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
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
3
Shapes and sizes# of amino acids, composition & sequences
Charges and polarity
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/
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
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.
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
8
Protein foldingSDS: Protein Denaturation
Protein denaturation
PAGE
SDS: minimize the impacts of protein charge and shape
Results:Separation by size!
9
Visualization
1. CBB2. Silver3. Fluorescent
dyes
More….
Example: determine purity
10
Protein size estimation
Human Proteins Size Distribution
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
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
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
14
Question: How will selected PTM affects protein pI?
2-Dimensional gel electrophoresis (2DE)
15
2DE
2DE Example
An effective tool for proteomics studies.
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
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
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
19
MS analysis of peptides: separation based on mass/charge (m/z) ratio
Mass Spectrometer
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
21
Mass Spectrometry – MSMeasuring peptide mass
MS spectrum
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
23
Mass difference between fragments can be used for peptide sequencing
V99 Y
163
G57
Amino acid properties
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
25
Example: Identification ofProteins in cellular organelles
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
27
For more information: Take advanced classes!
Introduction to genomics, proteomics and bioinformaticsAdvanced genomics, proteomics and bioinformaticsProtein courseAnalytical methods
Top Related