Post-transationally modified

Question 1

Describe enrichment and analytical approaches than can be used when investigating post-translationally modified (PTM) proteins.

Answer 1

1. General on PTMs
2. Phosphorylation
3. phosphorylation analysis via proteomis
4. phosphorylation drug target
5. ubiquitination
6. ubiquitination analysis techniques
7. ubiquitin drug target
8. crosslinking mass spectrometry (xl-ms)

Question 2

1. general on PTMs

Answer 2

1. why are they important?
   - crucial with respect with identifying potential biomarkers and therapeutic targets
2. How many are there and give examples
   - over 300 types of modifications
   - examples phosphorylation, acetylation, methylation, glycosilation and ubiquitination
3. Why are proteins modified?
   - regualtion of activity: modification may turn activity on/off or may generate a different function i.e might cause protein to fold slightly differently and allow a different protein to interact with it so it has a different function within the cell
   - protein-protein interaction: modification site may be a binding interface
   - subcellular localization: modification site may be a targeting signal/modification may be a membrane anchor
   (when modified, shuttled to different areas within the cell where they carry out their function e.g ubiquitination -> shuttling proteins to the cell surface)
   - ageing: modification may identify the protein for degradation/modification may target a protein a protein to be scavenged6

Question 3

2. phosphorylation

Answer 3

~ GENERAL INFO
- most common post-transational modification to proteins in eukaryotes
- 30-50% of proteins are phosphorylated at any given time
- phosphate derived from the hydrolysis of ATP is added to Serine (86.4%), Threonine (11.8%) or Tyrosine (1.8%) residues
- specific phosphorylation sites and asociated kinases are useful for their discriminatory power in:
1. molecular classification of cancers (diagnosis)
2. in predicting clinical outcome (prognosis)
3. response to drugs (therapy)

~ FUNCTIONS OF PHOSPHORYLATION
- plays a critical regulatory role in cells
1. important for glycolysis (1st step -> hydrolysis of ATP, release of phosphates used to phosphorylate glucose)
2. used for protein-protein interaction
3. used in protein degradation
4. regulates enzyme inhibition
5. maintains homeostasis by regulating energy-requiring chemical reactions

Question 4

3. Phosphorylation Analysis via Proteomics

Answer 4

1. Gel Staining
   - proteins are run on a 1/2D gel and the gel is fixed to freeze the positions of the protein
   - the first gel is stained with a total protein stain e.g SYPRO-Ruby Red, to stain every protein on the gel regardless of whether it is phosphorylated or not
   - the second gel is then stained with Pro-Q-Diamond, a phospho-specific stain that only stains proteins on the gel that have been phosphorylated (phosphoproteins)
   - the gels can be scanned at their respective excitation wavelengths for detection of total protein and phosphoprotein images
   - the signal abundance coming from phosphoproteins is directly associated with the level of phosphorylation on the protein
   - gives a comparitive profile of the total protein content and the phosphoprotein content
2. Phosphospecifc antibodies
   - phospho-specific antibodies are produced by immunizing the host animals with a short synthetic peptide containing the phosphorylated amino acid.
   - the animals can produce antibodies that not only recognise the modified amino acid.
   - can run western blots and protein arrays
3. Mass spectometry
   - for evaluating thousand of phosphorylated proteins in sample
   - before analysing by mass spectometry need to enrich for a peptides that are phosphorylated
   - most common technique for this is IMAC (immobilised metal affinity chromoatography) which is based on the high affinity of phosphate groups for metal ions such as Fe3+, Zn2+, Ga3+ and TiO2 (titanium dioxide)
   - carried out by conjugating titanium dioxide onto a bead (mictosphere matrix) and if phosphorylated will bind to the titanium dioxide
   - Quantitation of phosphopeptides
   1. SILAC (stable isoptope labelling with amino acids in cell culture) is a mass spectometry-based technique developed to detect difference in protein abundance between two (or more) samples. (cell culture)
   2. iTRAQ (isobaric tag for relative and absolute quantitation) is non-gel based technique used to identify and quantify proteins from different sources in one single experiment. (tissue/bio-fluids)
   3. label-free: protein quantification is generally based on two categories of meausrements (a) ion intensity changes such as peptide peak areas and (b) spectral counting of identified proteins after MS/MS analysis
      
      • to confirm that peptides are phosphorylated on mass spec check for 98 dalton mass loss due to loss of phosphate group, the peptide that has lost it’s phosphorylation site can then be sent for fragmentation into b and y ions to identify the peptide

Question 5

4. Phosphorylation drug target - Imatinib (Gleevec)

Answer 5

• in chronic myeloid leukemia (CML) the philadelphia chromosome leads to a fusion protein of abl (Abelson proto-oncogene) with bcr (breakpoint cluster region) termed bcr-abl.
• acts as a constitutively active tyrosine kinase driving tumorigenesis within this particular cancer
• Imatinib used to inhibit it’s activity by binding to the binding pocket in this fusion protein associated with the hydrolysis of ATP shutting down the enzyme’s ability to produce a source of phosphate groups (energy)

Question 6

5. Ubiquitination

Answer 6

• ubitination: ubiquitin a 76-amino acid protein is covalently added to lysine residues in target proteins
• catalyzed by over 500 E3 ligases in humans
• regulates many processes including protein degradation, cell signalling, trafficking and the DNA damage response
• ubiquitin modifications can be classified into 3 general types; monoubiquitylation, multi-monoubiquitylation and polyubiquitylation
• stepwise manner in which ubiquitination takes place:
  
  1. E1ubiquitin-activating enzyme binds to ubiquitin, there is an interaction between its sulfhydryl group and the c-terminus on the ubiquitin
  2. that is then transferred to E2 ubiquitin-conjugatin enzyme through the same interaction
  3. this is then transferred via the E3 ubiquitin-protein ligase to the target protein

Question 7

6. ubiquitin analysis techniques

Answer 7

• before quantitating the sample, need to enrich for peptides that are ubiquitinated
• this can be done via ubiquitin remnant profiling
• during digestion of protein using trypsin, trypsin cleaves ubiquitin after an arginine residue near its c terminus leaving a double glycine (covalently bonded) bound to a ubiquinated peptide
• mass spectrometry can then be used to quantify the obtained peptides using the same approaches used for phosphorylation

~ confirmation that peptides are ubiquitinated
- ubiquinated peptides can be confirmed on the mass spectra by checking for a 114 dalton attached to lysine.

Question 8

7. ubiquitination drug target: Bortezomib (Velcade)

Answer 8

• bortezomib (velcade) binds to the proteasome inhibiting its activity to degrade molecules
• as a results i kappa b (the inhibitory subunit of nfkb) is not degraded even tho it is tagged by the proteasome for degradation
• this leads to a dampening of nfkb and it is unable to produce factors associated with anti-apoptosis

Question 9

8. cross-linking mass spectometry (xl-ms)

Answer 9

• crosslinking mass spectometry relies on the introduction of a covalent bond between 2 spatially proximal amino acid residues by a chemical reagent e.g DSSO (membrane permeable and cleavable crosslinker)
• these artificially fixed interactions are capable of surviving denaturing conditions and can be analyzed using methods that normally would be destructive to non-covalent interactions
• can also be used to generated a connectivity map which can be used for identifying new therapeutic targets/using the formation of a complex as a biomarker for a particular disease