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

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

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

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)

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

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

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)

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

Lys and Arg are (+) at pH 7

His can be protonated/deprotonated

Polar side groups - acidic Negatively charged at pH >6

Negatively charged at pH >6

Polar side groups - neutral Terminal amide groups Can be de-amidated to Glu and Asp

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

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

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

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)

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

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

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”

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

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

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

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

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

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.

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 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

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

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

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.

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

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

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

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