Exploring Proteins and Proteomes. A collective name for the genes existed in an organism C. elegance...
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Transcript of Exploring Proteins and Proteomes. A collective name for the genes existed in an organism C. elegance...
Exploring Proteins and Proteomes
• A collective name for the genes existed in an organism
• C. elegance (roundworm) : 97 million bases, 19,000 genes
Drosophila melanogaster (fruit fly) : 180 million bases, 14,000 genes
Human : 3 billion bases, 25,000 genes
• Static and absolute information
Genome
• A collective name for the proteins expressed by the genome
• Dynamic and functional information
• It varies with cell type, developmental stage, and environmental condition
such as the presence of hormones.
• Regulation of mRNA synthesis, alternative splicing, mRNA stability, rate of
protein synthesis, post-translational modification, protein stability control,
protein degradation
Proteome
• The purification of proteins is an essential first step in understanding their
function.
• Purification should yield a sample of protein containing only one type of
molecule of interest.
• Proteins can be separated from one another on the basis of solubility, size,
charge, and binding ability.
• Assay : a test for some unique identifying property of the protein
• Specific Activity : the ratio of enzyme activity to the amount of protein in the
enzyme assay
Protein Purification
NADHcan absorb light
at 340 nm.
Nicotinamide adenine dinucleotide
Homogenation and Fractionation by Centrifugation
• Salting Out : protein solubility decrease by very high concentration of salt
• Salting In : protein solubility increase by low concentration of salt
• Dialysis : separation of small molecules from proteins through membrane with
pores such as cellulose membrane (cf. semi-permeable)
Salting Out & Dialysis
Ammonium sulfate for protein precipitation
Gel-Filtration Chromatography(Molecular Exclusion,
Size Exclusion,Molecular Sieve)
1. Thyroglobulin (669 kd)
2. Catalase (232 kd)
3. BSA (67 kd)
4. Ovalbumin (43 kd)
5. Ribonuclease (13.4 kd)
Anion ExchangerPositively Charged Column
Negatively Charged Proteins
Cation ExchangerNegatively Charged ColumnPositively Charged Proteins
Ion-ExchangeChromatography
Depend on local charge on proteins
LCLiquid Chromatography
FPLCFast Pressure Liquid Chromatography
HPLCHigh Pressure Liquid Chromatography
AffinityChromatography
(concanavalin A)
Highly specific- His tag
Gel Electrophoresis
v = Ez / fv : velocity of migrationE : electric field strength
z : net charge on the proteinf : frictional coefficient
f = 6r : viscosity of the medium
r : radius of the protein
Polyacrylamide gel electrophoresis
Polymer Formation of Acrylamide using Bis-Acrylamidefor PAGE (Poly-Acrylamide Gel Electrophoresis)
AmmoniumPersulfate
Sieving action!
SDS-PAGE : Denaturing Gel(Determination of the Molecular Weight of Protein)
Coomassie Blue Staining; > 0.1 g)(cf. Silver Staining: > 0.02 g)
Under BME, DTT
One SDS anion for every two a.a.
Except carbohydrate-rich proteins, membrane proteins
Mobility ; Log of MWResolution: 2% MW difference
Isoelectric Focusing & Two Dimensional Electrophoresis
pI : Isoelectric Point (pH with net charge zero)
-
-+
+
Evaluation of Protein Purification
As purification continues, relative presence of
contaminants should be decreased and
the proportional amount of the protein of
interest should be increased.
Centrifugation & Sedimentation Coefficient
• A more massive particle sediments more rapidly.
• A more compact shaped particle sediments faster.
(i.e. elongated particles sediments more slowly than do
spherical ones of the same mass. Frictional coefficient
f)
• A denser particle sediments more rapidly. Buoyant
force is smaller for the denser particle
• v < 1 : sink, v > 1 : float, v = 1 : no movement
s = m(1 - v) / fs : sedimentation coefficient
m : mass of the particle
v : partial specific volume; the reciprocal of the particle density : density of the medium
(1 - v) : buoyant force exerted by liquid medium
f : frictional coefficient; a measure of the particle shape
a. Seidmentation process in the cell
From Archimedes’ principleBuoyancy = weight of displaced fluid
Or fluid density x submerged vol x g
Principle of Analytical Ultracentrifugation
b.solute distribution in the cell
S Value for Various Proteins
Density and Sedimentation Coefficient
for Various Cellular Components
Gradient (Zonal or Band) Centrifugation:
Separation of Non-Denatured Proteins with different sedimentaion coefficients
(Size, Density and Shape)
Sedimentation equilibrium: centrifuged at low speed so that sedimentation is counter balanced by diffusion-Very accurate in mass determination without denaturing.- useful for large multimeric proteins.
Sedimentation velocity
Determination of Amino Acid Composition of the Peptide
1. Peptide hydrolyzation by heating it in 6N HCl at 100oC for 24 hrsAla-Gly-Asp-Phe-Arg-Gly
(Asp, Gly2, Ala, Phe, Arg)
2. Separation of amino acid hydrolysates by ion-exchange chromatography (e.g. sulfonated polystyrene resin; Dowex-50)
3A. Quantitation of Each Fraction by Ninhydrin;Yield Visible Color (usually blue except Pro for yellow);Detection Sensitivity = Microgram (10 nmol) of an Amino Acid
3B. Quantitation of Each Fraction by Fluorescamine;Yield Fluorescence;Detection Sensitivity = Nanogram (10 pmol) of an Amino Acid
Identification of N-Terminal Amino Acid
(FDNB) YieldFluorescentSulfonamide
FDNB, Dabsyl Chloride, or Dansyl Chloride
Can Specifically React with the N-terminal Amino Group, and
Yield DNB-Amino Acid, Dabsyl Amino Acid, or Dansyl Amino Acid, and
These Can Be Identified by Their Chromatographical Properties.
Determination of Amino Terminal Residue of a Peptideusing Dabsyl Chloride
Edman Degradation Sequentially Removes One Residue at a Timefrom the Amino End of a Peptide up to 50 times
Each round
can be complete
within 1 hr and
the Edman degradation
can be repeated
up to 50 cycles
in Practice.
Phenyl Isothiocyanate (PITC) Can Specifically React with
the N-terminal Amino Group, and Yield Phenyl Thiocarbamoyl (PTH) Amino Acid,
and This Can Be Identified by Its Chromatographical Property.
Separation of PTH-Amino Acids
Current Sensitivity ofPTH-AA Detection UsingGas-Phase Sequenator:
Picomole
Mild acidic condition
For sequencing of an entire Protein…??Divide and Conquer !!!
Deduction of Full Amino Acid Sequence of a Proteinby Overlapping the Sequences Obtained from individual Peptides
The Amino Acid Sequence Provides Insightsinto the Protein’s Function, Structure, and History
1. The sequence of a protein of interest can be compared with all other known
sequences to ascertain similarities. (Family, function prediction possible)
2. Comparison of sequences of the same protein in different species yields a wealth
of information about evolutionary pathway.
3. Amino acid sequences can be searched for the presence of internal repeats.
4. Many proteins contain amino acid sequences that serve as signals designating
their destinations or controlling their processing. (N-terminal 20 hydrophobic
residues, signal sequence, nuclear localization signal)
4 Repeating Motifs
in Calmodulin :
Each Unit Binds
a Calcium Ion
Antibody
• Antibody (immunoglobulin) is a protein synthesized by an animal in response to the
presence of a foreign substance (antigen).
• Antibodies have specific and high affinity against antigens.
• Proteins, polysaccharides and nucleic acids can be effective antigens.
• Epitope : a specific group or cluster (portion) of antigen to stimulate the synthesis of an
antibody and recognized by a specific antibody (antigenic determinant)
• Hapten : a small molecule containing epitope attached to a carrier
Antibody (continued)
• Each antibody producing cell synthesizes only one type of
antibody recognizing a single kind of epitope.
• The proliferation of a given antibody producing cell is
stimulated by the binding of its designated antigen to the cell
surface receptor of the antibody producing cell .
• Periodic injections of an antigen into the host animal can raise
the antibodies specifically recognizing the injected foreign
substance.
• Blood withdrawn from the immunized host animal
centrifugation separation of blood cells (pellet) and serum
(supernatant) anti-serum
• Anti-serum contains multiple kinds of antibodies each
recognizing a different surface feature of the same antigen.
• This heterogenic antibodies are called as polyclonal antibodies.
• This heterogeneity can complicate the use of these antibodies.
Monoclonal Antibody
• Monoclonal hybridoma cell lines can generate large amount of homogeneous antibodies.
• Monoclonal antibodies can serve as precise analytical, preparative and therapeutic reagents.(HCV, HIV, herceptin)
Immuno-Staining ofDrosophila
Embryousing
MonoclonalAntibodyagainst
Engrailed
Plasma cell
by antigen-antibody interaction
Monoclonal antibody drugs?
Marketable?
Figures from: http://www.patentdocs.org/2009/06/future-drug-sales-predictions-highlight-importance-of-followon-biologics-legislation.html
VEGF-A
VEGF
CD20TNF-A
TNFR
Antibody naming
Examples of anticancer monoclonal antibodyExamples of anticancer monoclonal antibody
Herceptin Binds to the C-terminus of Domain IV
Herceptin Fab
I
III
II
IV
N
C
HER2
Surface representations of EGFR and HER2 in Antibody-Bound Conformations
ELISA (Enzyme-Linked Immuno-Sorbent Assay)
Antibody detection, anti-HIV antibody
Antigen detection
Western Blotting
Radioactive secondary antibody
For protein expression and purification
Immuno-FluorescenceMicroscopy
Actin Filament Stainingusing -actin antibody
Immuno-ElectronMicroscopy
Detection of a channel proteinfrom the synaptic vesicles
using antibodies tagged withelectron-dense markers such as gold or
ferritin(Resolution better than 10 nm)
Fluorescence-labeled antibodies(resolution 200nm)
ex) Glucocorticoid receptor
Synthetic Peptides
• Synthetic Antigens for antibody formation• Receptor or Interacting Protein Isolation• Clinical Drugs (ex, vasopressin)• 3D Structure Study
MALDI-TOFMass
Spectrometry
MALDI : Matrix-AssistedLaser Desorption-Ionization
TOF : Time of FlightF=ma
Mass Spectrometry
Are Often Combined with
2D Electrophoresis
for Proteome Analysis
Electrospray Ionization (ESI)Electrospray Ionization (ESI)
Matrix Assisted Laser Desorption Ionization (MALDI)Matrix Assisted Laser Desorption Ionization (MALDI)
Primary Ionization Techniques for Molecules
LaserLaser
matrix + analytematrix + analyte
Sample supportSample support
aa
mm
mm
mm
mm
mm
mm
mmmm aa
aa
aa
aa
++
++
++
++mm
aamm++
Matrix Assisted Laser Desorption Ionization (MALDI)
Matrix Assisted Laser Desorption Ionization (MALDI)
h
Laser
+20 kV
Variable Ground Grid Grid
AH+
Sample plate1. Sample (A) is mixed with excess
matrix (M) and dried on a MALDI plate.
2. Laser flash ionizes matrix molecules.
3. Sample molecules are ionized by proton transfer from matrix:
MH+ + A M + AH+.
MALDI/TOF Mass SpectrumR
ela
tive
Abu
nda
nce
m/z
0
10000
20000
30000
40000
50000 100000 150000 200000
(M+H)+
(M+2H)2+
(M+3H)3+
Matrix
HO
CH C(CN)COOHOH
HO
COOH
HO
CH3O
CH3O
CH CHCOOH
-cyano-4-hydroxycinnamic acid 2,5-dihydroxybenzoic acid(2,5-DHB)
Sinapinic acid(3,5-Dimethoxy-4-hydroxy cinnamic acid)
HO
CH C(CN)COOHOH
HO
COOH
HO
CH3O
CH3O
CH CHCOOH
-cyano-4-hydroxycinnamic acid 2,5-dihydroxybenzoic acid(2,5-DHB)
Sinapinic acid(3,5-Dimethoxy-4-hydroxy cinnamic acid)
Sample Dilution/Concentration
Compound Concentration
Peptides and proteins 0.1 to 10 pmol/µL
Oligonucleotides 10 to 100 pmol/µL
Polymers 100 pmol/µL
•Dilute samples to the concentrations shown in the table below.
•If the sample concentration is unknown a dilution series may be needed to produce a good spot on the MALDI plate.
Note: highly dilute samples can be concentrated by Speed-Vac or Solid Phase Extraction.
Time of Flight (TOF)
Calibration of the mass scale
2
22
L
Kt
z
m
The mass-to-charge ratio of an ion is proportional to the square of its time of flight in the analyzer (“drift time”).
t = Drift timeL = Drift lengthm = MassK = Kinetic energy of ionz = Number of charges on ion
h1. Sample (M) is mixed with
excess matrix (X) and dried on a MALDI plate.
The plate is loaded onto the sample stage in the Ion Source
Ground Grid
Sample plate
to Mass Analyzer
Sample & Matrix
MALDI:MALDI: Matrix Assisted Laser Desorption Matrix Assisted Laser Desorption IonizationIonization
h
Laser
2. Laser flash produces matrix neutrals (X), matrix ions (XH)+, (X-H)- , and sample neutrals (M).
3. Sample molecules are ionized by proton transfer from matrix ions:
XH+ + M X + MH+.
X-H- + M X + M-H-
MALDI:MALDI: Matrix Assisted Laser Desorption Matrix Assisted Laser Desorption IonizationIonization
h4. Ion Extraction: High
voltage is applied to the sample plate, accelerating ions out of the Ion Source into the Flight Tube. MH+
+20 kV
MALDI:MALDI: Matrix Assisted Laser Desorption Matrix Assisted Laser Desorption IonizationIonization
Flight Tube
Detector
Ion Source
Time-of-Flight Mass AnalyzerTime-of-Flight Mass Analyzer
Principle: If ions are accelerated with the same potential at a fixed point and a fixed initial time and are allowed to drift, the ions will
separate according to their mass to charge ratios.
20-25 kV
++
Flight Tube
Detector
Ion Source
The ions enter the flight tube with the lighter ionstravelling faster than the heavier ions to the detector
+
+ +
Time-of-Flight Mass Analyzer
Flight Tube
Detector
Ion Source
The lighter ions strike the detector before the heavier ions.This “time of flight” (TOF) can be converted to mass
+
++
Time-of-Flight Mass Analyzer