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Protein Detection & Identification Methods

October 24, 2007MSB B554

Hong Liliho2@umdnj.edu973-972-8396

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

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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

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Shapes and sizes# of amino acids, composition & sequences

Charges and polarity

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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/

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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

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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.

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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

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Protein foldingSDS: Protein Denaturation

Protein denaturation

PAGE

SDS: minimize the impacts of protein charge and shape

Results:Separation by size!

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Visualization

1. CBB2. Silver3. Fluorescent

dyes

More….

Example: determine purity

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Protein size estimation

Human Proteins Size Distribution

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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

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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

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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

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Question: How will selected PTM affects protein pI?

2-Dimensional gel electrophoresis (2DE)

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2DE

2DE Example

An effective tool for proteomics studies.

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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

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2DE Protein Expression Analysis in Proteomics

B

Excise spot; elute; digest Extract peptides; MS analyze Protein identification

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Each amino acid has an unique mass

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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

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MS analysis of peptides: separation based on mass/charge (m/z) ratio

Mass Spectrometer

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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

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Mass Spectrometry – MSMeasuring peptide mass

MS spectrum

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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

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Mass difference between fragments can be used for peptide sequencing

V99 Y

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G57

Amino acid properties

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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

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Example: Identification ofProteins in cellular organelles

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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

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For more information: Take advanced classes!

Introduction to genomics, proteomics and bioinformaticsAdvanced genomics, proteomics and bioinformaticsProtein courseAnalytical methods