week 6 Flashcards
What is a proteome?
Entire complement of proteins (including post-translational modifications) present in a cell, tissue, organ or organism
What is proteome derived from?
Protein and genome
Define proteomics
large-scale study of a proteome using an array of proteomic methods
what does metabolome provide?
biological status
What are the data collected for proteomics?
- protein location
- abundance turnover
- Translational modifications
what is an example of post-translational modification?
- hyperphosphorylation of tau in Alzheimer’s disease
What do researchers use these 3 areas to study?
- protein activity
2. protein-protein interactions
What can alter protein function?
- phosphorylation by inducing conformational changes
2. affecting protein-protein interactions
What is phosphorylation state regulated by?
tightly concerted action of kinases and phosphatase
What can proteomic approach be used to study?
- post-translational modification and their impact in human health and disease
What are two major approaches of discovery phase?
- knowledge-based approach
2. unbiased approach
What is the biomarker workflow divided into?
3 main parts:
- Discovery
- Verification
- Validation
What is knowledge-based approach?
selection of biomarker candidate is based on existing molecular mechanism underlying disease initiation or progression
What is unbiased approach?
untargeted identification of differentially expressed proteins between 2 analysed groups
what does a small sample size at discovery phase lead to?
- overestimation of accuracy of biomarker performance and brings the reliability of findings into question
What does the classical proteomics work flow include?
protein separation using gel based or gel free techniques followed by identification by mass spectrometry
what can we look for in unbiased approach?
protein complement amongst 2 biological samples
compare and contrast and see what differs
what are examples of a proteomic discovery methods?
- Gel electrophoresis (e.g. 2D-GE)
- Matrix-assisted laser desorption/ionisation (MALDI)
- surface-enhanced laser desorption/ionisation (SELDI)
- Isobaric protein tagging
why is 2D-GE a widely-used method?
- high-resolution analysis of complex protein mixtures from a biological sample
What does 2D-GE separate proteins by?
- Isoelectric point (pI)
2. Size (MW)
Why may 2D-GE be an important method?
identifying between proteins that may be same molecular weight but have different isoelectric point
What is the advantage of 2D-GE?
ability to resolve thousands of proteins in a single gel
What is used to separate the proteins by isoelectric point?
- pH gradient
2. then the sample run via molecular weight (2D separation process)
What were researchers able to do on computer base?
pinpoint and zoom in on certain protein to look at them for further analysis
How are proteins distinguished in a complex mixture?
- proteins are separated in 2D isoelectric point and molecular weight
What is the disadvantage of 2D-GE?
- moderate reproducibility
2. limited detection capacity
What are the proteins that do not appear on the gel?
- Hydrophobic proteins
- Low abundance proteins
- Proteins that are above and below the pore size of gel
What is isoelectric focusing used to separate?
proteins in the first dimension
separates proteins based on their isoelectric point
What can isoelectric point be run in?
- tube gels
2. Fixed gradient gel strips (IPG strips)
What does IPG strips have?
- PH range - a researcher is able to select a specific PH range depending on their environment
What is PAGE (SDS-PAGE) used to separate?
proteins in the second dimension
this separates proteins on the basis of their size
What are examples of protein visualisation?
- Coomassie blue (quick stain) - most common
- Silver stain (sensitive stain)
- fluorescently labelled proteins (e.g. DIGE)
What is a disadvantage of Fluorescently labelled protein?
It uses fluorescent scanner to distinguish proteins which is very expensive equipment
In pharmaceutical company, what stain is more favoured in labs?
- Coomassie blue
2. Silver stain
What is gel scanning and computer software analysis?
- add gel to scanner
2. computer software captures whats on the gel and analyse it based on how dense a protein spot is compared to another
What are examples of protein analysis?
- comparison to master gels
- Densitometry
- Post translational modifications
Densitometry
increased and decreased proteins
Post-translational modification
- computer software - see PTM
- changes the properties of protein
- alter position of spot
- able to see if protein is phosphorylated or glycosylated
How are Gel spots excised and identified?
Mass spectrometry
What serves as a classical approach in analysis of differentially expressed proteins?
2D-GE followed by mass spectrometry
What is mass spectrometry?
analytical technique
samples are ionised into charged molecules
their mass-to-charge ratio is measured
What does a mass spectrometer have?
- Ion source
- Mass analyser
- Ion detector
What does the component of mass spectrometer vary on?
- Purpose of mass spectrometer
- Type of data required
- Physical properties of the sample
what is the principle of mass spectrometer?
- a sample is added to a mass spec
- ionized
- Accelarated
- undergoes electromagnetic detection
- Detected
- amplified into a signal
What does mass spec allow us to separate?
molecules based on mass-to-charge ratio
which allow us to identify them
What is Matrix-assisted laser desorption/ionisation (MALDI)?
mass spectrometric technique used to identify and analyse biomolecules
What are examples of MALDI?
- proteins
- peptides
- oligonucleotides lipids
- metabolites
What is the process of MALDI?
- The sample is mixed with a suitable matrix material and is applied to a metal plate
- A pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material
- Analyte molecules are ionised by being protonated or deprotonated in the hot plume of ablated gases
- Accelarated into mass-spec to analyse them
What are the 2 main functions of matrix for MALDI?
- Dilute and isolate protein peptide or general biomolecule
- Acts as a mediator to absorb energy via the laser ionisation
- Higher absorption capacity (protect sample from the laser ionisation)
What is the desorption process
- The matrix completely detaches from the surface
2. Undergoes desolvation- removal of matrix and ionisation
Why is the matrix a key?
- choose which analyte molecule you want to dilute
2. mediate energy absorption process
What are the 3 most commonly used for proteomics ?
- 2, 6-dihyroxyacetophenone
- alpha-cyano-4-hydroxycinnamic acid
- 2,5-dihydroxybenzoic acid
What is key to the method of MALDI?
- The matrix
2. Selection of matrix substances
What is surface-enhanced laser desorption/ionization (SELDI)?
- A soft ionization method in mass spec
- analysis of protein mixtures
- Variation of MALDI
- Proteins of interest in a sample become bound to a surface before MS analysis
What is SELDI used with?
- Time-of-flight (TOF) mass spectrometers
2. used to detect proteins in tissue samples, blood, urine, or other clinical samples
What is a key principle of SELDI?
- Sample is exposed to array chips with different active chromatographic surfaces that enrich certain groups of proteins
What is an example of a CHIP?
- Q10
2. useful for detecting anions
What is key difference between MALDI and SELDI?
- SELDI is able to use array chips to target certain groups of proteins not only via MW as in MALDI but target proteins that bind to metals
What does chromatographic surface arrays include?
- Immobilized metal affinity capture (IMAC)
- this targets proteins that bind to metal ions
- binding of metal ions is seen in a number of neurodegenerative disease states
C10
Cation exchange chip