1. Exploring Proteins and Proteomes

2. 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

3. 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
NADH
can absorb light
at 340 nm.
Nicotinamide adenine dinucleotide

4. Homogenation and Fractionation by Centrifugation

5. 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

6. Gel-Filtration Chromatography
(Molecular Exclusion,
Size Exclusion,
Molecular Sieve)
Thyroglobulin (669 kd)
Catalase (232 kd)
BSA (67 kd)
Ovalbumin (43 kd)
Ribonuclease (13.4 kd)

7. Ion-Exchange Chromatography
Anion Exchanger
Positively Charged Column
Negatively Charged Proteins
Cation Exchanger
Negatively Charged Column
Positively Charged Proteins
Ion-Exchange
Chromatography
Depend on local charge on proteins

8. LC FPLC HPLC Affinity Chromatography Liquid Chromatography
(concanavalin A)
Highly specific
- His tag LC
Liquid Chromatography
FPLC
Fast Pressure Liquid Chromatography
HPLC
High Pressure Liquid Chromatography

10. Gel Electrophoresis v = Ez / f
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

11. Polymer Formation of Acrylamide using Bis-Acrylamide
for PAGE (Poly-Acrylamide Gel Electrophoresis)
Ammonium
Persulfate
Sieving action!

12. SDS-PAGE : Denaturing Gel
(Determination of the Molecular Weight of Protein)
Coomassie Blue Staining; > 0.1 mg)
(cf. Silver Staining: > 0.02 mg)
Except carbohydrate-rich proteins, membrane proteins
Mobility ; Log of MW
Resolution: 2% MW difference
Under BME, DTT
One SDS anion for every two a.a.

13. Isoelectric Focusing & Two Dimensional Electrophoresis
pI : Isoelectric Point
(pH with net charge zero) + - + -

14. 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.

15. 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) / 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

16. a. Seidmentation process in the cell
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

18. b.solute distribution in the cell

20. S Value for Various Proteins

21. Density and Sedimentation Coefficient for Various Cellular Components

22. 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.

23. Determination of Amino Acid Composition of the Peptide
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)

24. 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

25. 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

26. Determination of Amino Terminal Residue of a Peptide
using Dabsyl Chloride

27. 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.

28. 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 of
PTH-AA Detection Using
Gas-Phase Sequenator:
Picomole
Mild acidic condition

29. For sequencing of an entire Protein…??
Divide and Conquer !!!

30. Deduction of Full Amino Acid Sequence of a Protein
by Overlapping the Sequences Obtained from individual Peptides

31. The Amino Acid Sequence Provides Insights
into the Protein’s Function, Structure, and History
The sequence of a protein of interest can be compared with all other known sequences to ascertain similarities. (Family, function prediction possible)
Comparison of sequences of the same protein in different species yields a wealth of information about evolutionary pathway.
Amino acid sequences can be searched for the presence of internal repeats.
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

32. 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

33. 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.

34. 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 of
Drosophila
Embryo
using
Monoclonal
Antibody
against
Engrailed
Plasma cell
by antigen-antibody interaction

35. Monoclonal antibody drugs?

36. Marketable?
VEGF-A
TNF-A
CD20
TNFR
VEGF
Figures from:

37. Antibody naming

38. Examples of anticancer monoclonal antibody
제품명
일반명
항체 종류
분자 타겟
회 사
적용 질병
OKT3
Muromomab
Murine
CD3
Orthobiotec/J&J
Prevention of acute kidney transplant rejection
ReoPro
Abciximab
Chimeric
GPIIb/IIIa
Centocor
Prevention of blood clot
Remicade
Infliximab
TNFα
Crohn's disease 외
Herceptin
Trazumumab
Humanized
HAA
Genentech/Roche
Metastatic breast cancer
Mylotarg
Gemtuzumab
CD33
Wyeth
Acute myeloid leukemia
Xolair
Omalizumab
IgE
Genentech
m-to-s persistent asthma
Raptiva
Efalizumab
CD11a
chronic m-to -s plaque psoriasis
Erbitux
Cetuximab
EGFR
Imclone
Colorectal cancer
Avastin
Bevacixumab
VEGF
metastatic cancer of colon or rectum
Tysabri
Natalizumab
VLA4
Biogen IDEC
Multiple sclerosis
Vectibix
Panitumumab
Human
Amgen/Abgenix
Lucentis
Ranibizumab
wet AMD

39. Herceptin Binds to the C-terminus of Domain IV
Herceptin Fab I
III II IV N C
HER2

40. Surface representations of EGFR and
HER2 in Antibody-Bound Conformations

41. ELISA (Enzyme-Linked Immuno-Sorbent Assay)
Antibody detection, anti-HIV antibody
Antigen detection

42. Western Blotting Radioactive secondary antibody
For protein expression and purification

43. Immuno-Fluorescence Microscopy Immuno-Electron
Actin Filament Staining
using a-actin antibody
Immuno-Electron
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)
Fluorescence-labeled antibodies
(resolution 200nm)
ex) Glucocorticoid receptor

44. Synthetic Peptides Synthetic Antigens for antibody formation
Receptor or Interacting Protein Isolation
Clinical Drugs (ex, vasopressin)
3D Structure Study

45. 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

46. Primary Ionization Techniques for Molecules
Electrospray Ionization (ESI)
Matrix Assisted Laser Desorption Ionization (MALDI)
현재 가장 주목 받고 있는 이온소스 두개
LC base에서 가장많이 사용하는 ESI 다양한 샘플에 접목시킬수 있으며
멀티플 차지를 띠기 때문에 고분자 분석에 용이함
MALDI 는 기존의 이온화 방식에 대비해서 고분자를 분석 가능하며 소프트 이온화 방식이라
기존 샘플의 fragmentation 없이 분자량을 측정할수 있음!!
가장중요한건 두가지 다 소프트 이온화 방식이라는것 !!

47. Matrix Assisted Laser Desorption Ionization (MALDI)
matrix + analyte
Sample support a m +
MALDI 가 이렇게 생겼다 이온화 방법은 다음 page에서

48. Matrix Assisted Laser Desorption Ionization (MALDI)
1. 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+.
Sample plate hn
샘플을 오천대 일에서 천대 일 정도의 비율로 석는다
레이져를 이용하여 메트릭스를 이온화 시키고
이온화된 메트릭스의 프로톤이 샘플로 트렌퍼 된다..
AH+
Variable Ground
Grid Grid
+20 kV

49. MALDI/TOF Mass Spectrum
(M+H)+
10000
20000
30000
40000
Relative Abundance
(M+2H)2+
말디 스펙트럼 대부분 일가 차지가 나오지만 이렇게 멀티플 차지가 나올수도 있다..
샘플을 하나 찍었는데 피크가 두개 이상 나왔다고 해서 거짓 정보가 아닐수도 있다..
Ex ei 에를 들어 fragmantation설명
(M+3H)3+
50000
100000
150000
200000
m/z

50. Matrix
메트릭스 종류 설명 하고 보통 벤젠 고리에 Coo- 233nm 파장을 가진다
N2 레이저가 같은 파장을 가진다

51. Sample Dilution/Concentration
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.
Compound
Concentration
Peptides and proteins
0.1 to 10 pmol/µL
Oligonucleotides
10 to 100 pmol/µL
Polymers
100 pmol/µL
시료 조합 비율 설명 보통 세츄레이션 시켜서 사용하다
기준을 삼을때는 대략 10mg/1ml 사용 이때 이온화를 돕기 위해서 이온첨가제
AGTFA 를 넣거나 메트릭스 클러스터 제거를 위해서 암모늄 바이 클로라이드 암모늄 포스페이트를 넣기도 함
Note: highly dilute samples can be concentrated by Speed-Vac or Solid Phase Extraction.

52. Time of Flight (TOF) 토프 아주 간단 그냥 단순한 긴 원통형 관
생성된 이온의 로스가 작아서 디텍션 효율이 적다
다른 어널라이져와 하이 브리드 시키기가 용이 하다 간단하니까

53. Calibration of the mass scale
The mass-to-charge ratio of an ion is proportional to the square of its time of flight in the analyzer (“drift time”).
E=이분의 일 엠브이에 제곱
t = Drift time
L = Drift length
m = Mass
K = Kinetic energy of ion
z = Number of charges on ion

54. MALDI: Matrix Assisted Laser Desorption Ionization
Sample plate hn
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 defines the end of the Ion Source and the beginning of the Mass Analyzer (Flight Tube)
to Mass Analyzer
Sample & Matrix
Ground Grid

55. MALDI: Matrix Assisted Laser Desorption Ionizationhn
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-

56. MALDI: Matrix Assisted Laser Desorption Ionizationhn
Ion Extraction: High voltage is applied to the sample plate, accelerating ions out of the Ion Source into the Flight Tube.
MH+
+20 kV

57. Time-of-Flight Mass Analyzer
Ion Source
Flight Tube
20-25 kV
Detector + +
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.

58. Time-of-Flight Mass Analyzer
Ion Source
Flight Tube
Detector + + +
The ions enter the flight tube with the lighter ions
travelling faster than the heavier ions to the detector

59. Time-of-Flight Mass Analyzer
Ion Source
Flight Tube
Detector + + +
The lighter ions strike the detector before the heavier ions.
This “time of flight” (TOF) can be converted to mass