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

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Proteins – Basics you need to know for Proteomics

by lwolberg on Jan 06, 2009

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