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Flashcards in Genomics and proteomics Deck (25)
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Define proteome

All the proteins present in a cell at a particular time


Define metabolome

The total number of metaboolites present in a cell (or tissue or organism)at a particular time


What are the transcriptome and the proteome linked by?

Codons in mRNA
- Generally considered standard
- Some exceptions e.g. mitochondrail genomes deviate slightly


What are the main features of the study of proteins?

- Study of purified proteins: function/biochemistry in isolation, separation based on charge, size, antigenicity, tags
- Study of their expression in vivo or in relation to disease


What is SDS-PAGE?

SDS - Polyacrylamide Gel Electrophoresis


What is SDS-PAGE used for?

One dimensional protein separation


How does SDS-PAGE work?

- Based on charge:mass ratio
- Applied electric field
- Stacking gel to ensure all proteins start at same point


What is SDS?

- A detergent used to keep all proteins soluble
- Denatures proteins


How are 2D-PAGE and SDS-PAGE used together?

- 2D separates proteins ofsimilar masses by number of charges within protein along pH gradient with applied electrical charge
- Then use SDS-PAGE to separate based on mass
- The strip used in 2D is used like a stacking gel , placed on top and separated by charge


What is the benefit of using SDS and 2D PAGE together?

Allows more separation of proteins that would usually overlap
- Allows identification of partial peptide sequences from protein samples


How can proteomes be compared?

- Compare matched gels for different samples
- Label proteins and run on same separation
- Differences in form/mass spec


What is a 2D separation modem used for?

- To go direct from protein mixture to identification
- Could be used in rapid diagnostics


What does the 2D separation modem rely on?

Knowing the sequences of proteins


How does the 2D separation modem work?

- IEF separation by HPLC in capillary tube
- Then protein determination by mass spec, matrix assisted time of flight (MALDI-TOF) or by ES electro-spray technology
- Utilises high performance liquid chromatography
- Range of solutions, column with solid matrix
- Start with 100% solution A, mix until 100% solution B
- As pH changes, proteins come off based on charge


How can direct comparisono of proteomes by 2D-PAGE be done?

- Overlay images
- Label proteins with colours, run on same gel


What are the limitations of SDS-PAGE?

- Based on charge:mass ratio
- Many proteins overlap in charge and size so poor resolution


What are the limitations of 2D-PAGE?

- Not all proteins will run in isoelectric focussing
- Amount of material limited
- Need to harvest organelles to measure proteomes
- Solutions to "stop cellular processes"
- Proteases may degrade protein of interest before it reaches the lab
- Large amounts of data
- No information on biochem


Give examples of protein analysis techniques

- Refractometry
- Protein array


What are the limitations of refractometry

- Affected by what else is in sample
- Does not say what is there but what kind of shift has occured (more or less "stuff" than normal)


What are the limitations of ELISA?

- Shows presence not amount
- Requires knowledge of what protein and correct antibody


What is the function of Western Blots?

Used to detect specific proteins by antibody


Describe the process of a Western Blot

- Transfer proteins from gel to filter
- Block filter to avoid non-specific binding
- Probe with antibody to protein of interest added
- Proteomics used to remove protein from cell, antibody to detect protein,then separate on normal gel


Give examples of uses of proteomics in clinical scenarios

- Western blots
- Snap tests for specific antigens and antibodies
- To gain more information about disease processes e.g. protein profiling of urine from dogs with renal disease
- Identification of cancer
- IDentificatino of causes/diagnosis
- Virulence/resistance mechanisms (requires hypotheses on what can be done, targets for control)


What are some uses of metabolomics in clinical scenarios?

- To establish why unpredictable effects may have occured
- To reformulate drug to avoid negative effects


How can DNA sequence beused to gain information about proteins?

- Genome = sequence, promotes and genes etc, converted to structural/messenger RNA
- Can be used to model the RNA
- RNA can be used to predict primary peptide sequences
- Structural modelling can be done based on similar primary sequences
- Structural prediciton based on primary peptide sequence
- However presence of RNA does not mean a protein will be expressed from it, ot that the protein will be functional