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Proteomics. Flashcards

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1
Q

Define Edman sequencing?

A

A method to determine the individual amino acids that make up a protein.

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2
Q

Define the isoelectric point?

A

The pH at which a molecule has a net charge of 0.

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3
Q

Define a phage coat protein?

A

A gene in the DNA of a bacteriophage that will code for a protein in the phage coat.

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4
Q

Define a phage display library?

A

A library made up of bacteriophages that contain foreign proteins that have been inserted into their protein coat.

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5
Q

Proteomics is the study of what?

A

It is the study of all of the proteins that are within a certain cell or body at a given time.

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6
Q

Is proteomics context dependent?

A

Yes.

As the number of proteins within a cell or body will change over time.

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7
Q

What is the goal of proteomics?

A

To get an integrated view of biology by studying all of the proteins within a cell rather than studying each protein individually.

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8
Q

What map does the study of proteomics allow us to create?

A

A 3D map that tells us the locations of all the proteins within the cell.

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9
Q

Proteomics will utilise information from which 3 disciplines?

A

Molecular biology.

Biochemistry.

Bioinformatics.

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10
Q

Will genomics tell us all of the products that can be produced by a particular gene?

A

No.

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11
Q

Why can genomics not tell us all of the products of a particular gene?

A

Because one gene may have multiple products.

Because some proteins may be modified after they have been produced.

Some proteins may be compartmentalised.

Some proteins will also undergo a process called proteolysis

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12
Q

What happens when a protein is compartmentalised?

A

When they are synthesised in one part of the cell and then transported to another location.

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13
Q

What is proteolysis?

A

When the original protein is cleaved to produce a functioning protein.

E.g. trypsinogen is cleaved to form trypsin.

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14
Q

The study of proteomics gives us what knowledge about specific genes.

A

Their functions.

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15
Q

What types of proteins does proteomics allow us to study?

A

The types of proteins are constructed by a particular cell type such as a bacterial cell or a cancer cell.

The type of proteins that are expressed by cells under certain conditions.

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16
Q

What systems allow us to view the proteins expressed by a cell?

A

Protein expression systems.

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17
Q

What must scientists do after they have extracted proteins from a cell?

A

They must then be separated into individual proteins.

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18
Q

When can proteins be individually analysed and characterised?

A

After the proteins have been separated from the protein mass.

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19
Q

What feature of proteomics will tell us the function of the protein.

A

The analysis and characterisation of proteins.

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20
Q

What process did scientists originally use to evaluate proteins?

A

Western blot.

Protein stains and assays.

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21
Q

Who developed 2D-gel electrophoresis in 1975.

A

Patrick O’Farrell and Leigh Anderson.

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22
Q

What does 2D-gel electrophoresis allow scioentists to do?

A

To analyse all of the proteins in a given cell at a given time.

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23
Q

Does 2D-gel electrophoresis allow for the identification of individual proteins?

A

No.

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24
Q

What can 2D-gel electrophoresis tell us about proteins?

A

The abundance of proteins.

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25
Q

What method of proetomics sorts proteins by the iso-electirc point?

A

Immobilised pH gradients.

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26
Q

What is the most common method of protein identification today?

A

Mass spectrometry.

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27
Q

Who developed the term proteomics in 1995?

A

Marc Wilkins.

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28
Q

What are the 5 most common techniques for separating individual proteins from a collection of proteins?

A

1D electrophoresis (1-DE).

2D electrophoresis (2-DE).

Protein digestion.

Purification.

Isotope coded affinity tags (ICAT).

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29
Q

What process is used in conjunction with 1 and 2D electrophoresis?

A

Western blot.

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30
Q

What will Western blot allow for the detection of?

A

The quantity of proteins within a sample.

To detect for the presence of a particular protein after 1 or 2D electrophoresis has been carried out.

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31
Q

What is the first step of 1D-E?

A

To extract proteins from cells.

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32
Q

What is happens during 1D-E after the proteins have been extracted from the cell?

A

The bonds within these proteins are broken via

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33
Q

Why are the bonds within the proteins broken during 1D-E?

A

It allows the protein to revert back to its primary structure which is the chain of amino acids.

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34
Q

What chemcials are used to break the bonds in proteins during 1D-E?

A

Chemicals such as mercaptoethanol.

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35
Q

What happens in 1D-E after the bonds within the proteins have been broken?

A

They are sorted into order of size.

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36
Q

What kind of process is used to sort the proteins into order of size in 1D-E electrophoresis?

A

SDS-PAGE gel electrophoresis.

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37
Q

What are the proteins coated with during SDS-PAGE gel electrophoresis?

A

The proteins are coated with SDS so they all have the same negative charge.

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38
Q

Why are the proteins coated with SDS during SDS-PAGE gel electrophoresis?

A

So that they all migrate towards the cathode during electrophoresis.

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39
Q

What is used to visualise the proteins after SDS-PAGE gel electrophoresis has been carried out?

A

By staining the proteins with a dye called coomassie blue.

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40
Q

What can be used immediately after SDS-PAGE gel electrophoresis to esitmate the molecular weights if proteins?

A

The readings are aligned next to a series of molecular weight markers which can be used to approximate the molecular weights of proteins.

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41
Q

What process should be carried out after SDS-PAGE gel electrophoresis to obtain more precise results?

A

Western blot.

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42
Q

What is the first step of Western blot?

A

To transfer the proteins from the gel onto a solid base such as a nitrocellulose or nylon membrane.

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43
Q

What machine will help to transfer the proteins onto the solid base during Western blot?

A

An electroblotter.

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44
Q

What process occurs during Western blot after the proteins have been trasferred to the solid base?

A

We can search the sample for antibodies that are specific to a certain protein.

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45
Q

How are the proteins edited after they have been added to the solid base during Western blot?

A

5% skimmed milk is added to the solid membrane.

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46
Q

Why is the milk added to the protein during Western blot?

A

The milk will attach to the membrane in all of the places where the target proteins have not attached.

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47
Q

What happens in Western blot after the milk has been added?

A

The blot is incubated with a primary antibody which will bind to specific proteins.

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48
Q

What happens in Western blot after the blot has been incubated with the primary antibodies?

A

The blot is incubated with secondary antibodies which will specifically bind to the primary antibody.

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49
Q

What is bound to the secondary antibodies that are used in Western blot?

A

The secondary antibody is bound to an enzyme called horseradish peroxidase.

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50
Q

What is the function of horseradish peroxidase when it is used in Western blot?

A

It creates chemical luminescence when exposed to hydrogen peroxide (H2O2) and luminol.

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51
Q

What feature of Western blot will inform the researcher that a particular protein has been found?

A

The luminescence of horseradish P creates a colour change.

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52
Q

What proteins can be seperated by 2D-E?

A

Proteins that have varying biochemical properties and have originated from a cell, tissue or protein extract.

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53
Q

How many steps are involved in 2D-E?

A

2 sequential steps.

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54
Q

How does the first step of 2D-E separate proteins?

A

It separates the proteins by their charge.

55
Q

What is used in step 1 of 2D-E to separate the proteins?

A

An immobilised pH gradient gel that ranges from pH 4 at one end to pH 10 at the other.

56
Q

How is step 1 of 2D-E carried out?

A

The proteins are placed into this gel and an electric current is applied.

This forces the proteins to migrate to the area of pH within the gel at which they will have no net charge.

57
Q

How does step 1 of 2d-E seprate the proteins?

A

By their charge or based on their isoelectric point.

58
Q

How does step 2 of 2-DE separate the proteins?

A

By their mass.

59
Q

What is the immobilised gel from step 1 of 2D-E placed into at the beginning of step 2?

A

Into an SDS-polyachrimyde gel.

60
Q

What occurs in step 2 of 2D-E?

A

A current is applied to the gel and the proteins will migrate towards the positive end of the gel.

This separates the proteins by molecular mass.

61
Q

How are the proteins seperated at the end of step 2 of 2D-E?

A

By molecular mass and by pH.

62
Q

What happens to spots of interest from after 2D-E has been carried out?

A

They can be removed from the gel and analysed by mass spectrometry to fully identify the protein.

63
Q

What can be used to identify a protein after 2D-E has taken place?

A

Western plot.

64
Q

What does the process of differential electrophoresis allow for?

A

For the comparison of proteins from a tissue or cell under different conditions.

65
Q

What is the first step of differential electrophoresis?

A

The proteins from each sample are labelled with different fluorescent markers and then undergo 2-DE.

66
Q

How are the 2 samples differentiated after 2D-E during differential electrophoresis?

A

Each sample will express different colours as they are stained with different markers.

67
Q

How does differential electrophoresis allow scientists to work out which proteins are expressed under which conditions?

A

Proteins that are expressed under 1 condition could be stained red and proteins that are produced under other conditions could be stained green.

When the 2 samples are combined, the presence of red areas indicates the expression of proteins from sample 1.

The expression of green areas would indicate the expression of proteins from sample 2.

68
Q

What colour indicates equal expression of both protins in differential electrophoresis?

A

Areas of yellow fluorescence would indicate equal expression of both proteins.

69
Q

What colour indicates no protein expression in differential electrophoresis?

A

Areas of orange fluorescence would indicate an up-regulation or down regulation of the proteins.

70
Q

Can mass spectrometers analyse very large proteins?

A

No.

71
Q

How are large proteins analysed by mass spectrometry?

A

They are digested into smaller fragments as mass spectrometers can analyse peptide fragments.

72
Q

How are large proteins digested into smaller fragments for mass spectrometry?

A

Proteases cleave the proteins at specific amino acid residues.

This makes peptide fragments that are around 16-20 amino acids long.

73
Q

What are some of the proteases that are used to break up large proteins for mass spectrometry?

A

Trypsin.

Chymotripsin.

Glu-C.

Lys-C.

Asp-N.

74
Q

What happens to the proteins after they have been cleaved for mass spectrometry?

A

They are purified.

75
Q

What are 4 methods of purifying the peptide fragments that are cleaved from the large proteins during mass spectrometry?

A

Liquid chromatography.

Capillary electrophoresis.

Cation exchange chromatography.

Reverse phase chromatography.

76
Q

What is a common method of performing the digestion and purification of proteins at the same time?

A

Column proteolytic digestion and column chromatography.

77
Q

What is the first step of column proteolytic digestion and column chromatography?

A

The protein sample and some enzymes are added to a test-tube.

78
Q

What happens to the protein enzyme combination after they have been added to the testtube during column proteolytic digestion and column chromatography?

A

The test-tube is spun to remove any impurities from the sample.

This causes the protein and enzymes to bind to a specific column within the tube.

79
Q

What happens during column proteolytic digestion and column chromatography after the sample has been spun and the protein/enzyme combination has bound to the column in the tube?

A

A solution that activates the enzymes is added.

The tube is spun again and a solution is added to elute the remaining peptide fragments.

These fragments can then be analysed by mass spectrometry.

80
Q

What does the process of using isotope coded affinity tags (I-CAT) allow for?

A

To differentiate between proteins that have been taken from 2 different sources.

E.g. proteins from 2 different cells or from the same cell under different conditions.

81
Q

How are the proteins from each sample differentiated during I-CAT?

A

The cysteine residues within the protein from cell A are all labelled with a solution called light ICAT.

The proteins from cell B are labelled with a solution called heavy ICAT.

82
Q

What isotope is used in light I-CAT?

A

Hydrogen.

83
Q

What isotope is used in heavy I-CAT?

A

Deuterium.

84
Q

What happens during I-CAT after the samples have been labelled with their various I-CAT solutions?

A

The 2 samples are combined with each other and with a selection of proteases that will digest the proteins.

85
Q

During I-CAT, what happens to the protein fragments after they have been digested?

A

They can be analysed by mass spectrometry.

86
Q

How are the different protein samples from I-CAT identified during mass spectrometry?

A

The 2 different isotopes give off different signals and this allows for differentiation between the 2 sets of proteins.

87
Q

What can I-CAT be used to determine about proteins?

A

It can determine what levels of a particular protein are expressed in a cell under certain conditions.

It can be used to see if a particular cell type will express more of a particular protein than another cell type.

88
Q

What are the 2 techniques that can be used to determine the individual amino acids that make up the structure of a protein?

A

Edman sequencing.

Mass spectrometry.

89
Q

Who invented Edman sequencing?

A

Victor Edman.

90
Q

Edman sequencing can be used after proteins have been separated by what processes?

A

Edman sequencing can be used after a proteins have been separated be 1D or 2D electrophoresis.

91
Q

What happens during Edmans sequencing after the proteins have been separated by electrophoresis?

A

The proteins are transferred to an inert nylon or nitrocellulose membrane.

92
Q

What happens during Edmans sequencing after the proteins have been transferred from the electrophoresis gel to the inert membrane?

A

The protein of interest can then be removed from the membrane and inserted into the Edman sequencer.

93
Q

What 2 things can happen during Edman sequencing after the protein of interest has been transferred to the Edman sequencer?

A

Cyanogen bromide (CnBr) can be used to cleave the peptide bonds that are formed from methionine residues.

Or skatole can be used to cleave peptide bonds at tryptophan residues.

94
Q

What happens during Edman sequencing after the protein of interest has been cleaved by CnBr or skatole?

A

It will be split into 3-5 peptide fragments.

95
Q

What will the number of peptide fragments that are formed from the use of CnBr or skatole willl be consistent with?

A

The amount of tryptophan or methionine residues in the protein.

96
Q

During Edman sequencing, what happens to the peptide fragments that are formed after CnBr or skatole cleavage?

A

They can be placed into the Edman sequencer.

97
Q

What happens during Edmans sequencing after the peptide fragments are placed into the Edman sequencer?

A

The sequencer will perform around 6-12 Edman cycles over 8-12 hours.

This will provide mixed data in the form of all the individual amino acids recovered after each cycle.

98
Q

How can the mixed sequencing data from the Edman sequencer be analysed so that the original protein can be identified?

A

The amino acids are submitted to FASTF or TFASTF algorithms so that the protein can be identified.

99
Q

What is the first step of mass spectrometry?

A

To send the protein samples for high performance liquid chromatography (HPLC).

100
Q

What does high performance liquid chromatography allow for during mass spectrometry?

A

It allows for the separation of the peptide fragments.

101
Q

What happens during mass spectrometry after the peptide fragments have been separated by HPLC?

A

The fragments enter a region of the spectrometer that has either;

Matrix assisted laser disruption ionisation (MALDI).

Or electro spray ionisation (ESI).

102
Q

What happens when the peptide fragments enter the region of the mass spectrometer that has MALDI or ESI?

A

They will be converted to their gaseous ions.

103
Q

What hapens during mass spectrometry after the peptide fragments have been converted to ions?

A

The ions are separated according to differences in mass to charge (m/z) and this allows for identification of the protein.

104
Q

What region of the mass spectrometer does identification of the ions take place in?

A

In the mass analyser region of the vacuum system.

105
Q

What are 3 techniques that are used to to identify or quantify the function of various proteins?

A

Protein function microarrays.

Phage display approach.

Yeast 2-hybrid system.

106
Q

Protein function microarrays are used to screen for what 6 types of protein interactions

A

Protein-protein interactions.

Protein lipid interactions.

Protein-DNA interactions.

Protein-drug interactions.

The identification of enzyme substrates.

The profiling of immune responses.

107
Q

What is the first step of performing a protein function microarray?

A

To purify different proteins or different protein domains and spot them onto nickel coated microscope slides.

108
Q

What happens during a protein function microarray after the proteins have been spotted onto the nickel coated microscope slides?

A

We can use probes that are labelled with specific molecules.

109
Q

What molecules are often used to label the probes used in a protein function microarray?

A

Nucleic acids.

Proteins.

Lipids.

Small molecules.

110
Q

What happens during a protein function microarray if a protein hybridises to the probe?

A

Then we know that the protein of interest interacst with the substance that was on the probe.

E.g. If the probe is labelled with a lipid then we know that the protein we are studying interacts with lipids.

111
Q

The interactions of proteins tells us what?

A

A lot about their functions and what processes they are involved in.

112
Q

What factor about proteins can be identified by the phage display apparoach?

A

Protein interactions.

113
Q

What is step 1 of the phage display apparoach?

A

We take vector DNA that contains a phage coat protein gene from a bacteriophage.

We then insert the DNA that codes for a particular protein into the bacteriophage DNA.

114
Q

What is step 2 of the phage display apparoach?

A

The bacteriophage infects a bacterial cell.

115
Q

What happens in the phage display apporach after the bacteriophage has infected a bacterial cell?

A

The bacterial cell will start to make phage proteins.

116
Q

What kind of protein will be syntheised by the infected bacteria during the phage display approach?

A

The insertion of foreign DNA causes the bacterial cell to make a fusion protein.

117
Q

What is the fusion protein that is made by the infected bacteriophage in the phage display approach?

A

It is half phage protein and half foreign protein.

118
Q

What happens to the fusion protein that is made via the phage display approach?

A

It is incorporated into the coat of newly synthesised bacteriophages.

The bacteriophage is then stored in a phage library.

119
Q

What kind of proteins do we want the bacteriphages in phage libraries to contain?

A

Known proteins.

120
Q

Why do we want the phages in phage libraries to contain known proteins?

A

As this means we can then extract a particular phage and use the foreign protein to test other proteins.

121
Q

What happens during the phage display approach after the phage has been added to the phage library?

A

We can take an unknown protein and mix it with a particular phage that contains immune proteins.

We can then see if the proteins from the sample will react with the immune proteins of the phage.

If an interaction occurs then we know that the foreign protein has an immune function.

122
Q

Why is the phage display approach performed using a microarray?

A

As the array has a test protein in all of the wells.

This means a different phage can be added to each well and we see if hybridisation has occurred.

This allows us to test the protein with multiple phages so that we can learn as much as possible about the function.

123
Q

What is the yeast 2 hybrid approach used for?

A

For testing for protein-protein interactions.

124
Q

What proteins are part of the yeast 2 hybrid approach experiment?

A

Activator proteins that control gene expression in eukaryotes.

125
Q

What is used to infect the yeast cells in the yeast 2 hybrid approach?

A

2 plasmids that contain the same gene. and introduces them into the yeast cell.

126
Q

What are the 2 plasmids that are used in the yeast 2 hybrid apporach?

A

1 plasmid is called HYBRID-1 and it contains the coding gene and a DNA binding domain.

The 2nd plasmid is called HYBRID-2 and it contains the coding gene and an activation domain.

127
Q

What protein is added to HYBRID-1 in the yeast 2 hybrid approach?

A

The known protein which will be fused to the DNA binding domain.

128
Q

What protein is added to HYBRID-2 in the yeast 2 hybrid approach?

A

A protein of interest which may or may not interact with the known.

129
Q

What happens in the yeast 2 hybrid approach if the proteins in the 2 hybrids are compatible?

A

They will interact.

This brings the 2 domains together and allows RNA polymerase to transcribe the gene.

130
Q

What happens in the yeast 2 hybrid approach if the proteins in the 2 hybrids are not compatible?

A

The proteins will not interact and the gene will not be transcribed.

131
Q

How are scientists informed of the protein interaction in the yeast 2 hybrid approach?

A

When the gene is expressed it will trigger a colour change within the cell.

132
Q

What are 6 major benefits of proteomics?

A

Protein expression profiling.

Detection of post translational modifications.

Detection of protein-protein interactions.

Detection of the structure of proteins.

Detection of the function of proteins.

The discovery of new proteins.

133
Q

What is a major challenge of proteomics?

A

That the proteins in the body are always changing.

Molecular Biology. (22 decks)

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