protein detection & identification methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/protein...
TRANSCRIPT
![Page 1: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/1.jpg)
1
Protein Detection & Identification Methods
October 24, 2007MSB B554
Hong [email protected]
Lecture notes: http://njms.umdnj.edu/proweb/lectures/note2007fall01.pdf
Objectives
1. Protein analysis to determine:• Purity, quantity and identity• Expression and localization• Post-translational modification• Induction and turnover
2. Principles behind the analytical techniques• Based on unique physical/chemical properties; size, charge, etc.• Assays are based on reactions producing light, color and radio activities
for detection
3. Techniques• Electrophoresis• Immunoblotting• Autoradiography• Mass spectrometry• Proteomics
![Page 2: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/2.jpg)
2
Purity, quantity and identity
Expression
Post-translational modification Induction and turnover
localization
Basic Principles for Analysis(How to differentiate one protein from another?)
Structure (Drs. Wang & Wah)Amino acid compositionPost-translational modification (Dr. Wagner)SizePolarity/Charge/HydrophobicityShapeAffinity (binding to other proteins/molecules)
Function: catalytic activities
![Page 3: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/3.jpg)
3
Shapes and sizes# of amino acids, composition & sequences
Charges and polarity
![Page 4: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/4.jpg)
4
Charges and polarityfrom post-translational modifications
Reactivities of amino acids
Physical/chemical reactions to facilitate colorimetric detectionExample: Protein concentration assays: Bradford, BCA, Lowry & Biuret, etc.
http://www-class.unl.edu/biochem/protein_assay/
![Page 5: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/5.jpg)
5
Bradford assay (Bio-Rad)
Based on a dye binding to basic and aromatic amino acidsCoommassie Brilliant Blue (CBB) G250Protein binding causes its maximum absorbance to shift from 465 nm to 595 nm (blue)
Bradford assay
![Page 6: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/6.jpg)
6
Techniques
Electrophoresis: separation by sizeSDS-PAGE
Isoelectric focusing: separation by charge2-Dimensional gel electrophoresisImmunoblotting: detection by specific affinity with antibodies (a special class of proteins)Autoradiography: radioactivityMass spectrometry: protein sequencing/identificationProteomics: high-throughput analysis
Electrophoresis
Physics: Charged particles in an electric field will migrate according to their charge-to-mass (size) ratioPositively-charged molecules migrates towards anode (negative pole) while negatively-charged molecules migrates toward cathode (positive pole)Electrophoresis medium creates frictions during the migration. Protein shape has an impact: globular proteins migrate faster while cylindrical proteins migrate slower.
![Page 7: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/7.jpg)
7
Sodium Dodecylsulfatepolyacrylamide gel electrophoresis(SDS-PAGE)
A method for protein separation and visualization based on size
Acrylamide polymerization & effect on protein separation
www.nationaldiagnostics.comwww.cas.vanderbilt.edu/bsci111a/protein-electro/supplemental.htm
![Page 8: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/8.jpg)
8
Protein foldingSDS: Protein Denaturation
Protein denaturation
PAGE
SDS: minimize the impacts of protein charge and shape
Results:Separation by size!
![Page 9: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/9.jpg)
9
Visualization
1. CBB2. Silver3. Fluorescent
dyes
More….
Example: determine purity
![Page 10: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/10.jpg)
10
Protein size estimation
Human Proteins Size Distribution
![Page 11: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/11.jpg)
11
Additional visualization methods
Immunoblotting: a method for specific detection of a protein based on the specific binding between protein of interest and a member of a class of proteins, called antibodiesAKA: Western Blotting
![Page 12: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/12.jpg)
12
Immunoblotting for specific protein detection
Using radioisotopes to label and detect proteins
32P-ATP can be used metabolically to label phosphoproteins
35S-Met can be used to label almost all proteins
![Page 13: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/13.jpg)
13
Isoelectric focusing (IEF)
A special type of electrophoresis for protein separation: based on chargeIsoelectric point (pI): the pH value at which a protein carries no net charge. (http://www.biology-online.org/dictionary/Isoelectric_point)pH < pI: net + chargepH > pI: net – chargeIEF matrix: a gel strip containing an immoblized pH gradient, charged proteins migrate to either cathode and anode crossing different pH stepsWhen migrate to their pI, protein will carry no net charge, therefore, no more mobility in an IEF device
Isoelectric point (pI)
pI=(2.1+3.9)/2=3.0
![Page 14: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/14.jpg)
14
Question: How will selected PTM affects protein pI?
2-Dimensional gel electrophoresis (2DE)
![Page 15: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/15.jpg)
15
2DE
2DE Example
An effective tool for proteomics studies.
![Page 16: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/16.jpg)
16
Proteomics
Simplified definition: systematic studies of protein structural and functional changes2DE: a tool for protein expression comparison between 2 systemsMass spectrometry: a method that uses an instrument, called mass spectrometer to determine the precise mass (size) and the sequence of proteins.
Changes in protein expression is important for cell function
![Page 17: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/17.jpg)
17
2DE Protein Expression Analysis in Proteomics
B
Excise spot; elute; digest Extract peptides; MS analyze Protein identification
A
Each amino acid has an unique mass
![Page 18: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/18.jpg)
18
tryptic digestioncleaves protein at R and K residues
PMF spectrum
Mass Spectrometer (MS) for Protein Identification
Sample Preparation
Gel Electrophoresis
Cut spots
2. Peptide sequencing by MS/MS
MS/MS Spectra
1. MS Analysis
Peptide Ions
Ions vs. Molecules
![Page 19: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/19.jpg)
19
MS analysis of peptides: separation based on mass/charge (m/z) ratio
Mass Spectrometer
![Page 20: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/20.jpg)
20
Peptide Mass Mapping
tryptic digestioncleaves protein at R and K residues
PMF spectrum
Mass Spectrometer (MS) for Protein Identification
Sample Preparation
Gel Electrophoresis
Cut spots
Peptide sequencing by MS
MS/MS Spectra
MS Analysis
Peptide Ions
![Page 21: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/21.jpg)
21
Mass Spectrometry – MSMeasuring peptide mass
MS spectrum
![Page 22: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/22.jpg)
22
Tandem Mass Spectrometry: peptide sequence determination
PMF spectrum
Peptide sequencing by MS
MS/MS Spectra
MS Analysis
MS is able to “fragment” a peptide into many smaller peptide ions
![Page 23: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/23.jpg)
23
Mass difference between fragments can be used for peptide sequencing
V99 Y
163
G57
Amino acid properties
![Page 24: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/24.jpg)
24
Question: How will selected PTM affects amino acid
mass?
+80 Da to ser
Q: Protein phosphorylation
If these two spots are the same protein but differ by phosphorylation, which one may be phosphorylated?
A B
![Page 25: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/25.jpg)
25
Example: Identification ofProteins in cellular organelles
![Page 26: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/26.jpg)
26
Protein detection and identification methods
1. SDS-PAGE: protein separation based on size2. IEF: protein separation based on pI3. 2DE: protein separation based on pI and size4. Coommassie Brilliant Blue: a dye for protein concentration
assay and general detection in gel electrophoresis5. Immunoblotting: a sensitive and specific method for
detecting interested proteins separated by gel electrophoresis6. Autoradiography: a sensitive and highly quantitative method for
studying dynamic changes of proteins separated by gel electrophoresis
7. Mass spectrometry: a method for protein sequencing and identification
8. Proteomics: systematic studies of protein structural and functional changes using all the tools described above
![Page 27: Protein Detection & Identification Methodsnjms.rutgers.edu/sgs/olc/core1/prot/2008/Protein Analysis Methods... · protein structural and functional changes 2DE: a tool for protein](https://reader030.vdocuments.us/reader030/viewer/2022040810/5e515a48b0e0283b762cd406/html5/thumbnails/27.jpg)
27
For more information: Take advanced classes!
Introduction to genomics, proteomics and bioinformaticsAdvanced genomics, proteomics and bioinformaticsProtein courseAnalytical methods