BIO2015: Lecture 10

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  • Created on: 13-05-14 13:29
what is proteomics?
The study of the full set of proteins encoded by a genome
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why is proteomics much more complicated than genomics?
constantly changing- one organism will have different protein expression patterns in different parts of its tissues, in different stages of its life cycle and under different environmental conditions.
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which is larger in eukaryotes- the genome or the proteome?
The proteome- . This is due to alternative splicing of the genes (isoforms) and posttranslational modifications (e.g. glycosylation, phosphorylation)
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what is glycosylation?
reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (glycosyl acceptor)
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what is phosphorylation?
a serine, a threonine or a tyrosine residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. Regulation of proteins by phosphorylation is one of the most common modes of regulation of protein function
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why is the proteome more complex than the genome?
the genome is defined by the sequence of nucleotides, the proteome is not limited to the sum of the sequences of the proteins present
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what are proteins?
primary gene products, biochemical compounds consisting of 1 or more polypeptides
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what is a polypeptide?
a single linear polymer chain of amino acids held together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues
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what is post translational modification?
when shortly after/during synthesis, the residues in a protein are often chemically modified- this alters the physical and chemical properties of the protein (folding, stability, activity) and thus its activity
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what is primary structure?
amino acid sequence
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what are secondary structures?
regularly repeating local structures stabilized by hydrogen bonds e.g. alpha helix, beta sheet, turns
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what are tertiary structures?
overall shape of the protein i.e. how it is folded- critical for protein structure
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what is tertiary structure stabilised by?
by nonlocal interactions, most commonly the formation of a Hydrophobic core, but also through salt bridges, hydrogen bonds, disulphide bonds, posttranslational modifications
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what are quaternary structures?
the structure formed by several protein molecules (polypeptide chains/protein subunits/single protein molecules) which function as a single protein complex (Not all proteins have a quaternary level of structure)
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what are reverse genetics?
identification of the gene from the gene products
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in proteome analysis what do we usually compare?
proteomes of cells in different states
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what is 2D electrophoresis used for?
the isolation of proteins for further characterisation by mass spectrometry- capable of simultaneously resolving thousands of proteins in one separation procedure
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which types of mass spectrometry are used after 2DE?
1) Peptide mass fingerprinting (MALDI/MS) 2) Sequence based identification (MS/MS)
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what are the gels in 2DE called?
proteome maps
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what is 2D chromatography?
a technique in which the injected sample is separated by passing through two different separation stages, where the second column has a different separation mechanism, so that bands that are poorly resolved from the first column may be completely sep
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what is 2D chromatography an alternative to?
2D electrophoresis
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what is 2D chromatography good for?
separating proteins that have a more extreme range of pIs (Isoelectric points) or a large range of molecular weights.
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what is an advantage of 2D chromatography?
it can be coupled directly to a mass spectrometer. In 2-DE, the samples must be cut from the gel for subsequent analysis.
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In 2D electrophoresis, how is sample preparation carried out?
1) Controlled conditions 2) Avoid contact with all potential contaminants (e.g. keratin) 3) Avoid sample degradation: a) minimise preparation time b) keep sample as cold as possible c) use appropriate protease inhibitors
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what are the reasons for applying protein precipitation procedures?
1) concentrate dilute protein samples 2) prevent/reduce proteolytic degradation
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what precipitation methods are used?
1) Ammonium sulphate precipitation 2) TCA precipitation 3) Acetone precipitation 4) TCA/Acetone precipitation 5) Ammonium acetate/method following phenol extraction
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in 2DE, what is the basis of separation of the first dimension?
Denaturing isoelectric focusing- proteins separated according to isoelectric point
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how does isoelectric focusing (IEF) work?
Proteins separated according to their isoelectric point (carry no charge)
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how does separation by 2-D SDS PAGE work?
Proteins separated according to their molecular weight
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in 2DE, what is the basis of separation of the second dimension?
SDS polyacrylamide gel electrophoresis (SDS PAGE)- Proteins separated according to the molecular weight
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what is DiGE?
a form of gel electrophoresis where up to 3 different protein samples can be labeled with fluorescent dyes (e.g. (for example Cy3, Cy5, Cy2) prior to two-dimensional electrophoresis.
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what are the advantages of Differential Gel Electrophoresis (DiGE)?
DiGE is a system for protein difference analysis: 1) very sensitive 2) do not need technical reps
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what are the disadvantages of Differential Gel Electrophoresis (DiGE)?
1) Need special scanner 2) Cypro dyes very expensive
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what is the process of DiGE?
The 3 samples are mixed and put in the same gel. After the gel electrophoresis, the gel is scanned with the excitation wavelength of each dye one after the other, so we are able to see each sample separately
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it overcomes limitations in traditional 2D electrophoresis that are due to what?
inter-gel variation- this can be considerable even with identical samples- Since the proteins from the different sample types (e.g. healthy/diseased, virulent/non-virulent) are run on the same gel they can be directly compared
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what is In Gel Protein Digestion usually carried out by?
trypsin
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when was 2DE introduced?
1975
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what are the advantages of 2DE?
1) Provides visual images that can be compared 2) Gives information on protein MW and pI 3) Can be stained to reveal subsets of proteins, e.g. phosphorylated and glycosylated 4) Can have high separation capacity
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what are the limitations of 2DE?
1) Hydrophobic proteins can't be analysed 2) Only most abundant proteins can be analysed 3) Proteins with very alkaline pI are poorly resolved on conventional 2D gels 4) Not possible to display all proteins in a single gel
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what are the size limitations?
1) Very high MW proteins (>250 kD)cannot enter the gel 2) very low MW proteins (
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what is the disadvantage of 2DE related to the pI of proteins?
pI of proteins range from pH 3-13; pH gradient from 3 to 13 does not exist (isoelectric strips pH 1 – 10)
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what must happen before peptides can be separated by mass spectrometry?
proteolytic digestion
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what does MALDI-TOF stand for?
Matrix Assisted Laser Desorption Ionisation-Time Of Flight- generates peptide mass fingerprints of the proteins
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what is Peptide Mass Finger-Printing?
1) protein consists of a unique sequence of amino acids 2) Enzymes cut at specific amino acids 3) Proteins cut with an enzyme will give a series of peptides of different masses 4) Different proteins will give a different series of peptides
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for example, where will trypsin cleave?
at Arginine (R) and lysine (K)
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Why do we need to compare proteomes?
Understand organism/tissue/cell response to changing environment
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how does comparing proteomes help in combating diseases?
In certain diseases or stages of development, the protein content of cells may change. If we understand these changes we may understand what is happening in the cell (Biomarkers)
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how can comparing proteomes be used in creating breeding programmes?
Differential response in plant tissues to biotic/abiotic stress
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how can comparing proteomes be used in aiding selection of nutrient use for efficient crops?
Differential response in plant tissues to nutrient exposure
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Card 2

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why is proteomics much more complicated than genomics?

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constantly changing- one organism will have different protein expression patterns in different parts of its tissues, in different stages of its life cycle and under different environmental conditions.

Card 3

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which is larger in eukaryotes- the genome or the proteome?

Back

Preview of the front of card 3

Card 4

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what is glycosylation?

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Preview of the front of card 4

Card 5

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what is phosphorylation?

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