proteomics terry kotrla, ms, mt(ascp)bb. human genome project launched 1990 took 13 years estimated...

Proteomics

Terry Kotrla, MS, MT(ASCP)BB

Human Genome Project

• Launched 1990 took 13 years• Estimated 100,000 human genes would be

discovered• Only 20,000-25,000 genes were needed.• Not much more than a worm

Proteins

• Genes encode proteins• Proteins determine an organism’s form,

function and phenotype• Proteins reflect changes in gene pool• Variations in protein profiles reflect

physiological adaptation to different environments

• Favorable – pass throughu

Evolution

• Humans have large genomes, 3 billion base pairs

• Worms have 100 million base pairs• Humans very complex• Single gene can encode multiple proteins with

very different functions• Accomplished by using a variety of DNA, RNA

and protein modification tools

Modification Tools

• Genes silenced by methylation of regulatory sequences

• Genes transcribed into RNA, the RNA transcript may be modified, edited or shuffled resulting in changes that affect protein synthesis

• Protein translated from RNA, many modifications possible

Proteomics Defined

• Study of the function, structure and interaction of proteins with each other and their environment

• Aims to completely describe all proteins in an organism, cell or under specific environmental conditions.

• Human Proteome Organization (HUPO) is an international attempt to catalog human proteins and their functions.

Proteome

• Collection of proteins that comprise a cell, tissue or organism.

• Differ from cell to cell, tissue to tissue and organism to organism.

• Constantly changing through biochemical and environmental interactions.

• Radically different protein expression in different cells and tissues and expression changes through out live and environment.

Antibodies

• Key proteins found in all animal immune systems.

• Generated to detect foreign invaders and tag them for destruction.

• Due to specificity for target makes them ideal for diagnostic tests.

• Immunodetection techniques widely used in proteomics research.

Western Blotting

• Immunodetection technique used to detect and quantify specific proteins in complex biological samples.

• Proteins extracted• Separated by electrophoresis• Transferred or “blotted” from gel onto paper

membrane• Add antibody which will bind to protein of interest.• Antibody attached to dye to make reaction visible.

Western Blot

• Can identify specific protein among hundreds or thousands.

• Based on 2 distinguishing features: molecular mass and antibody binding

• Still method of choice for HIV confirmation.

Hypothesis

• Proteins can be indicators of genetic and evolutionary relatedness

• Myosin major muscle protein essential for locomotion and survival in all animals.

• Essential structure and function of myosin has remained stable or “conserved” in all animals over time.

Process

• Protein gel electrophoresis and western blotting will be used to specifically identify a subunit of a myosin light chain from the many thousands of proteins comprising the muscle tissue of different fish.

• Compare myosin light chains of different species for variation, commonality and evolutionary divergences.

Conservation

• Mutation-> Variation -> Specialization -> Speciation -> Evolution

• Myosin is a protein essential for fight or flight, mutation affection function would decrease survival ability.

• DNA -> RNA -> Protein -> Trait -> Evolution• High degree of myosin conservation and stability

across the animal kingdom means that antibody against myosin protein in chickens will recognize myosin in trout.

Protein Structure

• Primary structure determined by linear amino acid sequence

• 20 common amino acids joined by peptide bonds to form specific polypeptide sequences.

• Polypeptide chains form primary structures.• All proteins exist in 3-D shape.

Protein Shape

• Determined by environmental factors such as:– pH– Temperature– Hydrophilic and hydrophobic interactions and– Protein-protein interactions

Secondary Shape

• Hydrogen bonding between side chains of individual amino acids

• Polypeptide chain bends and folds leading to secondary structural changes.

Tertiary Structure

• Caused by covalent modifications to polypeptide chains that also encourage 3 D shape.

Quaternary Structure

• Observed when multiple polypeptide chains come together to make a single functional protein.

• Myosin is a multi-subunit protein composed of 6 individual polypeptide chains.

Interactions

• All interactions occurring at primary, secondary, tertiary and quaternary level produce helices, pleated sheets and other 3D characteristics of biologically active proteins.

• Protein structure must be disrupted or denatured to accurately separate proteins by size.

Sample Prep

• Extract proteins from muscle tissues of different fish.

• Cell membrane has lipid bilayer• Lysis buffer used to break open or lyse muscle

cells contains detergent sodium dodecyl sulfate (SDS) and strong reducing agent dithiothreitol (DTT)

Sample Prep

• SDS coats proteins with negative charge.• DTT breaks disulfide bridges that contribute to

secondary, tertiary and quaternary structure.• SDS and DTT contained in lysis buffer

(Laemmli buffer)• Heating to 95C further denatures proteins.• Once extraction is complete proteins

uniformly coated with SDS carry equivalent negative charge density.

Sample Prep

• SDS-PAGE electrophoresis used to separate protein subunits or polypeptides based on size.

• Laemmli contains Tris – a buffer that maintains a constant pH, glycerol to add density to samples so they sin in wells when loaded and tracking dye, Bromophenol Blue, to help visualize sample during loading and allow for tracking of protein migration during electrophoresis.

Electrophoresis

• Proteins migrate through gel matrix based on size.

• Smaller peptides move more rapidly towards positive electrode.

• Larger take longer.• Denatured (linear) peptides can be more

readily analyzed via gel electrophoresis than large 3D complexes of proteins.

Polyacrylamide Gel Electrophoresis

• PAGE used to separate proteins using electric current.

• Negatively charged proteins migrate through gel towards positively charged anode.

• PAGE used to separate very small DNA fragments (<500bp) for DNA sequencing or PCR analysis.

• Agarose used to separate very large proteins.

PAGE

• PAGE resolves bands tighter• Uses 2 phases of polyacrylamide– Upper stacking gel typically 4%– Lower resolving gel (15% for this lab)

• Discontinuous system proteins migrate rapidly and compress at edge of denser resolving gel

• Allows thin bands of uniform size in each lane before they move into denser resolving gel and separate according to MW

PAGE

• Heat, ionic detergent and reducing agent completely disrupt the structures of proteins and protein complexes.

• Results in linear chains of amino acids.• These molecules snake through the gel at

rates proportional to their molecular masses.• Glycine in the buffer provides ions to transmit

current, SDS maintains denaturation of proteins.

PAGE

• Coomassie protein stain based on colloidal suspension.

• Once colloidal dye particles are near proteins in gel, dye is removed from colloids by nearby proteins due to high affinity of dye for proteins.

• If time permits wash gel 3x with DI water for 5 minutes before staining.

• Optimal staining is 1 hour with gentle shaking