Flashcards in Genomics and proteomics Deck (25)
All the proteins present in a cell at a particular time
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
- 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