Chapter 3
Exploring Proteins and Proteomes
Genome
• 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, ~20,000 genes
• Static and absolute information
Proteome
• 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
Protein Purification
• 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
NADH
can absorb light
at 340 nm.
Nicotinamide adenine dinucleotide
Homogenation and Fractionation by Centrifugation
Salting Out & Dialysis
• 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)
Ammonium sulfate for
protein precipitation
Gel-Filtration Chromatography
(Molecular Exclusion,
Size Exclusion,
Molecular Sieve)
1.
2.
3.
4.
5.
Thyroglobulin (669 kd)
Catalase (232 kd)
BSA (67 kd)
Ovalbumin (43 kd)
Ribonuclease (13.4 kd)
Ion-Exchange
Chromatography
Cation Exchanger
Negatively Charged Column
Positively Charged Proteins
Depend on local charge on proteins
Anion Exchanger
Positively Charged Column
Negatively Charged Proteins
Affinity
Chromatography
Highly specific
- Histidine tag
LC
Liquid Chromatography
(concanavalin A)
FPLC
Fast Pressure Liquid Chromatography
HPLC
High Pressure Liquid Chromatography
Gel Electrophoresis
Polyacrylamide gel electrophoresis
v = Ez / f
v : velocity of migration
E : electric field strength
z : net charge on the protein
f : frictional coefficient
f = 6r
 : viscosity of the medium
r : radius of the protein
Polymer Formation of Acrylamide using Bis-Acrylamide
for PAGE (Poly-Acrylamide Gel Electrophoresis)
Sieving action!
Ammonium
Persulfate
SDS-PAGE : Denaturing Gel
(Determination of the Molecular Weight of Protein)
Coomassie Blue Staining; > 0.1 mg)
(cf. Silver Staining: > 0.02 mg)
Under BME, DTT
One SDS anion for every two a.a.
Except carbohydrate-rich proteins,
membrane proteins
Mobility ; Log of MW
Resolution: 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
s = m(1 - v) / f
s : 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 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
Principle of Analytical Ultracentrifugation
a. Seidmentation process in the cell
From Archimedes’ principle
Buoyancy = weight of displaced fluid
Or fluid density x submerged vol x g
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 velocity
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.
Determination of Amino Acid Composition of the Peptide
1. Peptide hydrolyzation by heating it in 6N HCl at 100oC for 24 hrs
Ala-Gly-Asp-Phe-Arg-Gly
2. Separation of amino acid hydrolysates by ion-exchange
chromatography (e.g. sulfonated polystyrene resin; Dowex-50)
(Asp, Gly2, Ala, Phe, Arg)
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, 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.
(FDNB)
Yield
Fluorescent
Sulfonamide
Determination of Amino Terminal Residue of a Peptide
using Dabsyl Chloride
Edman Degradation Sequentially Removes One Residue at a Time
from 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
Mild acidic condition
Current Sensitivity of
PTH-AA Detection Using
Gas-Phase Sequenator:
Picomole
For sequencing of an entire Protein…??
Divide and Conquer !!!
Sequence Specific Cleavage by Cyanogen Bromide
Sequence Specific Cleavage by Trypsin
Deduction of Full Amino Acid Sequence of a Protein
by Overlapping the Sequences Obtained from individual Peptides
Sequencing of Multimeric Proteins
Denaturation :
Urea, Guanidium Chloride, SDS
Reduction of Disulfide Bonds :
b-Mercaptoethanol, Dithiothreitol (DTT)
Preservation of Reduced Sulfhydryl Groups :
Alkylation using Iodoacetate
Separation of Each Polypeptide Chain :
SDS-PAGE
Diagonal Electrophoresis
to Determine the Positions of the Disulfide Bonds
Reduction and Oxidation of Disulfide Bonds by Performic Acid
The Amino Acid Sequence Provides Insights
into 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 Repeating Motifs
in Calmodulin :
Each Unit Binds
a Calcium Ion
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)
Practical Usage of Amino Acid and DNA Sequences
•
Amino acid sequence data provide a basis for preparing antibodies specific for a
protein of interest.
•
Amino acid sequence are valuable for making DNA probes that are specific for the
genes encoding the corresponding proteins.
•
The nucleotide sequence of DNA (gene) directly reveals the entire amino acid
sequence of the protein encoded by the gene.
•
However, DNA sequence can not disclose the information regarding post-translational
modification.
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.
Plasma cell
• Monoclonal antibodies can
serve as precise analytical,
preparative and therapeutic
reagents.(HCV, HIV,
herceptin)
by antigen-antibody interaction
ImmunoStaining of
Drosophila
Embryo
using
Monoclonal
Antibody
against
Engrailed
Monoclonal antibody drugs?
Herceptin Binds to the C-terminus of Domain IV
HER2
Herceptin Fab
II
I
N
III
IV
C
Ribbon Diagram of Her-3 ECD
C
I
“Tethered”
IV
II
N
Fig. 14.28 pp397
Right-handed b helix
Laminin-like folds
III
Surface representations of EGFR and
HER2 in Antibody-Bound Conformations
Herceptin
ELISA (Enzyme-Linked Immuno-Sorbent Assay)
Antibody detection, anti-HIV antibody
Antigen detection
Western Blotting
Radioactive secondary antibody
For protein expression and purification
Immuno-Fluorescence
Microscopy
Actin Filament Staining
using a-actin antibody
Fluorescence-labeled antibodies
(resolution 200nm)
ex) Glucocorticoid receptor
Immuno-Electron
Microscopy
Detection of a channel protein
from the synaptic vesicles
using antibodies tagged with
electron-dense markers such as gold or
ferritin
(Resolution better than 10 nm)
Synthetic Peptides
•
•
•
•
Synthetic Antigens for antibody formation
Receptor or Interacting Protein Isolation
Clinical Drugs (ex, vasopressin)
3D Structure Study
• Increase water absorption
in the kidney; Aquaporin
• Caffeine decreases
release of AVP
Protection of Amino Group
by t-BOC
Activation of Carboxyl Group
by DCC
Solid Phase
Peptide Synthesis
(more than 100 amino acids)
Resin : Insoluble Matrix (Polystyrene Beads), HF : Hydrofluoric Acid
MALDI-TOF
Mass
Spectrometry
MALDI : Matrix-Assisted
Laser Desorption-Ionization
TOF : Time of Flight
F=ma
Mass Spectrometry
Are Often Combined with
2D Electrophoresis
for Proteome Analysis
Laser
Sample plate
hn
1. Sample (A) is mixed with
excess matrix (M) and
dried
on a MALDI plate.
2. Laser flash ionizes matrix
molecules.
AH+
3. Sample molecules are
ionized by proton transfer
from matrix:
MH+ + A  M + AH+.
+20 kV
Variable Ground
Grid
Grid
Why MALDI?
-Less sensitive to salts
-Lower PRACTICAL detection limits
-Easier to interpret spectra(less multiple
charges)
-Quick and easy
-Higher mass detection
-Higher Throughput(1000>samples per hour)
The Mass Analyzer: TOF
Time Of Flight(TOF)
Ion Source
Flight Tube
20-25 kV
+ +
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.
• Peptide can be sequenced by MS
Individual proteins can be identified by MS
• Protein cleavage, followed by chromatographic separation and MS
NMR (Nuclear Magnetic Resonance)
• Basis for Resolution : Electron (Proton) Chemical Shift in Magnetic Field
• Advantage
- Solving Protein Structures in Solution State
- Requires Smaller Quantity for Analysis than X-ray Crystallography
• Limitations
- Technical Difficulties for Bigger Size Proteins (> 40 kD)
- Technical Difficulties for Multi-Subunit Proteins
X-Ray Crystallography
• Basis for Resolution : X-Ray Diffraction and Electron Scattering
• Advantage
- No Size Limitation for Analysis
- Solve the Structure of Multi-Subunit Complexes
• Limitations
- Solving Protein Structures in Solid State
- Difficulties in Making Protein Crystals with Good Diffraction Properties