20.02.11 Protein based techniques

Question 1

Name 4 protein-based techniques

Answer 1

1. Western blotting
2. Immunoprecipitation
3. Immunohistochemistry
4. Mass spectrometry

Question 2

What does western blotting do

Answer 2

-Separates proteins by electrophoresis, which is then transferred to an adsorbent membrane where antibodies detect protein of interest

Question 3

Uses of western blotting

Answer 3

• Proves the absence/ presence of protein
• Compares protein levels across different tissues/ physiological conditions/ development.
• To assess purity
• Estimate relative molecular mass

Question 4

WB step 1- Sample preparation steps.

Answer 4

• Lyse cells/ tissue to release proteins. Often buffers have protease inhibitors to prevent degradation of proteins.
• If blot is being used for quantitative purposes then protein conc should be determined (BCA assay)
• Loading dyes used to track migration of sample through gel.

Question 5

WB step 2- Protein separation by SDS-PAGE

Answer 5

• PAGE= polyacrylamide gel electrophoresis
• Proteins separated by molecular weight. Done under denaturing conditions (using SDS, a detergent).
• SDS also gives proteins a negative charge (proportional to length). Therefore proteins separated by MW not charge.
• Non-denaturing conditions needed for antibody recognition. Here samples are not heated or exposed to SDS. Proteins usually have a negative charge in alkaline buffers so migration still occurs.
• 2D PAGE. When a gradient pH is present. pH changes the charge on the protein. Proteins run to their isoelectric point (pH where net charge is zero). The next separation is then based on MW. Used to separate complex samples.

Question 6

WB step 3- Blotting

Answer 6

• Proteins transferred from gel to membrane by diffusion, an electric field or a vacuum
• Gel and paper is sandwiched between absorbent material and submerged in transfer buffer and an electrical field applied. Proteins travel towards positive charged electrode and bind to membrane.

Question 7

WB step 4- antigen detection

Answer 7

• Membrane is washed with non-fat milk to prevent non-specific binding of antibody.
• Direct detection- primary antibody detects antigen on blot and is labelled
• Indirect detection. Primary antibody binds to antigen and a secondary labelled antibody binds to the primary.

Question 8

WB step 5- visualisation

Answer 8

• chomogenic= enzyme coupled antibodies, e.g. HRP (horseradish peroxidase). Substrates added which yield visible coloured products
• Chemiluminescent detection. Substrates produce a chemiluminescent product (CPD), which is detectable on X-ray film
• Fluorescent detection- secondary antibodies are coupled to fluorochromes.

Question 9

What is immunoprecipitation

Answer 9

• Antibody immobilized support that enriches/ purifies a protein from a sample.
• Purified proteins can then be quantified/ analysed by other techniques e.g. Western blotting

Question 10

Two methods for immunoprecipitation

Answer 10

1. Pre-immobilised approach. Antibody is pre-immobilised onto an insoluble support and incubated with lysate containing the target protein. Protein binds to antibody, remaining material washed away. The protein of interest is then eluted.
2. Free-antibody approach. Free unbound antibody forms immune complexes in lysate. Complexes are then isolated using beads. Beneficial if antibody has weak affinity for antigen or binding kinetics are slow.

Question 11

What is co-immunoprecipitation

Answer 11

• Co-IP can identify physiologically relevant protein-protein interactions.
• Protein specific antibodies indirectly capture proteins that bind to the target protein.
• Uses= identify new binding partners, binding affinities, biding kinetics, functions of target proteins.
• Pull down assays= not using antibodies, i.e. covalent attachment or affinity tags

Question 12

What is Chromatin IP

Answer 12

• Selective enrichment of a chromatin fraction containing a specific protein.
• Antibodies used to immunoprecipitated proteins of interest along with associated DNA.
• Uses= gives insight into gene regulation

Question 13

Application of IP in routine testing

Answer 13

• Useful to determine effect of missense mutations on protein-protein interactions, half-life, subcellular location.
• Not really amenable to routine testing.

Question 14

What is immunohistochemistry (IHC)

Answer 14

• Localises specific antigens in tissues using antibodies. Typically visualised using an antibody conjugated with an enzyme (peroxidase) which catalyses a colour-producing reaction.
• Gives evidence on presence/ absence/ localisation of a protein. Also tissue structure and cellularity.

Question 15

Difference between immunohistochemistry (IHC) and immunocytochemistry (ICC)

Answer 15

• IHC= uses sections of tissue (cells surrounded by matrix)

- ICC= uses cells with matrix removed, eg. cultured cells/ suspension/ smear.

Question 16

Difference between monoclonal and polyclonal antibodies

Answer 16

• Monoclonal= recognise a single epitope on an antigen. Less likely to cross react
• Polyclonal= recognises multiple epitopes on any one antigen. Cheaper and easier to produce.

Question 17

Advantages of IHC

Answer 17

• Fast TAT, cost effective

- Provides positional information

Question 18

Disadvantages of IHC

Answer 18

• Antibody cross-reactivity (can lead to false positives)
• Variability in fixation and processing (need to use controls)
• Not quantitative
• Not high-throughput
• Need pathologist expertise to analyse and interpret.
• Does not detect truncated or abnormal proteins with intact epitopes.

Question 19

Give examples of IHC in diagnosing genetic conditions

Answer 19

• IHC for MMR proteins in tumour tissues for Lynch syndrome.
• IHC with EGFR mutation-specific antibodies to detect responders to gefitinib in lung adenocarcinoma. Good for samples with low tumour cell content but has false positives and false negatives.

Question 20

What is mass spectrometry

Answer 20

• Measures mass-to-charge ratio (m/z) of one or more molecules. Calculates exact molecular weight of sample components.
• Can be used to identify unknown compounds, quantify known compounds, and to determine the structure and chemical properties of molecules.

Question 21

`What is a mass spectrometer comprised of

Answer 21

1. Ionisation source: Molecules converted to gas-phase ions so they can be moved by electric/magnetic fields
2. Mass analyser: ions sorted and separated according to m/z ratios
3. Ion detection system: separated ions measured and sent to data system for storage (along with their relative abundance).

Question 22

How are results of MS plotted

Answer 22

• m/z ratios of ions are plotted against their intensities.

- Each peak shows a component of unique m/z. Height shows relative abundance.

Question 23

How can MS be used for peptide/ protein analysis

Answer 23

• determine amino acid sequence
• Characterise post-translational modifications (phosphorylation/ glycosylation)
• determine absolute and relative protein quantities
• Identify proteins from complex samples
• Examine isoform expression

Question 24

What is SELDI-TOF MS

Answer 24

• Surface enhanced laser desorption/ ionisation time of flight mass spectrometry
• Biochemical characteristics of surfaces are used to bind specific protein/peptides (PPs) - i.e. hydrophobicity, electrostatic interaction, affinity chromatography

Question 25

How does SELDI-TOF work

Answer 25

1. The sample is applied to a modified chip surface, which binds specific proteins to the surface. 2. Contaminants and unbound proteins are then washed away. 3. an energy absorbing matrix is applied to the surface and allowed to crystallize with the sample 4. An irradiating laser ionizes peptides from crystals of the sample/matrix mixture to form ions. 5. The ions are then briefly accelerated through an electric potential and separated by their velocity differences. 6. mass-to-charge ratio of each ion determined (velocity of the ions is inversely proportional to the square root of the mass-to-charge ratio of the ion; ions with low mass-to-charge ratios are detected earlier than ions with high mass-to-charge ratios) 7. Combined protein spectra for each sample are compared to known controls.

Question 26

What is MALDI-TOF MS

Answer 26

- Matrix assisted laser desorption/ionisation time of flight mass spectrometry - Can be used for mutation detection

Question 27

Give two examples of protein microarrays

Answer 27

- Protein capture agents. E.g. antibodies. Used to assay abundance of proteins - Arrays containing proteins/peptides. Used to study protein interactions. - Can be used to detect disease, response to therapy (protein profiling of biological fluids), detect post-translational protein modifications.

Question 28

What is proteomics

Answer 28

Comprehensive, integrative study of proteins and their biological functions. Map of the proteome.

Question 29

WHat are some major research areas of proteomics

Answer 29

- Biomarkers (protein markers of disease) - Interactions (protein- protein, protein-drug) - Signalling pathways - Epigenetics (histone modification in development and disease) - Proteogenomics= discovery of novel protein-coding regions, new translation initiation sites etc. - issue with proteomics is that it relies on the assumption that current gene models are correct and that the correct protein sequences can be found using a reference protein sequence database

Question 30

Advantages of MS

Answer 30

- Comprehensive profiles of proteins without the need of protein separation - requires small amount of sample - High specificity and sensitivity - High throughput, cost effective (suitable for screening)

Question 31

Limitations of MS

Answer 31

- MS results could be affected by conditions of sample/ patient (e.g. freeze thaws, sample processing and storage, populations, nutrition, drugs) - Low level proteins may be masked by high level proteins - Preferential detection of proteins with a lower MW - Cost of commercial microarrays

Question 32

Use of MS in diagnosis of genetic conditions

Answer 32

- Protein chips used to classify cancers (benign hyperplasia vs cancer), early detection - Tandem MS for Newborn screening of metabolic disorders (PKU, MCADD)

Question 33

What is Tandem MS (or MS/MS)

Answer 33

- where two or more mass analyzers are coupled together using an additional reaction step - The molecules of a given sample are ionized and the first spectrometer (MS1) separates these ions by their mass-to-charge ratio. - Ions of a particular m/z-ratio coming from MS1 are selected and then made to split into smaller fragment ions - These fragments are then introduced into the second mass spectrometer (MS2), which in turn separates the fragments by their m/z-ratio and detects them. - The fragmentation step makes it possible to identify and separate ions that have very similar m/z-ratios in regular mass spectrometers.