Proteomics Flashcards
What does omics refer to?
Informally refers to genomics, proteomics or metabolomics
What is -ome used for?
To address the objects of study of such field, such as the genome, proteome or metabolome respectively.
What are the aims of omics?
The collective characterization and quantification of pools of biological molcules that translate into the structure, function and dynamics of an organism or organisms
What is the proteome?
All the proteins expressed at any one time
In which aspect relevant to omic technologies are proteins and DNA different?
DNA is specific to a certain species or individuals
Proteins can be specific at many levels - population, person, tissue, cell, organelle
What information can you get from the genome?
Functional information only
What information can you get from the proteome?
How much there is
Activated or not
State of activation
How much variation is there in the genome?
Little compared to the proteome
Arises only from the combinations of 4 nucleotides
How much variation is there in the proteome?
A lot
Arises from combination of up to 20 amino acids
Increased further by the post-transcriptional modification
How many genes are there in the genome?
Around 25 000
How many proteins are there in the proteome?
More than 1 million
Modification and splice variants increase the amount
The genome encompasses a —- code, whereas the proteomic code is highly —-
Static
Dynamic
What are prepro regions?
Proteins are often made as precursors or pro-proteins
Not active until they have undergone some form of post translational modification
They also contain a signalling peptide (pre-pro peptide) to signal the cell whether or not they should be secreted or incorporates into the plasma membrane
This is a cleavable peptide that must be removed before the protein is activated
What are examples of post translational modification?
Posphorylation
Acylation
Methylation
Ubiquitination
Glycolysation
Give an example where proteomics was used clinically
Ovarian cancer patients
Bioinformatics was combined with proteomics
This showed clusters identifying key genes involved, including Ubiquitin C and FGF4
Also showed gene outliers, which could be specific to the type of cancer
Which gene is specific and used clinically to diagnose ovarian cancer?
Mucin16
What type of field is bioinformatics?
Very multidisciplinary
What professions are involved in bioinformatics?
Biologists
Computer scientists
Mathematicians
Chemists
Statisticians
Physicists
What does bioinformatics entail?
The study of biological information with a computer
What information can bioinformatics provide us with regarding the proteome?
Structural
Find networks
Quantify a protein
Generate models: how changes in environment can affect protein modulation
What 3 tools can be used for bioinformatics?
BLAST
GO
Panther
Describe the BLAST bioinformatic tool
Takes a sequence and searches it
Compares the sequence to sequences in the database
Can be a protein or a gene
Uses this to guess the protein it is
Uses the conserved region of a protein to accurately predict its function
Can also give you the phylogeny information regarding that protein
Describe the GO bioinformatic tool
Gene ontology
Associates the function of a protein with a word
Creates a library of these proteins regarding their functions
Clusters proteins with similar functions together
Describe the Panther bioinformatic tool
Looks at a list of proteins described by GO and returns the average most enriched processes
Cancer cells: apoptotic processes, immune cells
Give an example of bioinformatic analysis done on a patient cohort
Asthma patients
Grouped patients into eosinophilic, neutrophilic and non-allergic
Showed that neutrophilic asthma formed 2-3 subgroups, where the effect of treatment and disease type were different
Identified groups which shared genes
What, in terms of hypotheses testing, are omic technologies useful for?
Omics are good for generating hypotheses
What are prescriptive vs non-prescriptive approaches in proteomics?
Prescriptive approaches have defined targets
Non-prescriptive approaches have unknown targets
What type of approach is used in protein arrays?
Prescriptive
Used to identify a predefined set of proteins
What technique are protein arrays based on?
Antibody recognition
Are protein arrays classified as omic technologies?
Not yet
We don’t have all the antibodies for all the proteins
Almost omic for yeasts
What type of approach is used in mass sectromertry ?
Non-prescriptive
Identifies all the detectable proteins in a sample
What are mass spectrometry results based on?
Peptide m/z values
Are mass spectrometers classified as omic technologies?
Yes
What are the two proteomic technologies used?
Protein arrays
Mass spectrometry
What is proteomic characterisation?
The characterisation and quantification of all polypeptide components present in a biological compartment
What are the three levels by which proteins are characterised?
Primary
Secondary and tertiary
Quarternary
What is the primary structure of proteins?
Amino acid sequence
What is the quarternary structure of proteins?
Protein-protein interactions
Interactome
What are the questioned addressed by proteomics?
Which proteins are translated from the genome/transcriptome?
Where are the proteins found?
Which proteins do the proteins bind to?
Are the proteins post-transcriptionally modified?
Does the protein concentration/localization/ interaction/ modification differ between samples?
What are the pros of mRNA profiling?
Fairly omic
Quick
Relatively cheap
What are the pros of proteomics?
Good for looking at ECM and fluids such as CSF, plasma, serum and urine
More informative and more accurate to focus on the active entities rather than their biosynthetic template to asses abundance, modification, turnover and localisation
What are the cons of mRNA profiling?
Bad at looking at certain ECM and fluids
What are the cons of proteomics?
Difficult to perform
Expensive
Not truly comprehensive
Do proteomes and mRNA profiles always correlate?
Some correlate well, some don’t
What are the two reasons for why we need sample fractionation?
Dynamic range
Complexity
Why does the dynamic range of a sample mean we need sample fractionation?
Some molecules very abundant in the serum
This is too large for a single detector
Why does the complexity of a sample mean we need sample fractionation?
A large number of protein sequences
1 000 000 protein sequences
This is too large for a single platform
What does proteomic mass spectrometry analyse?
Peptides produced by proteolytic digestion of samples
What important step is crucial to perform before fractionation?
Targeted depletion of abundant proteins
So they don’t interfere with results
Immunoaffinity and ligand binding
What are the two types of non-targeted fractionation?
Protein fractionation
Peptide fractionation
Two types of protein fractionation
1D or 2D electrophoresis
1D or 2D-liquid chromatography
Two types of peptide fractionation
Solution phase isoelectric focusing
2D HPLC
What is the key in fractionation?
Minimise experimental error
What are two ways to minimise the experimental error in fractionation?
High reproducibility
Avoid non-specific losses
What are the two types of variation that exist in experiments?
Technical variation
Biological variation
What is technical variation?
Experimental error
Occur whenever we prepare or analyze samples
What are ways to reduce technical variation?
Standard operating procedures
Quantifying using technical replicates
What are sources of biological variation?
Sample fidelity: are there components of your sample you are not aware of?
Anatomical heterogeneity
Longitudonal variation between periods of time
Individual variation between patients
What are ways to reduce biological variation?
Increase cohort size
Stratification - cluster variation
Quantify using biological variations
Biological samples are often impure
TRUE or FALSE
TRUE
Due to contamination and cell and tissue heterogeneity
What are the reasons we need validation?
Variation (biological and technical)
Proteomic results are based on probability
Important identifications require validations using orthogonal techniques
What does validation due?
Make sure the identity of the sample is correct and it localises to the tissue/cell/organelle
What are orthogonal techniques?
Completely different technique than the one used in the experiment
What are examples of orthogonal techniques?
Immunohistochemistry
ELISA
Labelling
Why are mass spectometers used over ELISAS and protein blocks?
ELISAs and protein blocks look at a protein at a time
Mass spectometers looks at many at the same time
These techniques can be applied to lipid and metabolomics as well
What are the steps of mass spectometers?
Preparation
Mass-spectometer
Protein identification
Analysis/bioinformatics
Validation
Describe the preparation steps taken before the sample is inserted into the mass spectometer
Proteins collected from urine tissue, lung lavage …
Reduce proteins through alkylation, which breaks the disulfide bonds between cysteine and then stop the bonds from forming
Digest using trypsin, which digests the C terminal of arginine and lysine
Extract
Label and pool
Fractionate
Give an example of a validation technique
Identify a biomarker for ovarian cancer
Look at serum of patients and compare it to healthy controls
Then validate these using orthognal techniques
Why do we need to digest the proteins?
In order to be able to fragment and ionise them
Can we identify a protein using its ionised form?
No
Can we identify a protein using its fragmented form?
No
Can we identify a protein using the ionised form of its peptide?
No
Can we identify a protein using the fragmented form of its peptide?
Yes
What is the challenge with ionising proteins?
Very high energy needed for direct desorption and ionisation
What is the challenge with fragmenting proteins?
Difficult to produced ordered series of daughter ions producing any amino acid sequence information
What is required in order to ionise peptides?
High energy
What is the usefulness of the fragmented peptides?
Peptide fragmentation data can be interpreted to deduce amino acid sequence information
What are we measuring in a mass spectrometer?
The mass to charge ratio of peptides fragmented from the proteins we want to analyse
Describe how liquid chromatography works in collaboration with mass spectometry
Start off with complicated sample
Separation technique is used, usually liquid chromatography
Break down proteins into peptides
Peptides are ionised through soft ionisation
Peptides get pushed into the mass spectometer
Mass spectometer measures the mass/charge ratio
Take peptide and push it through another mass spectrum following further fragmentation
Another spectrum is obtained, which helps identify what the peptide is and therefore quantify it
What does the mass filter do?
Guides the ions straight into the mass spec
What is the purpose of the separation techniques in mass specs?
Prevents the molecules from clustering together
Separates them by size or chemical properties
Why do we break proteins down into peptides in mass spec?
They are more stable
Can identify the peptides, which is used to quantify and identify whole proteins
What charge do proteins have following soft ionisation?
Positive charge
What is the benefit of the positive charge of proteins?
Helps filter out other substances
How do mass specs measure the mass to charge ratio of peptides?
Peptides are pushed into a vacuum
The time taken for the peptide to travel through is measured
We know the force needed to push the peptide through the vacuum, and the time
So we use this to calculate the mass
Singularly charged peptides moves in vacuums with half the force as peptides with double charges
TRUE or FALSE
TRUE
What are two techniques of soft ionisation?
Matrix-assisted laser desorption ionisation
Electrospray ionisation with water
Describe the process behind MALDI
Energy from a laser goes into the matrix
The matrix absorbs the energy and ionises some of the molecules
Time of flight is then measured
What does the time of flight in MALDI represent?
Amount of time taken for the ionised molecule to hit detector on the back
What are the two techniques used in ESI?
Quadrupole
Orbitrap
Describe the components of the quadrupole
Contains 4 poles
2 positive and 2 negative
How does a quadrupole work in identifying peptides?
The voltages are changed so that only one specific mass to charge ratio get through
The other ones crash and burn on the side
The positive rods remove molecules below a certain value
The negative rods remove molecules above a certain value
Describe the structure of the orbitrap
Outer barrel-like electrode
Coaxial inner spindle-like electrode that traps ions in an orbital motion around the spindle
How does the orbitrap work?
Ions are trapped because their electrostatic attraction to the inner electrode is balance by their inertia
What are the two types of mass spectra obtained following fragmentation?
Single stage MS
Tandem MS
What information is obtained from single stage MS?
Protein mass data, including purity and integrity
Peptide mass data using the fingerprint
What is the use of single stage MS?
Proteins are digested
Mass spectra are formed, and every possible protein in the human proteome is digested and fingerprints are made
We can then match protein samples to these fingerprints
What is the use of tandem MS?
We can measure the mass of the peptides
Can put a particular peptide and run it through a collision cell to fragment it in a predictable way
Then you measure the mass to charge ratios of all the fragment ions
We use these ions to measure the sequence
What charge do tryptic peptides possess normally?
Double
What types of ions are produced from the ionisation of tryptic peptides?
Single charged
b- and y- ions
Where are the b-ions retained?
In the N-terminal fragment
Where are the y-ions retained?
In the C-terminal fragment
What are 3 ways of quantifying peptides?
SILAC
Label free
Chemical mass tagging
What is SILAC?
Stable isotope labelling of amino acids in culture
Gold standard
How is SILAC carried out?
Grow cells in heavy medium
What is the disadvantage with SILAC?
Does’t work in animal or human experiments
Too fast or not possible
Examples of label free quantification mechanisms
iBAQ
Top3
EmPAI
What is the advantage of label free quantification mechanisms?
Does not modify proteins, so no risk of affecting ionisation or properites
What is the disadvantage of label free quantification mechanisms?
Have to do one sample at a time
What are examples of chemical mass tagging used to quantify proteins?
TMT
iTRAQ
Describe the mechanism behind chemical mass tagging
Process of chemically modifying peptides according to their mass
What is the advantage of chemical mass tagging?
You can have different weighted tags which makes it high throughput
What is the most ideal quantification technique?
There is no one ideal single method
Most use a combination of different methods
