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 NADH can 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) Thyroglobulin (669 kd) Catalase (232 kd) BSA (67 kd) Ovalbumin (43 kd) Ribonuclease (13.4 kd)

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

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

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

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

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.

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) / 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. 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

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 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 Current Sensitivity of PTH-AA Detection Using Gas-Phase Sequenator: Picomole Mild acidic condition

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

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

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

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

Monoclonal antibody drugs?

Marketable? VEGF-A TNF-A CD20 TNFR VEGF Figures from: http://www.patentdocs.org/2009/06/future-drug-sales-predictions-highlight-importance-of-followon-biologics-legislation.html

Antibody naming

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

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-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

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

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

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 !!

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

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

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

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

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.

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

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

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

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

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.

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

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