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Protein Detection & Identification Methods

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Protein Detection & Identification Methods Protein Detection & Identification Methods October 24, 2007 MSB B554 Hong Li [email protected] 973-972-8396 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 1 Purity, quantity and identity Post-translational modification Induction and turnover Expression localization Basic Principles for Analysis (How to differentiate one protein from another?) Structure (Drs. Wang & Wah) Amino acid composition Post-translational modification (Dr. Wagner) Size Polarity/Charge/Hydrophobicity Shape Affinity (binding to other proteins/molecules) Function: catalytic activities 2 Shapes and sizes # of amino acids, composition & sequences Charges and polarity 3 Charges and polarity from post-translational modifications Reactivities of amino acids Physical/chemical reactions to facilitate colorimetric detection Example: Protein concentration assays: Bradford, BCA, Lowry & Biuret, etc. http://www-class.unl.edu/biochem/protein_assay/ 4 Bradford assay (Bio-Rad) Based on a dye binding to basic and aromatic amino acids Coommassie Brilliant Blue (CBB) G250 Protein binding causes its maximum absorbance to shift from 465 nm to 595 nm (blue) Bradford assay 5 Techniques Electrophoresis: separation by size SDS-PAGE Isoelectric focusing: separation by charge 2-Dimensional gel electrophoresis Immunoblotting: detection by specific affinity with antibodies (a special class of proteins) Autoradiography: radioactivity Mass spectrometry: protein sequencing/identification Proteomics: high-throughput analysis Electrophoresis Physics: Charged particles in an electric field will migrate according to their charge-to-mass (size) ratio Positively-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. 6 Sodium Dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) A method for protein separation and visualization based on size Acrylamide polymerization & effect on protein separation www.nationaldiagnostics.com www.cas.vanderbilt.edu/bsci111a/protein-electro/supplemental.htm 7 SDS: Protein Denaturation Protein folding Protein denaturation PAGE SDS: minimize the impacts of protein charge and shape Results: Separation by size! 8 Visualization 1. CBB 2. Silver 3. Fluorescent dyes More…. Example: determine purity 9 Protein size estimation Human Proteins Size Distribution 10 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 antibodies AKA: Western Blotting 11 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 12 Isoelectric focusing (IEF) A special type of electrophoresis for protein separation: based on charge Isoelectric point (pI): the pH value at which a protein carries no net charge. (http://www.biology-online.org/dictionary/Isoelectric_point) pH < pI: net + charge pH > pI: net – charge IEF matrix: a gel strip containing an immoblized pH gradient, charged proteins migrate to either cathode and anode crossing different pH steps When 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 13 Question: How will selected PTM affects protein pI? 2-Dimensional gel electrophoresis (2DE) 14 2DE 2DE Example An effective tool for proteomics studies. 15 Proteomics Simplified definition: systematic studies of protein structural and functional changes 2DE: a tool for protein expression comparison between 2 systems Mass 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 16 2DE Protein Expression Analysis in Proteomics A B Excise spot; elute; digest Extract peptides; MS analyze Protein identification Each amino acid has an unique mass 17 Mass Spectrometer (MS) for Protein Identification Sample Preparation Cut spots Gel Electrophoresis tryptic digestion cleaves protein at R and K residues Peptide Ions 2. Peptide sequencing by MS/MS 1. MS Analysis PMF spectrum MS/MS Spectra Ions vs. Molecules 18 MS analysis of peptides: separation based on mass/charge (m/z) ratio Mass Spectrometer 19 Peptide Mass Mapping Mass Spectrometer (MS) for Protein Identification Sample Preparation Cut spots Gel Electrophoresis tryptic digestion cleaves protein at R and K residues Peptide Ions Peptide sequencing by MS MS Analysis PMF spectrum MS/MS Spectra 20 Mass Spectrometry – MS Measuring peptide mass MS spectrum 21 Tandem Mass Spectrometry: peptide sequence determination Peptide sequencing by MS MS Analysis PMF spectrum MS/MS Spectra MS is able to “fragment” a peptide into many smaller peptide ions 22 Mass difference between fragments can be used for peptide sequencing G V Y 57 99 163 Amino acid properties 23 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 24 Example: Identification of Proteins in cellular organelles 25 Protein detection and identification methods 1. SDS-PAGE: protein separation based on size 2. IEF: protein separation based on pI 3. 2DE: protein separation based on pI and size 4. Coommassie Brilliant Blue: a dye for protein concentration assay and general detection in gel electrophoresis 5. Immunoblotting: a sensitive and specific method for detecting interested proteins separated by gel electrophoresis 6. 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 26 For more information: Take advanced classes! Introduction to genomics, proteomics and bioinformatics Advanced genomics, proteomics and bioinformatics Protein course Analytical methods 27.
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