• Share
  • Email
  • Embed
  • Like
  • Save
  • Private Content
Proteins – Basics you need to know for Proteomics
 

Proteins – Basics you need to know for Proteomics

on

  • 9,086 views

Proteins – Basics you need to know for Proteomics

Proteins – Basics you need to know for Proteomics

Statistics

Views

Total Views
9,086
Views on SlideShare
8,847
Embed Views
239

Actions

Likes
2
Downloads
362
Comments
0

10 Embeds 239

http://www.chemist.gr 167
http://www.slideshare.net 24
http://scienceofthingsilike.posterous.com 19
https://blackboard.jhu.edu 15
http://axortagos.gr 7
http://www.axortagos.gr 2
http://www.arpaktiko.org 2
http://webcache.googleusercontent.com 1
http://www.lmodules.com 1
http://www.moschidis.eu 1
More...

Accessibility

Categories

Upload Details

Uploaded via as Microsoft PowerPoint

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment

    Proteins – Basics you need to know for Proteomics Proteins – Basics you need to know for Proteomics Presentation Transcript

    • Proteins – Basics you need to know for Proteomics
    • Outline for Rest of Course
      • Proteins and Exploring Proteins
        • Basics of protein structure
        • Basics of conventional protein analysis
      • PTMs of Proteins (Walsh)
        • Phosphorylation
        • Glycosylation
      • Intro to Proteomics (Simpson, Ch 1)
      • HPLC (Simpson, Ch 5)
        • RP-HPLC
        • 2D-HPLC
        • Lab Demo of nano-HPLC
      • Peptide Mapping (Simpson, Ch. 7)
        • Lab demo
      • Mass Spectrometry (Simpson, Ch.8)
        • Ionization – ESI, MALDI
        • Separation – TOF, Quad, IT, FT-ICR
        • Tandem MS
        • Fragmentation (p 577, and Mascot tutorial)
        • Lab demos
        • Bioinformatics for Proteomics
    • Objectives
      • Understand basics of protein composition and structure
      • 20 naturally occurring amino acids and their properties
      • Secondary structural motifs
      • Tertiary and Quaternary structure
      • Protein synthesis
      • Basic biochemistry techniques
    • Protein Structure and Function
      • Functions
        • Enzymes – catalysts of chemical reactions
        • Transport and storage – Fe, O 2 , CO 2 , Cu, etc.
        • Motion and motors – muscles, flagella
        • Structural support – collagen, elastase, actin
        • Immune protection – antibodies, cytokines
        • Stimuli response – vision, touch, smell, taste
        • Growth and differentiation – growth factors, hormones, cytokines
    • Aliphatic side chains (hydrophobic)
    • Proline
      • Imino acid
      • Side chain bound to  -carbon and N
      • Often found in bends of protein chains
      • Unique properties (more later)
    • Aromatic Side Chains
      • Very hydrophobic
      • Tyr – OH is reactive
      • Absorbance at 280 nm
    • Sulfur Containing AA’s – Met, Cys
      • Cys sulfur is highly reactive
        • Reduction/oxidation
        • Protein folding
      • Hydrophobic
    • Disulfide Bonds - Cys Reduced Oxidized Remember: LEO says GER
    • Hydroxyl side groups – Ser, Thr
      • More hydrophilic
      • Ser OH group can be reactive (serine proteases)
      • Lys and Arg are (+) at pH 7
      • His can be protonated/deprotonated
      Polar side groups - basic
    • Polar side groups - acidic
      • Negatively charged at pH >6
    • Polar side groups - neutral
      • Terminal amide groups
      • Can be de-amidated to Glu and Asp
    • Ionizable AA’s
      • pKa values must be memorized
      • pKa values are highly dependent on local environment
    • Polypeptides
      • Mnemonic: N C C- N C C- N C C- N CC
      • Always given with amino terminus (N-terminus) at beginning
      • 1 Dalton = 1 amu
      • Avg MW of aa is ~110 Da
    • Post-translational Modifications of AA’s
      • Some aa’s are chemically modified after protein synthesis
        • Often used to confer special biochemical properties essential to protein’s function
        • Examples
          • Acetylation of N-terminus
          • Hydroxylation of Pro (collagen)
          • Carboxylation of Glu (blood coagulation enzymes)
          • Phosphorylation of Ser, Thr, Tyr (cytokine receptors)
          • Sulfation of Tyr
          • Glycosylation of Ser, Thr, Asn
      • Proteins can also be proteolytically processed
        • Zymogen to active enzyme (trypsinogen/trypsin)
        • Fibrinogen to fibrin (to form blood clot)
        • Prohormones to hormones
      • Lots more on this later
    • tRNA Molecules
      • General features
        • 73 to 93 ribonucleotides (25 kD)
        • Methylated or dimethylated versions of bases (structure)
        • Cloverleaf structure with anti-codon loop
        • 5’ phosphorylated
      • Amino acid at 3’
      • IGC complementary to GCC (A)
    • 3-D structure
      • L-shaped molecule – anti-codon and amino acid attachment at opposite ends
      • CCA – conserved sequence for amino acid attachment
      • 4 helices
    • Ribosomes – molecular machines
      • Coordinate charged tRNA’s, mRNA, and nascent polypeptides during protein synthesis
      • Macromolecular complex
        • 50S subunit – 34 proteins + 2 RNA molecules: L1-L34; 23S and 5S RNA
        • 30S subunit – 21 proteins + 1 RNA: S1-S21; 16S RNA
        • S20 and L26 are identical
        • One copy of each RNA, two copies L7 and L12, 1 copy of all other proteins
          • L7 and L12 identical except L7 N-term is acetylated
    •  
      • Prokaryotes - Many ribosomes can translate a single mRNA at the same time
      • “ Polysome” or “Polyribosome”
    • Tunnel for growing polypeptide chain
    •  
    • Protein synthesis initiation
      • E. coli - Formylmethionyl tRNA
        • Removed from protein after about 10 amino acids in ~50% of proteins
      • Specific mechanism for f-Met synthesis and incorporation
    • Why Formylation of Met? Termination of translation if not formylated!
    • Animated versions
      • Narrated Animation
        • prokaryote protein synthesis
        • Prokaryote vs. eukaryote
        • Splicing
        • Protein secretion
    • Gel Filtration – molecular size
    • Ion exchange chromatography + + + + Cl - Cl - Cl - Cl - + + + + + + + + Cl - Cl - Cl - Cl -
    • Affinity Chromatography Equilibrate column Load feed onto column Then wash contaminants away Elute Product Clean Column and re-equilibrate column
    • Reverse Phase High Performance Liquid Chromatography - RP-HPLC
      • Used for both purification and as an analytical tool
      • High Resolution – able to separate proteins based on very small differences in structure
    • RP-HPLC Mechanism of Separation
      • Stationary phase – hydrophobic groups – C4, C5, C8, C18, phenyl
        • Proteins are dissolved in mostly aqueous buffer and injected onto the column
        • Molecules interact with the stationary phase
        • Organic solvent (modifier) is added to the mobile phase and kicks the molecules off the stationary phase
    • Purity by HPLC
      • HPLC is high resolution and can be used to assess purity of a protein sample
        • Compare with known reference standard of desired protein and potential contaminants
        • Can be quantitative – determine protein concentration by constructing calibration curve
    • Visualizing the effects of Purification - Electrophoresis
      • Gel Electrophoresis can be used to visualize the effectiveness of purification and estimate protein MW
      • Proteins migrate through the porous hydrogel based on molecular size – radius of gyration.
    •  
    • Isoelectric Focusing - IEF
      • IEF separates molecules based on their isoelectric points (IEP), or the pH at which they have a net neutral charge
      • IEF is usually used as an analytical tool to confirm a protein’s structure, but there are some semi-preparative devices that can be used for protein purification (difficult to run, though)
      • IEF is very powerful – proteins with IEP’s that differ by as little as 0.01 pH can be separated using specialized techniques
      • IEF can be run in a slab gel (similar to SDS PAGE format), in tubes, on paper strips, or in capillaries
      • IEF is also used a lot in proteomics research
    • IEF Basics
      • Consider a slab gel – polyacrylamide that is impregnated with “ampholytes”
      • Ampholytes are a mixture of synthetic polymers (usually amino acids) that vary in their IEP, say from pH 3 to pH 10. The ampholytes are usually pre-focused (but don’t have to be) to set up a pH gradient in the gel
      • The samples for analysis are NOT treated with SDS or reductant – they are added to the IEF gel in their native state with a small amount of ampholytes
      pH 3 pH 10
    • Running an IEF Gel pH 3 pH 10
      • Samples are added to the Gel and gel is placed in apparatus. The upper chamber is filled with anolyte (10 mM phosphoric acid) and the lower chamber is filled with catholyte (20 mM NaOH). The electric field is then turned on.
      upper chamber – phosphoric acid (+) positive electrode lower chamber - NaOH (-) negative electrode power supply
      • The ampholytes and sample proteins will migrate until their charge is neutral, then they don’t migrate any more. Thus a stable pH gradient is set up, and molecules are separated based on their differences in IEP
      • The proteins are then stained for visualization
    • IEF Examples Monoclonal Antibody Production
    • 2-D SDS PAGE
      • Two-dimensional separation
        • 1 st dimension – IEF (paper strips, tube gels)
        • 2 nd dimension – SDS PAGE
      • Lay paper strip down on top of gel; run 2 nd dimension
    •  
    • Immunological Assays
      • Use of antibodies to detect, purify, and qunatify your protein of interest
      • Antibodies
        • Immunoglobulins that have a high affinity for their respective antigen
      • Antigen – substance that elicits an immune response
        • Proteins, oligosaccharides, small organic molecules (hapten)
        • Epitope – structural portion of the antigen that directly interacts with antibody
      • Ab/Ag interaction ~ lock and key
        • NONCOVALENT – a combination of H-bonds, van der Waals, and ionic interactions with surface topology
    • IgG’s
    • Ag/Ab interactions
    •  
    •  
    • ELISAs
    • Western Blots
    • Immunohistochemistry, Immunoelectron microscopy