KcsA
potassium
channel
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Detail of the electron
density map of
Deacetoxycephalosporin
C synthase from
Streptomyces clavuligerus.
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4,Nuclear magnetic resonance
(NMR) spectroscopy
Recent and prospective advances in NMR
spectroscopy that will allow structure
determination of larger proteins as well as
increase the accuracy and speed of the analysis,
These advances include,
nullPartial deuteration of protein samples,
nullNew magnets (up to 1 GHz),
nullSuperconducting probes,
nullSoftware for resonance assignments and
structure determination,
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5,Comparative model building
null Templete selection
null Comparative modeling
null Function prediction
10/27/2005 Chaoqun Wu,Fudan University 38
Templete
selection
Protein fold assignment.
A genome-encoded amino acid
sequence (center) is tested for
compatibility with a library of
known 3D protein folds.
An actual library would contain
of the order of 1000 folds; the
one shown here illustrates the
most common protein folding
motifs,
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High-accuracy comparative models are based on
more than 50% sequence identity to their templates,
Comparative modeling
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Before Modeling
1,Identification of modelling templates,
2,Comparative protein modeling requires
at least one sequence of known 3D-
structure with significant similarity to the
target sequence,
3,Aligning the target sequence with the
template Sequence,
10/27/2005 Chaoqun Wu,Fudan University 41
Step By Step Work
Secondary Structure and Backbone
Conformation
Super-secondary structure
Side Chain Conformation
Tertiary Protein Structure and folds
Quaternary Structure
Comparative protein modelling
To evaluate the quality of a model
Secondary Structure and Backbone
Super-secondary structure
Side Chain Conformation
Tertiary Protein Structure and folds
Quaternary Structure
Comparative protein modelling
To evaluate the quality of a model
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Secondary Structure and Backbone
Conformation
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Detail from a three-
dimensional
structure of space-
grown insulin
crystals,
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Framework construction
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Completing the backbone
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Super-secondary structure
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Side Chain Conformation
Here are some conformations
that can be adopted by
Arginines:
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Adding side chains
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Tertiary Protein Structure and folds
The lone helix The helix-turn-helix motif
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The four-helix bundle
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Quaternary Structure
Tertiary domains
Aggregating
together
to form a unit,
The photoreaction
centre is a good
example,
10/27/2005 Chaoqun Wu,Fudan University 53
6,Function prediction
The similarities and differences between two plant and archeal
members of a family of glycosidases that includes a protein
implicated in ageing,
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7,Datebase and software
Databases
Several different databases will be useful in
facilitating the structural genomics project,These
databases will be used for,
1,Target selection,
2,Coordination of the experimental efforts,
3,Archiving of the new structures,
4,Dissemination of the results,
5,Performing new research with the old and new
data,
10/27/2005 Chaoqun Wu,Fudan University 56
Using the structures
Development of the databases and software
will be necessary to integrate raw structural
information and structure-derived results with
other information,such as:
null Genetic and biochemical analysis,
null Gene expression patterns,
null Mutational analysis,
null Binding data from protein chips,
null Interaction data from two-hybrid systems,
null Metabolic pathway information,
and so forth,
10/27/2005 Chaoqun Wu,Fudan University 57
蛋白质水平的分析蛋白质水平的分析蛋白分子的等电点分析蛋白分子的亲水性分析蛋白分子的抗原性分析进化树分析氨基酸糖基化位点预测同源性分析同源性分析功能域分析功能域分析结构域分析结构域分析膜蛋白分析膜蛋白分析分泌蛋白分析分泌蛋白分析立体结构分析立体结构分析蛋白分子建模蛋白分子建模
10/27/2005 Chaoqun Wu,Fudan University 58
结构分析结构分析蛋白质的结构对于蛋白质发挥功能非常重蛋白质的结构对于蛋白质发挥功能非常重要，现在已经有许多蛋白质的三级结构被要，现在已经有许多蛋白质的三级结构被测定，可以通过同源模建的方法分析目标测定，可以通过同源模建的方法分析目标蛋白的三级结构。
蛋白的三级结构。
工具：
工具：
EXPASY的蛋白分析工具或的蛋白分析工具或
NCBI的的
STRUCTURE。
。
10/27/2005 Chaoqun Wu,Fudan University 59
A computational method has been developed for the assignment
of a protein sequence to a folding class in the Structural Classifica-
tion of Proteins (SCOP),Current data places the number between
1,000 and 5,000 distinct protein folds.
This method uses global descriptors of a primary protein seque-
nce in terms of the physical,chemical,and structural properties of
the constituent amino acids.
The method is applicable for the fold assignment of any protein
sequence with or without significant sequence homology to known
proteins,A WWW page for predicting protein folds is available at
URL http://cbcg.lbl.gov/.
Structural Classification of Proteins
(SCOP)
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EXPASY
This is the ExPASy (Expert Protein Analysis System)
proteomics server of the Swiss Institute of
Bioinformatics (SIB),This server is dedicated to the
analysis of protein sequences and structures as well
as 2-D PAGE (Disclaimer
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8,Applications of Strutural Genomics
nullFunction Studies
nullDrug Development
nullMetabolic Pathway
10/27/2005 Chaoqun Wu,Fudan University 68
iNOS,Cys
194
,Glu
371
.
Oxidase-domain eNOS
nNOS
Example
10/27/2005 Chaoqun Wu,Fudan University 69
iNOS,
Cys
194
-heme binding,
Glu
371-
Arginin binding.
10/27/2005 Chaoqun Wu,Fudan University 70
Example:
Mechanism-based
Design of a protein
kinase inhibitor
nature structural biology?
volume 8 number 1?
january 2001 37
Keykavous Parang et al..
10/27/2005 Chaoqun Wu,Fudan University 71
Protein kinases play a critical role in cell signaling
Pathways by catalyzing the transfer of the
γ-phosphoryl group from ATP to the hydroxyl groups
of protein side chains,The protein kinase superfamily
is one of the largest as ～2% of eukaryotic genes encode
them,Because of their importance in contributing to a
variety of pathophysiologic states,including cancer,
inflammatory conditions,autoimmune disorders,and
cardiac diseases,intense efforts have been made to
develop specific protein kinase inhibitors as biological
tools and as therapeutic agents,
γ
～
10/27/2005 Chaoqun Wu,Fudan University 72
Example:
Crystal structure of the binary
complex between cIRK and the
bisubstrate inhibitor,
a,Overall view of the binary
complex in which cIRK is shown in
surface representation and the
bisubstrate inhibitor in stick
representation,
b,Stereo view of the Fo - Fc electron
density for the bisubstrate inhibitor
computed after simulated annealing
(1,000 K),
c,Stereo view of the interactions
between the inhibitor and key
catalytic residues,
10/27/2005 Chaoqun Wu,Fudan University 73
Accuracy and application of
protein structure models.
The different ranges of applic-
ability of comparative protein
Structure modeling,threading,
and de novo structure prediction,
The corresponding accuracy of
protein structure models; and
Their sample applications,
The accuracy of the models
decrease significantly in going from
(A) to (E),but the overall structure
is still roughly correct,
10/27/2005 Chaoqun Wu,Fudan University 74
Part VII.
Platforms
for proteomics
10/27/2005 Chaoqun Wu,Fudan University 75
Pillar Proteomic Technologies
Amino Acid Composition
Array-based Proteomics
2D PAGE ( LC 2D)
Mass Spectrometry
Structural Proteomics
Informatics (and the challenges facing the
Human Proteome Project)
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Platforms for proteomics and functional
genomics,Methodology is shown in the outer
columns,resultant data sets in the middle
columns,and model systems in the centre,
Nature 422,193 - 197 (13 March 2003)
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A,2D gel analysis
2D-gel of
erythroleukemia
cell
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双向凝胶电泳
（two-dimensional gel electrophoresis,2-DE）
基本原理是蛋白质首先根据其等电点在pH
梯度胶中等电聚焦，然后按照它们的分子量大小进行SDS-PAGE第二次电泳分离。
第一向等电聚焦条件和方式可根据待分离的蛋白质分子特性的不同而变化。例如pH梯度
(immobilized pH gradients)中，pH梯度范围可按所需设置。
10/27/2005 Chaoqun Wu,Fudan University 79
2D PAGE
2-D gel electrophoresis is a multi-step
procedure that can be used to separate
hundreds to thousands of proteins with
extremely high resolution,
It works by separation of proteins by their
pI's in one dimension using an immobilized
pH gradient (first dimension,isoelectric
focusing) and then by their MW's in the
second dimension.
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2D PAGE
2-D gel electrophoresis process consists of
these steps,
Sample preparation
First dimension,isoelectric focusing
Second dimension,gel electrophoresis
Staining
Imaging analysis via software
10/27/2005 Chaoqun Wu,Fudan University 81
Advances in 2D gel electrophoresis are
revolutionizing proteomics by providing a
means to separate different proteins for further
analysis.
Protein analysis uses a diseased or treated
sample and a control sample,2D gel
electrophoresis is performed for each sample to
separate proteins based on their molecular
weight and charge,
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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 83
Two-Dimensional Gel
Electrophoresis
Protein separation
Identification of
the proteins
corresponding to
the selected
spots
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Examples of 2D gels
molecular weight (kDa)
pH
2D separation of
human lymphoma
proteins
Many sources of proteins possible
10/27/2005 Chaoqun Wu,Fudan University 85
2D map of WI-38 human lung fibroblasts
IEFNEpHGE
I.P,I.P.
S
S
S
D
S
M
.
W
.
(
k
D
)
M
.
W
.
(
k
D
)
D
10/27/2005 Chaoqun Wu,Fudan University 86
B,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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Drawbacks of 2D PAGE
Technique precision lacks reliable
reproduction.
Spots often overlap,making identifications
difficult.
More of,an art” than,a science.”
Slow and tedious.
Process contains may,open” phases
where contamination is possible.
10/27/2005 Chaoqun Wu,Fudan University 89
Advantages vs,Disadvantages
Good resolution of
proteins
Detection of post-
translational
modifications
Not for
hydrophobic
proteins
Limited by pH
range
Not easy for low
abundant proteins
Analysis and
quantification are
difficult
10/27/2005 Chaoqun Wu,Fudan University 90
2-D凝胶电泳的挑战和问题
1.低丰度蛋白质的检测、灵敏度不够
2.不同实验间蛋白质所在位置的漂移
3.不能全面代表膜蛋白或疏水蛋白的真实情况
5.某些蛋白质在等电聚焦缓冲液中低溶解度(Insolubility)
6.特大分子量的蛋白质的检测难
7.特小分子量的蛋白质的检测难
8.某些蛋白质带电荷的微小差异(Charge microheterogeneity)
9.容易受污染(Susceptibility to contamination)
10.处在变性的条件(Requirement for denaturing conditions)
10/27/2005 Chaoqun Wu,Fudan University 91
C,2-D analysis by liquid
chromatography(LC)
Gel filtration
Ion-exchange
Hydroxyapatite columns
Hydrophobic
Immobilized reactive dyes
Affinity
Chromatofocusing
HPLC
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Typical proteomics experiment using gel
electrophoresis
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2D - LC/LC
(trypsin)
Peptides all bind
to cation
exchange column
Study protein
complexes
without gel
electrophoresis
Successive elution
with increasing salt
gradients separates
peptides by charge
Peptides are
separated by
hydrophobicity on
reverse phase
column
Complex mixture is
simplified prior to
MS/MS by 2D LC
10/27/2005 Chaoqun Wu,Fudan University 94
Reverse Phase column
Polypeptides enter the column in the mobile phase…
…the hydrophobic,foot” of the polypeptides adsorb to the
hydrophobic (non polar) surface of the reverse-phase
material (stationary phase) where they remain until…
…the organic modifier concentration rises to critical
concentration and desorbs the polypeptides
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2D - LC/MS
10/27/2005 Chaoqun Wu,Fudan University 96
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But 2D-PAGE is not a best choice for proteome analysis
because of individual extraction,digestion,and analysis of
each spot from 2D-PAGE is a tedious and time-
consuming process.
The largest 2D-PAGE-based proteome study to date
identified 502 unique proteins for the Hemophilus
influenzae proteome.
Fundamental limitation of 2D gel electrophoresis is its
failure to display the entire protein complement of sample
on the gel surface.
10/27/2005 Chaoqun Wu,Fudan University 98
Large proteins and hydrophobic proteins such as
membrane proteins have difficulty entering the gel.
Acidic proteins(PI<4) or basic proteins(PI>10) are poorly
resolved,Most importantly,the levels of low-copy-number
proteins present in low abundance are below the detection
limit of silver staining techniques.
This limitation is probably most critical because many of the
regulatory proteins that might be involved in the disease
process and are candidates for many drugs are present in
the cell in low concentrations.
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Liquid Chromatography
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Liquid Chromatography
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nullProteins Fractionated using 2D Chromatography
nullProteins resolved by both chromatofocusing and
reversed phase chromatography
nullProteins unique to diseased state - Isolated
*
*
*
*
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ICAT（Isotope Coded Affinity Tages,同位素标记亲和标签）
10/27/2005 Chaoqun Wu,Fudan University 104
Differential LC/MSMS
10/27/2005 Chaoqun Wu,Fudan University 105
Advantage of 2 dimensional liquid
chromatography
When reversed phase is preceded by ion exchange
1,The combined effect of the two chemistries
procedures better purification than either
chemistry alone.
- Ion exchange and reversed phase are
orthogonal separation method,The effects
of the two methods are synergistic; final
resolution is greater than with either
method alone
10/27/2005 Chaoqun Wu,Fudan University 106
2,The ion-exchange chromatogram
can aid locating the desired product
- Sometimes reversed-phase analysis of a
crude synthetic peptide does not clearly
indicate a main peak,When ion exchange run
first,the last major peak on the ion-exchange
chromatogram usually contains the peptide of
interest.
10/27/2005 Chaoqun Wu,Fudan University 107
3,Hydrophbic reaction products are
removed by the ion-exchange step
- This reduces the loading capacity required
for the reversed-phase separation,In addtion,
fouling of the reversed-phase column that
may require cleaning with IPA or stronger
solvents is eliminated.
10/27/2005 Chaoqun Wu,Fudan University 108
4,Strongly acidic cleavage reagent
are removed prior to reversed-phase
- HF and TFA reagents used in cleavage
reactions can attack a silca-based reversed-
phase column,An initial step of ion-exchange
on a chemically resistant polymer-based
column removes these reagents,thereby
protecting the reversed-pahse column.
10/27/2005 Chaoqun Wu,Fudan University 109
Advantages vs,Disadvantages
Determination
of MW and aa,
Sequence
Detection of
posttranslational
modifications
High-throughput
capability
High capital costs
Requires sequence
databases for
analysis
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MudPIT (multidimensional protein
identification technology)
多维液相色谱与串联质谱仪在线联用
Consist of
1,Multidimensional liquid chromatography
2,Tandem mass spectrometry
3,Database searching by the SEQUEST
algorithm
10/27/2005 Chaoqun Wu,Fudan University 111
Yates’ group suggest non-gel-based
chromatography systems to resolve and identify
thousands of proteins from biological sample.
The acidified complex peptide mixture is applied to
a strong cation exchange(SCX) chromatography
column,and a discrete fraction of the absorbed
peptides are displaced onto a reversed-phase(RP)
chromatography column using a salt step gradient.
Peptides are retained on the RP column,but
contaminating salts and buffers are washed away
and diverted to waste.
The peptides are then eluted from the RP column
into the mass spectrometer using a gradient of
increasing acetonitrile.
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Multidimensional protein identification technology (MudPIT)
10/27/2005 Chaoqun Wu,Fudan University 113
Peptides all bind to cation
exchange column
Peptides are separated by
hydrophobicity on reverse
phase column
Successive elutions with
increasing salt gradients
separates peptides by
charge
Complex mixture is
simplified prior to
MS/MS by 2D LC
“MudPIT”
(trypsin)
Study protein
complexes
without gel
electrophoresis
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IEX-HPLC,Ion-exchange high-performance liquid chromatography
离子交换高效液相色谱法
RP-HPLC,Reverse-phase high-performance liquid chromatography
反相高效液相色谱法
MudPIT
Trypsin
+ proteins
p53
IEX-HPLC
RP-HPLC
10/27/2005 Chaoqun Wu,Fudan University 115
Additions to protein solvents
Class examples purpose
Buffer stability
Salts KCl,NaCl,stability
Detergents SDC,stability,
solubility
surfactant Triton X-100 stability,
solubility
Glycerol,sucrose stability
Sodium azide
bacteriostatic
Metal chelator ETDA,EGTA stability
Sulfhydryl agents DTT stability
ligands metals,ATP
stability
Protease inhibitors PMSF stability
10/27/2005 Chaoqun Wu,Fudan University 116
D,Array-based Proteomics
Offer a high-throughput technique for
proteome analysis.
These small plates are able to hold many
different samples at a time.
Current research is ongoing in an attempt
to interface array methodologies with
Mass Spectrometry at ORNL.
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Array-based Proteomics
10/27/2005 Chaoqun Wu,Fudan University 118
Protein Chips Offer Powerful
Method for Probing Protein
Function
10/27/2005 Chaoqun Wu,Fudan University 119
Functional protein microarrays are
critical for the next phase of proteomics
research.
"Profiling proteins will be invaluable,
for example,in distinguishing the proteins
of normal cells from early-stage cancer
cells,and from malignant,metastatic
cancer cells that are the real killers," said
HHMI investigator Stuart L,Schreiber.
10/27/2005 Chaoqun Wu,Fudan University 120
Like DNA chips,protein chips will be able
to analyze thousands of samples
simultaneously,leading the way towards
a complete map of the entire complement
of human proteins,
But,unlike DNA,proteins are not so easy
to attach to chips,Where DNA is robust,
and able to withstand harsh experimental
conditions,proteins are fragile and will
denature if they aren't treated gently,
10/27/2005 Chaoqun Wu,Fudan University 121
Functional protein microarrays are consi-
dered critical to progress in proteomics
research,And,not surprisingly,academic
and industrial researchers alike are expen-
ding considerable time and energy to come
up with an operable system,
Trends in Biotechnology Vol,20,No,4,April 2002
10/27/2005 Chaoqun Wu,Fudan University 122
Protein biochip
analytical system
will enable high-
throughput protein
discovery,protein
profiling,protein
structure and
activity analyses,
as well as protein-
protein and
protein-small
molecule
interaction studies.
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Proteins are extremely sensitive to the
physical and chemical properties of the
particular substrate,It is difficult to come
up witha so-called generic solid phase
material,
Proteins must remain in a liquid environ-
ment to retain their activity,
Proteins have one-,two- and three-dimen-
sional configurations as they transform
from a straight chain of amino acids into a
functional unit,
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The arrayer picks up about a microliter of sample from
four wells of a 96- or 384-well plate and deposits about
1 nanoliter of each sample at defined locations on a series
of glass microscope slides,The arrayer uses a pin and
ring system,
the samples are picked up in
small rings that each hold
about 1 microliter and a solid
pin (150 um diameter) then
punches repeatedly through
the ring to deposit the proteins
on the slides,To prevent
evaporation of the nanodroplets,
we include 40% glycerol in the
protein samples,Nanoliter
droplets of 40% glycerol remain
hydrated,even when left
exposedto the atmosphere
overnight.
1,Arraying the proteins
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Arraying Proteins on Glass Slides
The proteins were spotted on BSA-NHS
slides following a 3 hr incubation in a
humid chamber at room temperature,the
slides were inverted and dropped onto a
solution of PBS,pH 8.0 containing 500 mM
glycine,After 1 min,the slides were turned
right side up and immersed in the glycine
solution for 1 hr at room temperature with
gentle agitation,
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Chemically Derivatized Glass Slides
Protein chip slides,displaying activated
amino and carboxyl groups on the
surface of an immobilized layer of
bovine serum albumin (BSA),were
fabricated,
10/27/2005 Chaoqun Wu,Fudan University 130
2,Protein Array typing
(1) Hydrophobic Protein Arrays
Hydrophobic arrays bind proteins through
hydrophobic surface interaction with amino
acids such as alanine,valine,leucine,
isoleucine,phenylalanine,tryptophan and
tyrosine,
Binding occurs in aqueous,high salt
conditions and binding is reduced by
decreasing salt and increasing the
concentration of organics.
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(2) Hydrophilic Protein Arrays
Hydrophilic arrays bind proteins through
electrostatic and dipole-dipole interactions
as well as hydrogen binding,Proteins with
hydrophilic and charged surface animo acids
such as serine,threonine and lysine bind well.
Binding occurs in aqueous buffers with a water
wash prior to analysis.
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(3) Anion Exchange Protein Arrays
Anionic arrays bind proteins through
electrostatic interaction of negatively charged
amino acids such as aspartic acid and glutamic
acid,
Binding occurs at high pH with low salt and
binding decreases as pH decreases and salt
concentration increases.
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(4) Cation Exchange Protein Arrays
Cationic arrays bind proteins through
electrostatic interaction of positively charged
amino acids such as lysine,arginine and
histidine,
Binding occurs at low pH with low salt;
binding decreases as pH and salt
concentration increase.
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(5) Immobilized Metal affinity Protein Arrays
Immobilized metal affinity capture (IMAC) arrays
bind proteins and peptides which have affinity
for metals; proteins with exposed histidine,
tryptophan and/or cysteine typically bind to
metals immobilized on these chip surfaces,
Binding occurs under pH 6-8 and high salt and
decreases as the concentration of imidizole and
glycine increase.
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(6) Preactivated Protein Arrays
ProteinChip contains a carbonyldiimidazole
surface which covalently reacts with amine
groups,DNA and proteins,including antibodies,
can be immobilized on the slids surface.
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(7) 2-D gel is also a Protein Arrays
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(8) Gel- based protein chips
The gel is aqueous,porous (creating a
large surface area) and dimensional
(eliminating solid-liquid interfaces),
The gel substrate is truly three-dimensional
and porous," Burke explained,
It is speculated that the gel provides a
hydrophilic environment that lends itself
to protein-protein interactions,and that
these proteins behave more naturally.
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(9) Silicon- silane based protein chip
Coat the chip surface with a film of silicon
dioxide,Then the molecules of interest
are attached covalently via a silane
coupling reagent to offer ready-to-use
chips as well as pre-activated chips,
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(10) Protein-antibody based chips
The protein-antibody pairs selected will
be used to develop a new generation of
microarrays for detecting proteins,For
instance,imagine creating biochips for
membrane proteins,
……
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3,Classes of capture molecues
for protein microarray
An attachment tag is added to the top layer,
and a protein capture agent (such as an
antibody fragment,a peptide or even a novel
scaffold of antibody-like molecules) gets
bound to its free end,These protein capture
agents can be tailored specifically to snag
the proteins of interest during the actual
experiment.
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Classes of capture molecues
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Classes of capture
Molecues for protein
microarray
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(1) Protein-Protein Interactions
Protein microarrays can be used to screen rapidly for protein-protein
interactions,As a demonstration of this application,selected three
pairs of proteins that are known to interact,
protein G and IgG;
p50 (of the NF-kappa-B complex) and I-kappa-B-alpha;
FRB domain of FRAP and FKBP12,
While the first two interactions occur without any special
requirements,the third interaction in dependent on the presence of
the small molecule rapamycin,When array the first protein of each
pair in quadruplicate on five identical aldehyde slides and probed
each slide with a different fluorescently labeled protein,The results
are shown below
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Detecting protein-protein interactions on
glass slides,
(A) Slide probed with BODIPY-FL-IgG.
(B) Slide probed with Cy3-I-kappa-B-
alpha.
(C) Slide probed with Cy5-FKBP12 and
100 nM rapamycin,
(D) Slide probed with Cy5-FKBP12 and
no rapamycin.
(E) Slide probed with a mixture of
BODIPY-FL-IgG,Cy3-I-kappa-B-alpha,
Cy5-FKBP12,and 100 nM rapamycin,
In all panels,BODIPY-FL,Cy3,
and Cy5 fluorescence were false-
colored blue,green,and red,
respectively.
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A single slide holding 10,800 spots,Protein G was spotted 10,799
times,with a single spot of FRB in row 27,column 109,The slide
was then probed with BODIPY-FL-IgG,Cy5-FKBP12,and 100 nM
rapamycin,BODIPY-FL and Cy5 fluorescence were false-colored
blue and red,respectively.
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(2) Kinase-Substrate Interactions
Protein microarrays can also be used to detect enzyme-substrate
interactions,as long as the enzyme modifies its substrate in a way
that can subsequently be detected,Since kinases modify their
substrates by adding a phosphate group,it is possible to screen for
kinase-substrate interactions by detecting the phosphorylation
event,
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(3) Characterized kinase-substrate
nullPKA and Kemptide (a peptide substrate for PKA);
null casein kinase II (CKII) and protein phosphatase inhibitor 2 (I-2);
null p42 MAP kinase (Erk2) and Elk1.
The protein substrates of each pair were spotted in
quadruplicate on three BSA-NHS slides and each slide
was incubated with a different kinase in the presence of
gamma-33P-ATP,To detect phosphorylation,the slides
were dipped in a photographic emulsion,incubated in the
dark for 4-10 days,and then developed by hand,This
resulted in deposition of silver grains directly on the
slide surface,which were then visualized with an
automated,scanning light microscope,
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Detecting the substrates of
protein kinases on glass
slides,
(A)Slide incubated with the
catalytic subunit of PKA.
(B) Slide incubated with CKII.
(C) Slide incubated with p42
MAP kinase (Erk2).
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(4) Small Molecule-Protein Interactions
Protein microarrays can be used to identify the protein
targets of biologically active small molecules.
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Three proteins for which small molecule ligands were
available,
Anti-DIG — a mouse monoclonal antibody that binds the
steroid digoxigenin;
Streptavidin — a bacterial protein that binds the common
vitamin biotin;
FKBP12 — a human immunophilin that binds the synthetic
pipecolyl alpha-ketoamide AP1497,
Small molecule ligands,
1,a derivative of
digoxigenin;
2,biotin;
3a,AP1497;
3b,AP1767;
3c,AP1780,
The compounds were
coupled to BSA through
their carboxyl groups
(via a flexible linker).
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Detecting small molecule-protein
interactions on glass slides.
(A) Slide probed with Alexa488-
BSA-DIG.
(B) Slide probed with Cy5-BSA-
biotin.
(C) Slide probed with Cy3-BSA-
AP1497.
(D) Slide probed with a mixture of
Alexa488-BSA-DIG,Cy5-BSA-
biotin,and Cy3-BSA-AP1497,
In all panels,Alexa488,Cy3,and
Cy5 fluorescence were false-colored
blue,green,and red,respectively.
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(5) Preparing Protein Microarrays by Soft-
Landing of Mass-Selected Ions
The use of mass spectrometry as a separation
method is expected to provide high selectivity
because components having different molecular
formulae,including isotopomers,can be separated
and individually soft-landed,
This should include separate glycoforms of
proteins,individual polysulfonated forms,and other
post translationally modified variants [ the mass
difference due to acetylation of an immunoglobulin
G antibody can be resolved with a mass resolution
of 3000 m/z].
Science,Vol,301,1351-1354,September 5,2003
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4,Data analysis
Proteins are to be labeled with Cy3 and
Cy5,respectively,using monofunctional
reactive dye.
To visualize fluorescence,the slides are to
be scanned using an fluorescence slide
scanner
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The three steps involved in data
interpretation are,
1,data normalization,
2,data filtering
3,pattern identification.
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Analysis using software
2D gels are transformed into high
resolution digital protein maps using
staining,imaging,and bioinformatics
software.
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Proprietary technologies and software allow common features
to be matched from gel to gel,enabling protein expression profiling
across different samples in a biological system,
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Bioinformatics Analysis
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5.蛋白质芯片具有巨大的应用前景
null蛋白质功能分析研究
null检测与疾病相关的蛋白质,进而用于疾病诊断
null药物筛选,
null检测蛋白质突变
null其他
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(1) In general,protein chips miniaturize many
types of protein measurements.
including,
nullprotein profiling,
nullprotein discovery,
nullprotein activities,
nullprotein structure,
nullidentification of protein-protein
nullprotein-small molecule interactions
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(2) Protein Biomarker Applications
Robust,highly reproducible data on biomarkers will
advance
many fields of medicine and drug discovery (see picture),
Toxicological biomarkers signal adverse responses to
medication,enabling clinicians to measure drug safety
and prescribe drugs to patients who will tolerate them,
Disease biomarkers enable
researchers to develop
pre-symptomatic diagnostic
tests,If the disease biomarker
has a direct role in a cellular
disease pathway,it also
constitutes a new drug target
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Potential diagnostic markers for
detection of prostate,bladder,
colorectal and ovarian cancers
were discovered in hours or
days,directly from biological
fluids or tissue samples,In a
recent study,researchers at the
National Cancer Institute and the
FDA used Ciphergenas
technology to generate highly
reproducible protein,fingerprints”
correlated with specific phases
of prostate,breast and colorectal
cancer progression,
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E,Two-Hybrid Assay
Modern
Genetic
Analysis.
Interaction
between two
proteins
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F,Structural Proteomics
Pioneering work is undergoing by Baumeister et
al,which can significantly reduce the amount of
painstaking labor in the crystallization of proteins.
Current techniques are not considered,high
throughput” within the structural realm.
Novel solutions combine current technologies,
such as NMR and XRC.
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G,Informatics
Significant improvements are needed in:
– Data presentation standards and formatting
– Software infrastructure
ISB - have created many powerful software packages that
interpret data from different techniques.
EBI and HUPO have come together to promote uniform data
storage and analysis:
– http://psidev.sourceforge.net
The proteomics community has,over the course of the past ten
years,become slightly,less proprietary.” Ron Beavis of U,
Manitoba has developed x! tandem,an open-source search
algorithm as an alternative to SEQUEST.
Development of novel software for both analysis and strategies [for
biologists?] to manage the data are two fronts that we can see as
opportunities for folks with a CS background.
