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Comparative Proteomics Lecture; Protein Isolation Lab Activity . SDS-PAGE and Western Blot Lab Activity
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15 mins ago Comparative Proteomics:
Pre-lab Activity;
Protein Extraction;
Protein Electrophoresis
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Student Manual
Does molecular evidence support or reMe the theory of evolution? DNA gets a lot of
attention, but proteins do all the work. Proteins determine an organism’s form, function, and
phenotype. As such, proteins determine the traits that are the raw material of natural
selection and evolution.
In this lab you will use protein gel electrophoresis, the technique most widely used in
biotechnology research, to examine muscle proteins from closely and distantly related fish
species, and to identify similarities and differences in these organisms’ protein profiles, or
fingerprints.
Analogous in principle to DNA fingerprinting, protein profiles can also reveal genetic
similarities or differences, and from such mole~ular data it is possible to Infer relatedness.
Each protein band that a fish has in common with another fish is considered a shared
characteristic. A fish family tree, or cladogram, can be constructed based on protein bands
that the fish have in common. Cladistic analysis assumes that when two organisms share a
common characteristic, they also share a common ancestor with that same characteristic.
Muscle protein consists mainly of actin and myosin, but numerous other proteins also make
up muscle tissue. While actin and myosin are highly conserved across all animal species,
the other proteins are more diverse, varying even among closely related species.
During this laboratory-based scientific investigation you are asked: Can molecular data
show similarities and differences among species? You will compare the similarities and
differences in the protein profiles of various fish species, create a cladogram (family tree)
from your own gel results, and compare your data to published evolutionary data. Then you
will be asked: Do the data agree? Why or wh:v not? What explanations can you suggest?
Molecular biology has unlocked secrets of mystifying new diseases, given us the premier
tools for defining biological identity, and created a pillar of data to support Darwin’s theory
of common descent. In short, molecular biology and its elegant techniques have
revolutionized our understanding of life’s origins and mechanisms.
Is it just genes that determine what proteins will be made? Current research in the field of
proteomics suggests not. The following section is designed as a review of important
background Information for this laboratory investigation.
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Background
Proteomics
The central dogma of molecular biology of DNA • RNA • protein has given us a comfortable
explanation of how the information encoded by our DNA is translated and used to make an
organism. It describes how a gene made of DNA is transcribed by messenger RNA and
then translated into a protein by transfer RNA in a complex series of events utilizing
ribosomal RNA and amino acids. New discoveries about alternative roles for RNA, multiple
forms of proteins being encoded by single genes in our cells, and changes to proteins after
translation are changing this comfortable scenario and we are finding that things (as ever in
biology) are not so simple. Although in essence the ~entral dogma remains true,
investigations into genomics and proteomics are revealing a complexity that we had never
imagined.
In 1990, a massive research effort took place to sequence what was estimated to be the
100,000 genes that coded for each protein synthesized by humans (the human genome).
This study, the Human Genome Project, took 13 years to complete. When the study began,
scientists estimated that there were over 100,000 human genes. Now, years after the
genome has been sequenced, there is still no consensus on the actual number of human
genes, but the current estimate is down to around 22,000 human genes, this is only a few
thousand more genes than encodes the genome of a much simpler organism, C. e/egans,
a nematode worm that has around 19,000 genes.
So why are a similar number of genes required to make a worm and a person? Importantly,
a human has a much larger total genome (3 billion base pairs) than a worm (1 00 million
base pairs) suggesting that the total amount of DNA rather than the actual number of
genes may be what gives rise to complexity. In addition, recent developments have shown
it is quite common in complex organisms for a single gene to encode multiple proteins.
Moreover, changing when, to what level and where a protein is expressed, or changing a
protein after it has been translated (posttranslational modification) can result in proteins
with very different functions. This realization of the importance and diversity of proteins
started a whole new field termed proteomics.
Proteomics is the study of proteins, particularly their structures and functions. This term
was coined to make an analogy with genomics, and while it is often viewed as the “next
step”, proteomics is much more complicated than genomics. Most importantly, while the
genome is a rather constant entity, the proteome differs from cell to cell and is constantly
changing through its biochemical interactions with the genome and the environment. The
entirety of proteins in existence in an organism throughout its life cycle, or on a smaller
scale the entirety of proteins found in a particular cell type under a particular type of
stimulation, are referred to as the proteome of the organism or cell type, respectively. One
organism will have radically different protein expression in different parts of its body, in
different stages of its life cycle, and in different environmental conditions.
Proteomics was initially defined as the effort to catalog all the proteins expressed in all cells
at all stages of development. That definition has now been expanded to include the study
of protein functions, protein-protein interactions, cellular locations, expression levels, and
posttranslational modifications of all proteins within all cells and tissues at all stages of
development. Thus, it is hypothesized that a large amount of the noncoding DNA in the
human genome functions to highly regulate protein production, expression levels,
posttranslational modification etc., and it is this regulation of our complex proteomes, rather
than our genes, that makes us different from worms.
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To catalog all human proteins and ascertain their functions and interactions presents a
daunting challenge for scientists. An international collaboration to achieve these goals is
being coordinated by the Human Proteome Organization (HUPO).
Research in the proteomic field has discovered a number of modification systems that
allow one gene to code for many proteins and mechanisms that finely regulate the sub- and
extracellular locations and expression levels of proteins. These include alternative splicing
of exons, use of different promoters, posttranscriptional modification, translational
frameshifting, posttranslational modification, and RNA editing.
Evolution
The term evolution probably brings to mind Charles Darwin and the Theory of Natural
Selection. In short, this theory states that there are more organisms brought into the
environment than can be supported by the environment. Each of these individuals are
different- even among the same species. The environment selects organisms best suited
to survive and reproduce based on those differences. Adaptations are the differences that
make one organism more suited to the environment than another individual. These
adaptations are phenotypic (physical) characteristics such as finch beaks that are determined
by a genetic component. The genetic component is inherited from the parent in the form of
genes.
Variations in an organism’s proteins may reflect physiological adaptations to an ecological
niche and environment, but they originate as chance DNA mutations. Such random mutation
events, if favorable, persist through the natural selection process and contribute to the
evolution of new species -with new specialized functions.
The discovery of the chemical structure of DNA by Watson, Crick, Wilkins, and Franklin
and our understanding of how the triplet code of nitrogen bases leads to the synthesis of
proteins (which is the phenotypic expression) convinced us that adaptations are the result
of changes in the DNA code (mutations). However, current research in the field of proteomics
is leading some scientists to question whether or not DNA is the final determining factor in
the synthesis of proteins and thus the determining factor in evolution.
Student Manual
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Lesson 1: Introduction to Protein Electrophoresis and
SDS..PAGE
How Can We Study Proteins Found in Muscle?
Eoly.§crylamide gel .e,lectrophoresis (PAGE) can be used to separate small molecules such
as proteins. Understanding protein structure is important to understanding how we can use
PAGE for protein analysis.
Proteins are made of smaller units (monomers) called amino acids. There are 20 common
amino acids. The sequence and interaction between these different amino acids determine
the function of the protein they form. Amino acids are joined together by peptide bonds to
form polypeptide chains. Chains of amino acids constitute a protein. In tum these chains
may interact with other polypeptides to form multi-subunit proteins.
Amino acids can be combined in many different sequences. The sequence of the amino
acids in the chain is referred to as the primary protein structure. All amino acids have the
same basic structural component (Figure 15).
0
OH
R
Fig. 15. Chemical structure of an amino acid.
The “R” group may be charged or uncharged, or may be a long side chain. Thus, each
amino acid has different properties and can interact with other amino acids in the chain.
Hydrogen bonding between these side chains, primarily between the C=O and the N-H
groups, causes the protein to bend and fold to form helices, pleated sheets, reverse turns,
and non-ordered arrangements. Disulfide bonds between methionines can also bend and
loop the amino acid chain. This is considered the secondary structure of the protein.
The tertiary structure of the protein is determined by the interaction of the hydrophilic and
hydrophobic side chains with the aqueous environment. The hydrophobic regions aggregate
to the center of the molecule. The hydrophilic regions orient themselves toward the exterior.
These ordered bends and folds make the protein compact. Examples of tertiary protein
structure are structural and globular proteins.
The quaternary structure of proteins is achieved from the interaction of polypeptide chains
with others. Multiple polypeptides can combine to form complex structures such as the
muscle protein myosin, or the blood protein hemoglobin, which are both composed of four
polypeptide chains. These complex proteins are often held together by disulfide bonds
between cysteines. In fact, PAGE analysis was first carried out in 1956 to show the genetic
disease sickle cell anemia is caused by a change to a single amino acid of the hemoglobin
protein (Ingram 1956).
Prior to electrophoresis, the proteins are treated with the detergent sodium dodecyl sulfate
(SDS) and heated. SDS and heat denatures (destroys) the protein tertiary and quaternary
structures, so that the proteins become less three dimensional and more linear. SDS also
gives the protein an overall negative charge with a strength that is relative to the length of
its polypeptide chain, allowing the mixture of proteins to be separated according to size.
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Fig. 16. The combination of heat and the detergent SDS denatures proteins for SDS-PAGE analysis.
The proteins, in their 80S-containing Laemmli sample buffer, are separated on a gel with a
matrix that acts to sieve the proteins by size upon addition of an electric current. A
polyacrylamide gel is positioned in a buffer-filled chamber between two electrodes, protein
samples are placed in wells at the top of the gel, and the electrodes are connected to a
power supply that generates a voltage gradient across the gel. The 80S-coated,
negatively charged proteins migrate through the gel away from the negatively charged
anode toward the cathode, with the larger proteins moving more slowly than the smaller
proteins. This technique was developed by U.K. Laemmli, whose 1970 Nature paper has
received the highest number of citations of any scientific paper. 808-PAGE is still the
predominant method used in vertical gel electrophoresis of proteins.
As soon as the electric current is applied, the 80S-coated proteins begin their race toward
the positive electrode. The smaller proteins can move through the gel more quickly than the
larger ones, so over time, the proteins will be separated according to their sizes.
Protein size is measured in dattons, a measure of molecular mass. One dalton is defined
as the mass of a hydrogen atom, which is 1.66 x 1o-24 gram. Most proteins have masses
on the order of thousands of daHons, so the term kilodatton (kO) is often used to describe
protein molecular weight. Given that the average weight of an amino acid is 11 0 daHons,
the number of amino acids in a protein can be approximated from its molecular weight.
•
Average amino acid = 110 daltons
•
Approximate molecular weight of protein
=number of amino acids x 110 daltons
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Fig. 17. A quaternary prolaln complex denatured wllh reducing agents, heat, and 806, can be eaparaled
Into Individual proteins and resolved by size using SDS-PAGE.
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In this investigation, you will use SDS-PAGE to separate and analyze the protein profiles of
the muscle tissue of different fish. By comparing the protein profiles of different fish species
you can test the hypothesis that protein profiles are indicators of genetic and evolutionary
relatedness.
VIsualizing your proteins
Proteins in your samples are not visible while the gel is running. The only visible proteins
will be those in the Precision Plus Protein Kaleidoscope standard that have been
prestained with covalently attached dyes. You should see these proteins resolve into
multicolored bands that move down the gel as power is run through the gel. If the electric
current is left on for too long, the proteins will run off the bottom of the gel. To guard against
this and to show you the progress of your gel if you did not have the standards, a blue
tracking dye is mixed with the Laemmli sample buffer used to prepare your protein samples.
This blue dye is negatively charged and is also drawn toward the positive electrode. Since
the dye molecules are smaller than the proteins expected in most samples, they move
ahead of the proteins in the gel.
After turning off the electric current and removing the gel from the glass plates that hold it in
place, the gel is placed in a stain. The stain used in this technique was originally developed
to dye wool, which, like your own hair, is composed of protein. This stain binds specifically
to proteins and not to other macromolecules such as DNA or lipids. After destaining,
distinct blue bands appear on the gel, each band representing on the order of 1012
molecules of a particular protein that have migrated to that position: the larger the amount
of protein, the more intense the blue staining.
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1
Lab. Comparative Proteomics:
1. Pre-lab Activity – How closely related are different fish species?
2. Protein Extraction
3. Protein Electrophoresis
1. Pre-Lab Activity: Predict how closely related different fish species are using different sources of
information
Your instructor has assigned you five different species of fish.
You will fill in a provided ‘Fish Data Sheet ‘ for each fish. Use information from the following database
and material provided in this handout in the section labeled ‘Taxonomic Classification’.
Go to http://www.fishbase.net
First follow the example below to become familiar with how the database works. The database may
have changed slightly from the example.
Example – Rainbow Trout
Type “rainbow trout” into the Common Name field on the FishBase home page.
This search term returns at least 22 different types of fish called rainbow trout. All species are the same.
Click on the species Oncorhynchus mykiss from the USA. Use the web page this link takes you to in order
to fill in the Fish Data Sheet. Note: some information, such as swim type, may need to be reached by
following an additional link on the page.
Common Name: Rainbow trout
Scientific Name: Oncorhynchus mykiss
Taxonomic Classification:
Family: Salmonidae (Salmonids)
Order: Salmoniformes (salmons)
Class: Actinopterygii (Ray-finned fishes)
Size: Max weight 25.4 kg
Environment: Benthopelagic; anadromous freshwater; brackish; marine; depth range 0-200 m
Biology: Survive better in lakes than streams. Needs fast flowing well oxygenated waters for spawning.
Can adapt to sea water if necessary.
Swim type: moves body and caudal fin
Definitions of unfamiliar terms: Benthopelagic – feeds on bottom, midwaters, and near surface. Hovers
near bottom.
Anadromous – ascend rivers to spawn
Now it is time to investigate your own fish species. Enter the common name of one of the species of fish
you will be investigating into the FishBase search field. Then click on the scientific name link and use the
information it brings up to fill in the fields of your Fish Data expressed in fish muscles, feel free to add it
2
to your data sheet in the additional factors Sheet for each fish species you will be investigating. If you
find terms you are unfamiliar with, find out what they mean and write definitions in the field for
definitions of unfamiliar terms. Hint: performing an Internet search with the unfamiliar term and then
writing “definitions” after it (for example. “benthopelagic definition”) usually brings up dictionary
definitions of terms.
Once your Fish Data Sheets are filled in, use the information you have gathered to answer the questions
below and develop your hyphotheses about the relatedness of the fish species.
Focus Questions
1. Which of the fields you have filled in on your Fish Data Sheets (taxonomic classification, environment,
biology, swim type and additional factors) do you think will help you most in predicting which fish have
the most similar muscle protein profiles?
2. For each field you have chosen, state why you think these fields will help you to make these
predictions, and for those fields you haven’t chosen state why you don’t think these will help.
3. Predictions: Using the fields you have stated above to help you predict which fish will have similar
muscle protein profiles, predict which two fish will have the most similar protein profiles. Describe how
you have come to this prediction.
4. Predictions: Similarly, predict which two fish will have the most contrasting protein profiles, i.e., the
fewest proteins in common. Describe how you have come to this prediction.
Develop your hypotheses.
Use your predictions to write out two hypotheses:
Hypothesis 1: Which fish species are most closely related?
Hypothesis 2: Which fish species are least closely related?
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Taxonomic Classification:
An evolutionary tree shows the evolutionary lineages of different species over relative time.
Evolutionary trees, (also called cladograms), can be based on many different types of data. Some trees
are constructed using a single type of data and some trees use multiple types of data. The traditional
way of constructing evolutionary trees was to look at the physical morphology of organisms, including
sizes, shapes an ddevelopmental structures of both living organisms and fossils. Today, similarities and
differences in protein and DNA sequences are being used. Both methods are valuable and often
complement each other but the…
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