Proteomics Flashcards
Which is capable of identifying a higher proportion of the proteome accurately? Peptide-centric or protein-centric approaches? Why?
• Peptide-centric approaches are capable of identifying a higher proportion of the proteome than protein-centric
o Many more protein identifications and parameters (e.g. peptides) per protein
o Every peptide generated becomes an individual parameter that represents a protein- important for quantitation
There is a lot of statistical power of using peptides to represent their parent proteins
What is the difference between relative and absolute quantitation?
o Relative vs absolute quantitation
Relative quantitation- how much there is in sample 1 vs in sample 2
Absolute quantitation- how many micrograms of each protein are present within a sample
What is the aim of comparative quantitative shotgun proteomics?
• Measures relative abundance across two different samples (one control and one test)
What are 3 methods to perform comparative quantitative shotgun proteomics?
- Method A-metabolic stable isotope labelling
- Method B- isotope tagging by chemical reaction
- Method C- stable-isotope incorporation via enzyme reaction
Describe the process of metabolic stable isotope labelling for quantitative shotgun proteomics and when it can be used
• Method A-metabolic stable isotope labelling
o Cells growing in culture or living tissues
o During the growth of the cell, incorporating a stable isotope
Stable isotopes used to quantify changes in protein abundance e.g. 2H or 13C
o Isotope is added to growth medium and cells take it up and incorporate it when they undergo protein translation
In this approach, an isotope labelled amino acid e.g. (leu or lys- light and heavy) must be incorporated during protein synthesis
• Cell will make proteins with light (or normal) isotope and proteins with heavy isotope
o Isotopes have identical chemical properties
o The proteins are then digested-should get a light version of the peptide and a heavy version of the peptide, which creates a mass difference
o Do chromatography and mass spectrometry-> the heavy and light versions of the peptides will appear as pairs: one from the control and one from the test
o Peptides tagged with differing isotopes will appear adjacent on a mass spectrum
Separated by known mass difference of the light and heavy amino acids as it is incorporated
o Area under each peak equate to relative abundance of the two peptides
Can use spectral intensity of the peaks in the mass spectrometer as a means of measuring the abundance difference (relative abundance)
What is an example of a tag/method used for metabolic stable isotope labelling
- Stable isotope labelling by amino acids in cell culture (SILAC)
What is the process of stable isotope labelling by amino acids in cell culture (SILAC) and what are its requirements?
• Process-
o Labelled peptides-> optional protein purification-> combine control and test samples and digest with trypsin-> quantitation by MS
• Grow cells in presence of a labelled amino acid for which the cell cannot biosynthesise
o Incorporation of SILAC label over time
• No labelling step as label incorporated during synthesis
o E.g. deuterated leucine (Leu)
o 100% Leu labelled, approx 50% peptides
Leucine is the most common amino acid, and lysines are extremely common in tryptic peptides
• Allows differentiation of leucine and isoleucine
• Needs cells grown in culture
Describe an example of proteomics of B cell differentiation using SILAC quantitative proteomics
- Define B cells
- Differentiation of B cells
- Study details
• Example- proteomics of B cell differentiation using SILAC quantitative proteomics
o B cells are immune cells (lymphocytes)
o Differentiate into plasma cells that secrete antibodies against non-self antigens (eg. bacteria)
o Stimulate differentiation by treating with bacterial lipopolysaccharide (LPS)
o 234 protein expression changes, including a cluster involved in antibody production
o Looked at how protein expression changed over time (samples were collected over several days)
o Performed MS-based quantitation based on unlabelled vs 13C6-Leu
Dynamic quantification shown at the single peptide level during B cell differentiation
Control- day0/day0: this should be identical
o Cluster analysis and cellular location of 234 differentially abundant proteins
Enables for formation of hypotheses
What is cluster analysis?
Cluster analysis-task of grouping a set of objects in such a way that objects in the same group are more similar to each other than to those in other clusters
What are the advantages of stable isotope labelling by amino acids in cell culture (SILAC)?
- Very reproducible
- Generally high labelling efficiency
- Multiple peptides per protein- therefore good statistical confidence
- Compatible with PTMs
- Compatible with most cell-based systems
What are the disadvantages of stable isotope labelling by amino acids in cell culture (SILAC)?
- Need cells growing in culture
- Some cells are fussy- poorly incorporate label
- Human based Tissue studies
- Blood/plasma don’t contain metabolism cells
- Also results in 2 ions or more per peptide in MS scans
Describe the SILAC mouse, how it is produced and why it is so useful
• The SILAC mouse
o For tissue studies
o Take a mouse-> give it a lysine free diet supplemented with either a light or heavy version of the amino acid-> make a SILAC mouse (with either light or heavy amino acid) depending on supplied amino acid
Good incorporation over a period of time for many proteins except for haemoglobin (red blood cells)
o SILAC mouse can reveal information about protein turnover
What is a disadvantage of the SILAC mouse and how is it overcome? What is a potential problem with the solution?
Extended labelling times however do not result in 100% labelling efficiency- most likely due to more complex recycling of amino acids
• This can be overcome by feeding mice over several generations
o Newly born mice only ever know the light/heavy diet
o Results in close to 100% incorporation
o However, sometimes the mothers can eat their young so have to be careful
What is the SILAC fly useful for and how is it produced?
• The SILAC fly
o For tissue studies
o Make yeast in culture that incorporates light or heavy version of amino acids and exclusively breed one set to eat heavy or light yeast food
What is the SILAC worm useful for and how is it produced?
• The SILAC worm
o For tissue studies
o Grow SILAC worm on E.Coli.agar plate that either has heavy or light amino acid
Worms eat the E.Coli and either become labelled with heavy or light amino acid
o Compare the proteomes of the worms
What is the procedure for isotope tagging by chemical reaction in comparative shotgun proteomics and when can it be used?
o Can be used on all cell and tissue types as incorporated after protein synthesis
o Addition of a chemical tag specific for a handle in peptide sequences
Use a specific chemical handle to add a mass tag to peptides
Handles most commonly the primary amines of peptide N-termini or Lys residues or the -SH (thiol) group of Cys
Isotopes/chemical tags have identical chemical properties but are different in mass or are isobaric
o Area under each peak equates to relative abundance of the two peptides
What are isobaric tags in isotope tagging by chemical reaction in comparative shotgun proteomics and why are they useful?
• Isobaric- has the same mass
o Enables for quantitation at tandem mass spectrometry level
o Peptides tagged with differing isotopes will appear adjacent on a mass spectrum
What are tags used in isotope tagging by chemical reaction in comparative shotgun proteomics?
- Tags- Isotope coded affinity tags (ICAT)
- Tags- Isobaric affinity tags (iTRAQ-Isobaric tags for relative and absolute quantitation)
- Tags- Tandem Mass tags
Describe the process of isotope coded affinity tags for isotope tagging by chemical reaction in comparative shotgun proteomics and the quantification process
o Process:
Sample-> lysis-> label mix samples and digest-> 2-DLC separation -> MS scan-> selected for MS/MS
o Label proteins via cysteine residues
o Two tags therefore pair-wise comparisons
o Quantification procedure
Mix label sample 1 and label sample B in equal amounts-> combine and proteolyze-> avidin affinity enrichment-> MS1 scan -> get sequence-> protein identification and quantification
1-3 peptides per protein are used to accurately quantify the relative abundance of each protein
• Limited by amounts of cysteines
Non-cys containing peptides discarded > reduces sample complexity, but also loses information such as phosphorylation
Describe the structure of the isotope coded affinity tags for isotope tagging by chemical reaction in comparative shotgun proteomics and the purpose of this structure
o 3 components-
Thiol-specific group enables protein/peptide binding (functional group)
Linker of differing mass to differentiate control and test groups
• Chemically inert
Affinity tag for purification (biotin)
o Light tag (H) or heavy (D) tag used: differ by 8 Da
o Has a biotin molecule for affinity purification
What are the advantages of ICAT (isotope coded affinity tags) for isotope tagging by chemical reaction in comparative shotgun proteomics
- Reduces complexity
- Easy analysis
- Reproducible
What are the disadvantages of ICAT (isotope coded affinity tags) for isotope tagging by chemical reaction in comparative shotgun proteomics
-Not all proteins contain Cys (20-30% cannot be analysed)
Cysteines are the second least common coding amino acid (only makes up 1.1% or 1.3% of all known coding amino acids in the protein database)
Hence, this tag doesn’t bind many peptides (drawback)
-Those that do contain Cys may have only 1 or 2, hence replicates are needed for statistical analysis
-8 Da ICAT tag difference may complicate MS spectra- by doubling the number of ions in the MS scan
Describe the process of using isobaric affinity tags (iTRAO) for isotope tagging by chemical reaction in comparative shotgun proteomics and the process of quantification using these tags
• Tags- Isobaric affinity tags (iTRAQ-Isobaric tags for relative and absolute quantitation)
o Can do many samples (8 or more)
o Process:
Samples-> lysis-> digest-> label-> mix samples and 2-DLC-> MS scan shows several peptides from same protein-> MS/MS fragmentation cleaves reporter allowing quantitative comparison and identification
o Exploits primary amines
o Only does relative quantitation
o Differentiation at mass spectrometry level-
All peptides are tagged-> no affinity purification
Perform multi-dimensional liquid chromatogaphy
The 4 tags have the same mass so only one peak seen in precursor/MS scan
During MS/MS, tag fragments revealing 4 reporter ions
Reporter mass beneath those seen for y-1 ion
• Intensity of each peak is relative to total abundance of the protein
Allows four way comparison
When undertake fragmentation, remove peptide reactive group, get neutral loss at balance region and release reporter ions-
• Allow for relative quantification at MS:MS level and peptide sequence
Describe the components of isobaric affinity tags (iTRAO) for isotope tagging by chemical reaction in comparative shotgun proteomics and their uses
o 3 components to tag- Peptide reactive group • Amine specific (Lys and N terminal) Isobaric tag (total mass= 145) • Balance (mass 31 to 28) o Neutral loss in MS:MS o Balances the mass change of reporter to maintain a total mass of 145 • Reporter (mass 114 to 117) o Charged o Gives strong signature ion in MS:MS o Gives good b- and y-ion series o Maintains charge state o Maintains ionization efficiency of peptide o Signature ion masses lie in quiet low mass region
Describe an example of using isobaric affinity tags (iTRAO) for isotope tagging by chemical reaction in comparative shotgun proteomics for characterisation of head and neck squamous cell carcinoma. Describe:
- The cancer
- The method
- The result
o Example- head and neck cancer biomarkers
Head and neck squamous cell carcinoma (HNSCC) (includes tongue, larynx, pharynx etc.)
6th most common cause of cancer-related deaths
Lack of early biomarkers (50% diagnosed at advanced disease)
Compared 15 patient samples (cancerous and non-cancerous) and a pooled non-cancerous control
• Aligned samples to pooled non-cancerous control to create heat maps
o Aligned mean fold change found in cancer patients relative to normalized control
• Pooled non-cancerous control enables measurement of individual differences in proteins that may lead to false positives
Several proteins induced/repressed in cancerous tissue
Validation using immunohistochemistry, RT-PCR (transcriptomics) and Western blots
Tested marker specificity to different kinds of cancer (YWHAZ, Stratifin, S100A7, B-actin)
What are the advantages of using isobaric affinity tags (iTRAO) for isotope tagging by chemical reaction in comparative shotgun proteomics
- Many peptides identified per protein so statistical confidence is high
- Requires fewer replicates
- All labels have same mass so improved analysis time (MS scan)
- Suitable for all biological systems
- Suitable for post-translational modification analysis
What are the disadvantages of using isobaric affinity tags (iTRAO) for isotope tagging by chemical reaction in comparative shotgun proteomics
- iTRAQ tends to underestimate n-fold changes
- iTRAQ labels may induce super-charging of peptides [particularly 8-plex iTRAQ] resulting in reduced protein identifications
- -True with high numbers of reagents
How do tandem mass tags for isotope tagging by chemical reaction in comparative shotgun proteomics work?
• Tags- Tandem Mass tags
o Work like ITRAQ tags: isobaric tags where quantification is performed at mass spectrometry level
Describe how stable-isotope incorporation via enzyme reaction works in comparative shotgun proteomics
o Can be used on all cell and tissue types as incorporated after protein synthesis
o Exploits the enzyme reaction (e.g. trypsin digest) to incorporate a stable isotope
Tags- dimethylation
o Trypsin digest adds water to the C-terminus of the newly created peptide (Lys/Arg C-terminus). Perform trypsin digest in light or heavy (deuterated) water to incorporate a mass difference
Isotopes have identical chemical properties
o Peptides tagged with differing isotopes will appear adjacent on a mass spectrum
o Area under each peak equates to relative abundance of the two peptides
What is the surfaceome and why is it useful to understand?
• Surfaceomes- proteins (or parts thereof) located on the surface of a cell
o Understanding what is on the surface of organisms can be very important in an applied way
Vaccines
How two cells interact with each other
o Membrane and surface associated proteins are important in vaccine design and we can look specifically at those surface proteins
• Surfaceomes are important for vaccine development
• Looks at proteins located on surface of cell or on the membrane
• Look at which protein/peptide epitopes are surface-exposed
What is the secretome?
• Secretomes- proteins secreted from a cell into the extracellular space
What are organellar proteomics?
• Organellar proteomics- nuclear proteins, ribosomal proteins, etc.
How is a good vaccine made/ what are requirements of a good vaccine?
• A good vaccine is made through:
o Exposed to the immune system
o Can use whole attenuated (non-pathogenic) live or dead organisms (e.g. Mycobacterium bovis BCG for tuberculosis)
Most vaccines these days are designed against specific biomolecules
• Liposaccharides are highly immunogenic but aren’t specific
• Proteins are generally specific
o Elicit an immune response (antigenic/immunogenic)
o Ideally an immune response that persists
o Then it needs to capture the whole organism upon infection so that it cannot proliferate
Who pioneered pathogen genomics for reverse vaccinology?
o Pioneered by Dr. Rino Rappuoli
What are the phases of pathogen genomics for reverse vaccinology?
Exploit genome sequencing
Sequencing of many strains/serovars/subtypes etc. to create a representative pan genome with genes conserved across all representatives
• Pan genome- the entire set of genes for all strains within a clade
Perform in silico predictions to identify potential antigens (and/or proteomics to determine their expression under different conditions)
Synthesize best candidates [can be many!] as recombinant proteins
• High copy plasmid replicated in E.Coli
Determine protection in animal models
• Take antigens and test them
Human trials
What are the symptoms of meningitis and sepsis?
o Inflammation of the lining of the brain (the meninges) or the blood (sepsis)
o Flu-like symptoms but can cause death in hours in infants and young adults
What is neisseria meningitidis?
o Neisseria meningitidis- Gram negative bacterium with five major serotypes (antibody binding): A, B,C,Y and W135
Why was serotype B of meningitidis difficult to make a vaccine of?
o No current vaccine for serotype B which causes 45-80% of N.meningitidis cases (driven in part by successful vaccination campaigns against the other serotypes)
Serotype B doesn’t work with other vaccines because the outer capsule (polysaccharide) is an autoantigen
• Serotype B is not recognised as a foreign organism in humans
How was reverse vaccinology used to make a vaccine for serotype B of meningitidis? Describe the difficulties in the process and the end result
o Genome (sequenced through whole-genome sequencing)
Sequenced a strain of the meningococcus genome
o 350 proteins successfully over-expressed into E.Coli
Not all successfully over-expressed in E.Coli because some might be toxic to E.Coli, some will form inclusion bodies and not be purifiable
o Raised antibodies in mice and screen for those that bind to the bacteria (7 proteins)
o The strongest binders were chosen for clinical trials
o Most did poorly due to:
Antigenic variability across strains
Some antibodies were missing in hypervirulent strains
Phase variability
Low copy number
o Current strategy is to use a multicomponent vaccine based on 4 proteins
Open reading frames put together and expressed as a single construct
Ensures that at least some reading frames are expressed across strains
o Completed phase 4 trials- and is effective across many B strains
o Became Bexsero- a multicomponent vaccine for prevention of meningococcal disease (available in Australia in 2015)
What elements are contained in the pan genome of serotype B N.meningitidis? Describe their significance
• 2 pathogenicity islands
• Highest number of phase variable genes
o Phase variable genes- genes that can be switched on and off- not good vaccine candidates
• Polysaccharide synthesis pathway
• 570 ORFs encode potential surface-exposed or secreted proteins (that is vaccine candidates)
What are pathogenicity islands?
o Pathogenicity islands- little bits of DNA acquired from other microorganisms or from the environemtn that are not shared with all the strains
What is the process of reverse vaccinology and the time frame?
o Process of reverse vaccinology-
Start with genome sequence-> predict candidates with bioinformatics-> prepare high copy number plasmid and recombinant protein-> test in animal models
Takes 1 to 2 years
What are the essential components for reverse vaccinology to be successful?
o Reliance on:
Genome sequence
Predictive tools to identify antigens
Guarantees that those antigens do not have human homologs
• Due to autoantigens
Proteins must be amenable to recombinant expression (often difficult with membrane proteins)
Requires scale ($$$/person years)
What is the process of conventional vaccinology and at what scale is it? What is its time frame?
o Conventional vaccinology Generally small scale Tests patient response first and works from that Purification of antigenic components Expression of those proteins o Process- Look at organism and host response to organism (small scale)->remove blood cells and plasmids-> take preparation of proteins/biomolecules and test to see whether there are antibodies against these using convalescent patient serum-> anything that human response is positive for, purify proteins and start testing in animals Takes 5-15 years
What is western blotting based on and how can it be used in vaccine development?
o Western blotting
Based on the simple principle of antibody-antigen reactivity where in this case bacterial protein lysates (or sub-cellular fractions) are the potential antigens
• One assay per gel/per blot
Patient sera will contain a series of primary antibodies against specific antigens- the secondary will be a labelled anti-human antibody
• Secondary antibody used for visualisation
What is pre-fractionation of the proteome and why is it useful? What cells/tissues can be pre-fractionated?
• Selecting specific subset of proteins to look at
• Used to answer a specific question about the organism
o Allows for focusing on specific question and better/more specific manner in which to answer it/gain data for it
o Allows for increased specificity and “zooming” on a particular part of the proteome to gain more detailed data
• More complex cell/tissues can also be fractionated:
o Surfaceome
o Mitochondria
o Nucleus
o Vacuoles
o Endoplasmic reticulum
o Golgi body
o Myofilaments
What are the advantages of pre-fractionation of the proteome for study?
- Specific analysis of a particular set of proteins (e.g. no need to look at cytoplasmic proteins if you are interested in adhesion)
- Reduces the complexity of the proteome and allows more lower abundance proteins to be visualised
What are the disadvantages of pre-fractionation of the proteome for study?
- Technically more challenging
- –Very fast ultra-centrifugation approaches
- Difficulty in achieving purity of samples
- –Enrichment only- no fractionation is ever 100% pure (abundant cytoplasmic or other proteins will always contaminate)
- Great number of samples to view entire proteome
What are the two main modes of study of the surfaceome and how good are they at studying the surfaceome?
o 2-DE gels are poor for looking at hydrophobic proteins, especially those with more than 3 transmembrane domains
o 2-DLC/MS-MS (shotgun proteomics) can also be used- relies on peptides and hence hydrophobicity is not an issue
What is the process of using 2-DE gels to study the surfaceome and what is a problem with this approach?
Process: bacterial cells-> sodium carbonate precipitation and ultracentrifugation-> membrane protein-enriched fraction-> Solubilization in 2DE specific buffer+ ASB-14-> excise spots, digest and identify by PMM MALDI-MS+ MS/MS
2-DE approach under-represents hydrophobic proteins with many TMR
• This is because the total protein is hydrophobic-not soluble
What is the process of using shotgun proteomics to study the surfaceome and why does this approach work so well with the surfaceome?
o 2-DLC/MS-MS (shotgun proteomics) can also be used- relies on peptides and hence hydrophobicity is not an issue
Hydrophobicity not an issue as trypsin only cleaves in soluble regions and since it relies on peptide analysis, that’s ok
Process: bacterial cells-> sodium carbonate precipitation and ultracentrifugation->membrane protein-enriched fraction-> digest complex membrane protein-enriched fraction with trypsin-> separate by SCX+RP and identify by MS/MS (shotgun approach)
Advantages of 2-DLC approach:
• Improved protein/proteome coverage
o More proteins identified
o Minimized dynamic range effects
o High mass and/or basic proteins
o Hydrophobic proteins and those with many TMR
• Rapid process
• Compatible with quantitation (iCAT, iTRAQ, etc.)
What are epitopes?
o Epitopes- regions of proteins that can trigger a cellular immune response mediated by T or B cells
What is a way of studying surfaceome epitopes and why would we do so?
o Mode of study-
Cell shaving-
• Surface-exposed epitopes are those most likely to interact with pathogens or conversely, with the immune system
o Characterisation and identification of vaccine candidate proteins
Can attempt to use surface-exposed peptides as protective antigens (essentially means that humans make antibodies to either the protein or the exposed epitope that can then ‘protect’ against subsequent infection with the organism
Used cell shaving approach
Describe the process of cell shaving for surfaceome epitope study
• Proteinase-K can be used: these peptides/proteins are generally in their native forms and not all amino acids of the protein will be exposed-get nice short peptides
o Trypsin can also be used
• Process A:
o Whole cells are incubated in the presence of proteinase-K/trypsin
o Proteinases shave off surface-exposed parts of the protein
Anything that is within the membrane and cytoplasm, as well as any non-protein components of the surface, remain untouched
o Peptides subjected to MS/MS
o Issue with this process- cells do not like being in incubation solution and they will lyse. To avoid this, can employ process B for false positive identification
• Process B: False positive identification
o Whole cells are incubated without proteinase-K/trypsin
o Cells removed by centrifugation and trypsin inserted in the supernatant for digest to occur
o Peptides subjected to MS/MS
Anything seen in the MS/MS result must be a cytoplasmic protein (contamination from cell lysis) due to the lack of trypsin in the incubation solution
What is the secretome?
• Secretome- all the proteins that are released into the extracellular environment
Why is it important to look at the secretome and what is the proteomic advantage/disadvantage of looking at this -ome
- Amenable to proteome analysis as obviously highly soluble
- Tend to be lower in mass as they are secreted from a secretion system -easier to deal with through proteomics
- Important virulence factors- toxins, proteases, siderophores, as well as non-protein factors
- Cell-cell communication
- However, they are technically challenging to collect
Describe the process of examining nuclear proteins/how they are collected and visualised
- Isolate the nucleus using differential centrifugation (organelle separation)
- Examine purity using microscopy/staining
- Solubilize proteins and perform 2-DE or digest/2-DLC
- Second round of enrichment using DNA-affinity chromatography- enriches for proteins that bind DNA (e.g. transcription factors)
Why is it important to enrich transcription factors when looking at nuclear proteins?
• Nuclear proteins+ DNA-binding proteins (should be enriched for low abundance transcription factors etc.)
o Transcription factors are amongst the most least abundant proteins found in a eukaryotic cell (5-20 copies per cell)- hence this is important
What types of environments do cells, tissues and organisms respond to and how?
• Cells, tissues and organisms respond to changes in their internal (genetic) and external environment by molecular adaptation
What is molecular adaptation?
o Molecular adaptation is a process involving changes in the expression of genes (and thus proteins) needed for survival under those altered environmental conditions
What are regulons?
- Regulons (regulome)- those genes/proteins that respond to a change in internal environment/genetic conditions
- Regulon- a group of genes/proteins that are controlled (promoted or repressed) by a single regulatory gene/protein
What are stimulons?
- Stimulons (stimulome)-those genes/proteins that respond to a change in external environmental conditions (e.g. temperature, nutrient availability, oxidative stress)
- Stimulon- a cluster of genes/proteins that respond to a change in the external environment (or a stimulus)
What information is shown on genomic heat maps?
• Shows how genome responds:
o Some genes are rapidly switched off and then gradually return to near normal (reduced) levels
o Some genes are rapidly switched on and then gradually return to near-normal (elevated) levels
o Some genes are gradually repressed over time
o Some genes are gradually induced over time
• Shows on a temporal time scale for dynamic visualisation
What does transcriptomics measure?
o Transcriptomics measures the relative levels of mRNAs in a given sample
Describe and explain the components influencing the correlation between transcript and protein
o An increase in transcription (mRNA) does not necessarily lead to an increase in translation (protein)
mRNAs have varying half-lives- a small amount of a long-lived mRNA can lead to much translation, while a large amount of short-lived mRNA can do the opposite
Proteins have varying half-lives
o Poor correlation between transcriptomics and proteomics when observed at a single time point
mRNA, protein and metabolites are temporally dissociated-
• mRNA responds first, followed by protein, followed by the product of that protein
• A single time-point is not representative of the temporal nature of genomic response
Transcriptomics fold changes have larger magnitude than proteomics
What influences the protein abundance?
Stability of mRNA/protein, protein abundance relates to degradation as much as synthesis [energy required to synthesise a protein]
What are problems with examining transcriptomics alone to solve a protein problem?
o Problems with looking at transcriptomics alone
Body fluids have no mRNA
Can’t examine subcellular fractions
Transcriptomics also does not account for protein post-translational modifications
How can the regulon/regulome be analysed/studied?
• Transcriptomics and proteomics can be applied to determine the regulome controlled by known regulators
o Members of such regulons change in their expression (up- or down-regulated)
• Analyse by
o i) gene knock-out- proteins that increase in abundance are repressed by the regulator, those that decrease in abundance are promoted or positively regulated
Can also silence/repress it if complete knockout is lethal
o ii) gene over-expression- proteins that increase in abundance are promoted, those that decrease are repressed
• Look for global regulators (in bacteria two-component systems, in mammalian systems transcription factors)
• Use of transcriptomics and/or proteomics
What is the role of regulatory proteins
• Role of regulatory proteins-
o If a certain repressor is mutated (deleted) or a transcription factor (activator) is over-expressed, proteins that are part of their regulons should change in expression
What is Tup1?
• Responsible for the repression of glucose-repressed genes
What is Yap1?
• Activator of environmental stress genes
What symbol represents deletions?
• Deletions have a Δ in front of them
What symbol represents overexpressions?
• Overexpressions have a +++ in front of them
What is sarA in S.aureus and what is its role?
Regulons- sarA in S.aureus
• SarA- staphylococcal accessory regulator
• Global regulator of virulence factor gene expression in S.aureus
o Secreted proteins- high in virulence factors
• Either induces (positive regulation) or represses (negative regulation) target gene expression
How is sarA studied for possible interventional targets and describe the success of this/ the reason for this level of success
• Genetic ‘knock-out’ of gene [sarA] encoding regulatory protein [SarA] enables detection of SarA-regulon using proteomics
• Lip1 is not a good interventional target for S.Aureus treatment-
o Lip1- a lipase that breaks down host cell lipids (appears in WT and SarA mutant)
o Lip2- backup for Lip1 if SarA is turned off
Describe hypoxia-inducible factor 1 (HIF-1) under normoxia (normal oxygen conditions)
• Normoxia-under normal oxygen conditions
o HIF-1 alpha is sensing oxygen and is hydroxylated by proline hydroxylase that adds the hydroxyl groups to two prolines
o Von Hippel-Lindau protein (VHL) binds to HIF-1
o Ubiquitination of the HIF-1 protein
o Allows targeting to proteasome (cellular garbage compactor) and subsequent proteasomal degradation, which makes peptides that can be recycled by the cell
Describe hypoxia-inducible factor 1 (HIF-1) under hypoxia (not enough oxygen conditions)
• Hypoxia- not enough oxygen
o Proline hydroxylation doesn’t occur to HIF-1 as there is no oxygen for hydroxylation
o Instead HIF-1 alpha goes to the nucleus and combines with its nuclear cofactor HIF-1 beta
o Protein complex binds as transcription factor to target DNA
Target DNA is referred to as a hypoxia response element (HRE)
How does HIF-1 stimulate glycolysis in hypoxia and why is this important?
o In hypoxia, HIF-1 stimulates glycolysis (which is important in tumourigenesis)
Cells sense a change in oxygen availability and increase transcription of HIF-1 (in hypoxic conditions, HIF prolyl-hydroxylase is inhibited, since it uses oxygen as a co-substrate)
HIF-1 binds to the promoter regions of glycolytic genes, increasing their expression to maintain production of ATP
Similarly PDH kinase is increased, phosphorylates and inactivates PDH and flux into the TCA cycle (slows TCA cycle as don’t want production of reactive oxygen species-> can lead to damage DNA), while stimulating LDH expression and production of lactate (decreasing pH)
Increased protease degrades mitochondrial cytochrome oxidase and both decreased TCA and COX reduce ROS (reactive oxygen species) accumulation
How does HIF-1 allow tumour cells to survive?
• HF1 and cancer
o Tumour cells divide rapidly and therefore need fuel in the form of glucose
o At the same time, growing tumours are poorly vascularized and therefore are poorly oxygenated
o HIF-1 responds to this hypoxia and increases expression of GLUT glucose transporters and glycolytic enzymes
o Tumours are also tolerant to low pH (lactate production)
o HIF-1 positively regulates expression of the hormone VEGF (vascular endothelial growth factor) which stimulates angiogenesis-> this can mean that the tumour is vascularised to keep it alive
Angiogenesis- making blood vessels
o In reality, tumour HIF-1 expression is a gradient based on oxygen availability
Describe 3 manners of visualising proteomics data and how these were used to visualise the HIF-1 regulome?
• Proteomics demonstrates the HIF-1 regulome
o Volcano plots
Fold change in x axis
P value in y axis
o Functional cluster
Which proteins does HIF regulate that we know are in the same functional pathway
o Pseudo-Western validation of known and putative HIF-1 targets
How can the stimulome be studied?
• Omics approaches can also be used to study how cells, tissues and organisms respond to a change in the external environment
What is the stimulome required for and how does it behave?
• Genes/proteins that respond to such a change (again up- or down- regulation) are part of the stimulome required to adapt to such an environmental change
• The individual components of the stimulon may change differently or identically (up-or down-regulation)
• Environmental stress (heat shock, oxidative stress, hypoxia, temperature, nutrient limitation, etc.) are all potential stimulomes
• Environmental stress-
o Over time, in order to survive, the organism must adapt to environmental changes
Can be sudden or subtle environmental changes
Describe the yeast stimulome and how it was found, and why the stimulome is such
o In yeast, one group looked at 142 different stresses/environmental conditions and used transcript-based microarrays to determine there were 900 genes in the yeast environmental stress response (about 15% of the genome)
At least in yeast, response to a variety of stresses is somewhat conserved (that is each gene responds in a near identical way irrespective of the type of environmental change)
Only 1/3rd of genes show a response specific to a given environmental change
• This is the environmental stress response
Yeast conserves how it responds to stresses by switching on/off essential and non-essential components respectively
Environmental stress response allows yeast to adapt to a change in environment
What is the magnitude of the stimuloe response dependent on?
• The greater the shock that is provided to the cell, the greater the magnitude of the genomic response
Does the stimulome adapt to environmental conditions? What happens when it does so and what is this change regulated by?
• Over time, the organism reaches a new steady state where certain genes remain elevated as part of the adaptation process
o The new steady state is a function of the size of the original environmental change
What is the role of peroxiredoxin?
• Peroxiredoxin: uses the free thiols on its cysteines as oxidant mops
o Proteins most likely mopping free radicals
Does a single gene give rise to only a single protein? Why/why not?
• A single gene may give rise to more than one functional protein due to the influence of chemical or physical modifications to proteins
o 6 different PTMs possible at different sites in the protein- 64 possible combinations of gene-products=64 different possible functions
However, most proteins encoded within the human genome have more than 6 PTM sites
Will probably never know how many proteins the human genome can encode
o It may be possible that a site in the protein can be modified by more than 1 PTM
o The capacity for an organism to post-translationally modify its proteins is commensurate with its genome size
o PTMs alter protein/peptide chemical composition and thus change the properties used to separate these biomolecules in proteomics
What are two different proteins that can result in the Nox1 gene and how? What are the purposes of each of these variants?
o Example- Nox1
Full-length nox1 gene is transcribed to produce a full-length mRNA, which in turn is used to produce the full-length integral membrane protein NADPH oxidase (NOX-1L)
• Is part of the electron transport chain in mitochondria
• Has 6 transmembrane domains
Produced by alternative splicing, NOX-1S does not contain the NADPH binding site, but is able to transport H+ ions
• Has 4 transmembrane domains
• Can act as proton transporter
What are post-translational modifications?
• PTMs are additions or subtractions to translated proteins that can alter the chemical structure of a protein and therefore modify its function
What is the function/role of post-translational modifications?
• PTMs can act as molecular switches to turn on (or off) enzyme activity
• By influencing structure, a PTM may influence how proteins interact with other proteins, DNA, RNA and ligands
• Post-translational modifications change the structure and function of these proteins without any reference back to the original genome
• Some PTMs are rapid and transient, while others are irreversible
• Functions of PTMs
o Alter protein structure/function relationship
o Influence protein-protein, protein-DNA, protein-ligand interactions
o Activate or repress activity (recycling)
o Mark proteins for degradations
What is the purpose of protein-protein interactions?
Protein-protein interactions
• Many proteins need to interact with other proteins, ligands, nucleic acids to function
What occurs when proteins interact with each other?
• Where two or more proteins interact with each other they may temporarily form a complex
o A protein complex consists of several proteins and the complex itself provides the function- proteins within the complex may be structural (support or transport) or enzymatic
What are protein tertiary structure and protein-protein interactions determined by?
• Protein tertiary structure and protein-protein interactions are largely determined by hydrophobic interactions, charge-based interactions and hydrogen-bond interactions, with other influences including disulfide bonds
o PTMs can alter these relationships
What protein-protein interactions are influenced by regulatory PTMs
Regulatory PTMs based on altering interactions within or between proteins • Inducible interactions • Cooperative interactions • Multi-site switches • Sequential interaction • Mutually exclusive interaction • Antagonistic modifications • Intramolecular regulation interactions • Convergent interactions
What are inducible protein-protein interactions influenced by regulatory PTMs?
o A phosphorylated residue is recognised by its interaction partner e.g. (Src-homology-2 (SH2) domain) while the non-modified residue does not interact
What are cooperative protein-protein interactions influenced by regulatory PTMs?
o Multiple domains bind in an obligatory fashion to two phosphorylated residues (both must be modified to proceed)
What are multi-site switches protein-protein interactions influenced by regulatory PTMs?
o Phosphorylation required on several sites before interaction can occur
What are sequential protein-protein interactions influenced by regulatory PTMs?
o The recruitment of a modifying protein to a phosphorylation site (step 1) precedes, and is required for, the transfer of a second modification to a target protein (step 2) which is then recognized by interacting proteins (step 3).
What are mutually exclusive protein-protein interactions influenced by regulatory PTMs?
o Modification by phosphorylation allows binding as per 1 however when the site is modified by different PTM (e.g. O-GlcNAc) the interaction is inhibited
What are antagonist protein-protein interactions influenced by regulatory PTMs?
o Modification by phosphorylation allows binding as per 1 , however this inhibits modification at a second site proximal to the first.
What are intramolecular regulation protein-protein interactions influenced by regulatory PTMs?
o Autoinhibition by self-modification and interaction between domains within the protein
What are convergent protein-protein interactions influenced by regulatory PTMs?
o A single PTM can be recognized by different types of protein interaction domain.
What are the two main ways in which PTMs are produced?
• Enzymatic vs non-enzymatic PTM
What are enzymatic PTMS? What PTMs are enzymatic? Describe.
o Enzymatic addition of a modification and subsequent enzymatic removal of PTM-> incredibly dynamic PTM process
Kinases (add phosphate)/phosphatases (remove phosphate)
• Around 150 kinases coded in the genome, only know what about 50 of them do
Acetyltransferases/acetylases
• Acetyltransferases- Transfers an acetyl group to lysines on proteins
• Acetylases- remove acetyl group
Methyltransferases/methylases
• Methyltransferases- add a methyl group to lysines or arginines
• Methylases-remove methyl group
O-GlcNAc transferase/O-GlcNAcase
• O-GlcNAc transferase- transfers a modification called O-linked N-acetylglucosamine on nuclear proteins
• O-GlcNAcase-removes the O-linked N-acetylglucosamine
• Only one copy of each in the human genome
What PTMs are non-enzymatic? Describe
o Non-enzymatic
Oxidation of proteins in the presence of reactive oxygen species (generally Cys [Cys-SOH, SO2H, SO3H], sometimes Met)
Deamidation
What are the major types of PTMs?
o Phosphorylation o Acetylation o Glycosylation o Redox (redox biology and protein oxidation) o Methylation o Deamidation o Proteolysis o Ubiquitination (Ubq)
Describe the phosphorylation PTM in terms of:
- Definition
- Role
- Enrichment methods
- Impact on the protein
o Phosphorylation (signalling)
PO4 on serine, threonine, histidine and tyrosine
• On bacteria, seen on histidine and aspartic acid
Molecular on-/off switch to regulate enzymatic activity
Very dynamic PTM, involved in virtually all cellular processes
More than 100,000 sites reported for the human proteome
Multiple enrichment methods
Addition of 80Da mass, slight acidic shift
Describe the acetylation PTM in terms of:
- Definition
- Types
- —–Definition
- —-Role
- —-Enrichment methods
o Acetylation (influence how proteins act with each other and how they are digested with proteases)
Acetyl groups on lysine
N-acetylation:
• Attachment of an acetyl group to Lys
• Mediates protein-protein interaction
• Enrichment using acetyllysine-specific antibodies
N-term acetylation:
• Acetylation on mature protein N-termini, often after removal of initial Met
• Linked to protein stability, localization and interaction
• Enrichment using SCX, COFRADIC, TAILS, ChaFRADIC
Describe the glycosylation PTM in terms of:
- Definition
- Abundance
- Role
- Enrichment methods
- Impact on the protein
- Study methods
- Motif
o Glycosylation (cell-cell communication)
N-linked (asparagine) and O-linked (serine or threonine) sugars (large, acidic structures)
Very complex PTM, structure as information carriers
Predominantly on extracellular domains involved in cell-cell interaction and recognition, involved in protein folding and chaperon recruitment
Various cancer biomarkers are glycoproteins
More than 25% of the human proteins appear to be glycosylated
Multiple enrichment methods
Addition of carbohydrate structures to proteins
• Not necessarily adding a known mass/subunit to protein-adding large structures to proteins
Combined structure and site-specific glycoproteomics remains challenging
• For 2DE, difficult to coat glycosylated proteins with SDS as the negatively charged carbohydrate repels the negatively charged SDS-> teardropping occurs
Motif for glycosylation-
• Asparagine residue- any amino acid but proline- serine/threonine
Describe the methylation PTM in terms of:
- Definition
- Role
- Enrichment methods
o Methylation
Methyl groups on several amino acids (Lys, Arg)
Regulates RNA processing, transcription, DNA damage repair, protein translocation (mostly Arg) and epigenetic regulation of gene transcription
Enrichment using antibodies
Describe the deamidation PTM in terms of:
- Definition
- Role
- Impact on the protein
o Deamidation (molecular clocks)
Asparagine to aspartic acid or glutamine to glutamic acid, acidic shift
• Results in pI shift and a one mass unit shift
Describe the proteolysis PTM in terms of:
- Definition
- Role
- Enrichment methods
- Impact on the protein
o Proteolysis (proteases and degradomics) Removal of (poly)peptide Enzymatic cleavage by exo- or endopeptidases Irreversible PTM: determines localization, function, activity and turnover Can change activity of protein Released peptides can exhibit biological functions Large change in mass and pI Enrichment using COFRADIC, TAILS, ChaFRADIC
Describe the ubiquitin PTM in terms of:
- Definition
- Role
- Enrichment methods
o Ubiquitination (Ubq)
Ubiquitin, a 8.5kDa protein, is attached to Lys via an isopeptide bond
Mono and poly (branched/linear) Ubq possible
Mediates protein degradation, signalling, trafficking
Upon tryptic digestions, a Gly-Gly residue remains at the Lys and modified peptides can be enriched using anti-diGly-antibodies
Describe the redox PTM in terms of:
- Definition
- Role
- Enrichment methods
o Redox (redox biology and protein oxidation) Oxidation • Addition of 16(O),32(O2),48(O3)Da on cysteine, tryptophan or methionine Redox reactions involving Cys sulfur oxidation to disulfides or higher oxidation states • Disulfide bonds- oxidative PTM where two cys free thiols come together to form double sulfur linkage o Mostly intramolecular o Stabilizes protein structure o Currently no large-scale enrichment method available
How is the diversity of protein modifications studied?
• Gaging the diversity of protein modifications
o 2-DE gels can be used to estimate the ability of an organism to post-translationally modify proteins
Different spots that are close together on 2DE gels are commonly the same protein with different post-translational modifications
How are PTMs characterised? Describe the principle behind this method and any adjustments sometimes required.
• Characterization of PTMs
o The addition or subtraction of chemical compounds results in a mass shift generally predictable and observable using mass spectrometry (MS)
o Each chemical modification has a characteristic mass and charge
Except glycosylation-at hundreds of different possible structures at the N-linked level and O-linked level
• However, glycans are comprised of predictable monosaccharides that the masses are known
o Proteins modified by physical or proteolytic cleavage will also be apparent using comparative MS
o More specific techniques may be used to enrich for modified proteins/peptides prior to MS
This is because modifications are stoichiometrically considered to be low abundance
What are different modes of PTM crosstalk? Describe them
• Two or more modifications influencing the protein structure- function relationship
o Competition (2 PTMs target the same site; mutually exclusive)
o Recruitment (A PTM is needed to enable a second site to be modified; sequential)
o Antagonism (A PTM inhibits a second PTM at a proximal site)
• Large-scale Crosstalk
o A PTM is needed to induce e.g. kinase or acetyltransferase activity (or phosphatases / acetylases)
Describe how acetyl-K can induce structural flexibility allowing phosphatase activity with the kinase consensus motif KXXS
• Example
o Acetyl-K induces structural flexibility allowing phosphatase accessibility
Kinase consensus motif KXXS
The KxxpS variant is in a fixed conformation due to the formation of a phosphate-lysine salt-bridge
• Phosphorylated serine at position 9 and lysine at position 6
Acetyl-K inhibits the salt-bridge and results in a more flexible conformation, which is more amenable to phosphatase activity (serine).
What is signalling and what does it involve?
• Signalling is a biochemical mechanism that mediates sensing of the environment to facilitate a cognate response by the genome
o Sense change in environment-> signal is rapidly transduced by phosphorylation cascade-> results in changes in gene expression
o Signaling involves the binding of a signal molecule (e.g. hormone) to a specific receptor (GPCRs and RTKs)
What are the two main pathways through which signalling can occur?
o Signals can diffuse into the cell and bound by cytosolic receptors and only bound receptor ligand complex can pass into the nucleus to activate gene expression
o Receptor binding in the membrane- cell surface receptor is bound by ligand which results in activation cascade, ultimately resulting in change in effector protein that can move to the nucleus and modify gene expression+ activate/deactivate pathways
Changes can occur independent of genome level
There is always a negative feedback mechanism that enables signalling to stop in the absence of continued stimulation by the original signal
What extracellular signals/stimuli do cells respond to?
• Cells respond to extracellular signals/stimuli that activate membrane-associated or cytosolic receptors
What receptor categories do membrane receptors include?
o Membrane receptors include G-Protein coupled receptors (GPCRs) and Receptor Tyrosine Kinases (RTKs)
About how many G protein coupled receptors are encoded in the genome?
About 800-900 G protein coupled receptors
About how many receptor tyrosine kinases are encoded in the genome?
About 60-70 Receptor Tyrosine Kinases
What are the possible roles of activated receptors?
• Activated receptors can act as direct transcription factors (cytosolic receptors) or regulate downstream pathways via protein phosphorylation and/or intracellular second messengers
What does protein phosphorylation/dephosphorylation regulate?
• Protein phosphorylation by kinases and dephosphorylation by phosphatases regulate protein activity in the cellular pathways and can amplify intracellular signalling
o Amplification- each kinase in the pathway can phosphorylate more than one target
What amino acids do protein kinases target in eukaryotes?
• In eukaryotes, protein kinases target serine, threonine and tyrosine residues
Describe what types of receptors an external signal can interact with
- G protein-coupled receptor
- Receptor enzyme (tyrosine kinase)
- Gated ion channel
- Nuclear receptor
How do G-protein coupled receptors work?
- G protein-coupled receptor: External ligand (L) binding to receptor (R) activates an intracellular GTP-binding protein (G), which regulates an enzyme (Enz) that generates an intracellular second messenger (X)
o X is a second messenger (e.g. cAMP) which can activate or inhibit specific protein kinases
How do receptor tyrosine kinases work?
- Receptor enzyme (tyrosine kinase)- ligand binding activates tyrosine kinase activity by autophosphorylation
o Kinase activates transcription factor (T) altering gene expression
How do gated ion channels work?
- Gated ion channel- Channel opens or closes in response in response to concentration of signal or membrane potential
How do nuclear receptors work?
- Nuclear receptor- Hormone binding allows the receptor to regulate the expression of specific genes
How do bacterial two-component regulatory systems work?
• A stimulus is detected by the sensing domain of the HK, which initiates signalling by autophosphorylation (protein phosphorylates itself at a histidine)
• The phosphate group is transferred to the RR which is activated by phosphorylation and passes this signal to its output/signal domain enabling it to bind cognate promoter regions of target genes to initiate (or repress) their transcription
o RR is phosphorylated at a conserved aspartic acid
What are the components of the bacterial 2 component regulatory system?
• The 2 components are a SENSOR histidine kinase (HK) and a response regulator (RR)
Describe the structure of the histidine kinase in the bacterial 2 component regulatory system
o HK is located in the membrane- has external sensor domain, and part of the protein is located in the plasma membrane and cytoplasm
Describe the structure of the response regulator in the bacterial 2 component regulatory system and the role of these structures
o Response regulator consists of 2 domains
The N-terminal-receiver domain (containing the ASP/ASP-P)
• Conserved (the same)
The C-terminal DNA binding or signal/output domain
• The output domain facilitates DNA-binding and is structurally variable such that specific promoters can be targeted
• DNA binding areas specific for different sequences of DNA
• Facilitate which genes will be part of the response regulon
What is the purpose of the PhoP-PhoQ system and how does it work?
PhoP-PhoQ
• Example of a bacterial two-component regulatory system
• Salmonella PhoP-PhoQ TCS regulates magnesium transport
• At low magnesium (neutral pH), PhoP-PhoQ (sensing domain in the histidine kinase) is activated (by autophosphorylation) and the PhoP- RR (which was activated by histidine kinase) binds the mgtA promoter to increase expression and ultimately translation of the MgtA transporter to increase Mg2+ uptake
• Pho-P also increases expression of rstA (another transcription factor)
What is a protein kinase
• Protein kinase- protein/enzyme that phosphorylates another protein or itself
Describe how a protein kinase works
o When it adds the phosphate group, it uses the phosphate from ATP
ATP dependent process
Describe how a protein phosphatase works
o Protein phosphatase removes phosphate group but leaves target with a hydroxyl group
Reason there is a hydroxyl group is that, in mammalian systems, phosphorylation occurs on amino acids that have a hydroxyl group
What can activated kinases do?
o Activated kinases phosphorylate multiple target proteins at consensus motifs to alter their structure/function
o Activated kinase signal transduction ultimately leads to a change in gene transcription
Where do activated kinases phosphorylate?
o Phosphorylate at a particular sequence motif
Are kinases very specific to a particular site or are they more promiscuous? Discuss
o Proteins can be phosphorylated at more than one site by more than one kinase
Kinases target multiple sites
o Some are specific for one protein, others responsible for phosphorylating many proteins (e.g. Protein Kinase A[PKA] and Protein Kinase C[PKC])
Is phosphorylation only stimulatory?
o Phosphorylation can be stimulatory and inhibitory
Do phosphorylation effects only affect a single protein?
o Phosphorylation can have cascade effects
Describe the time frame of phosphorylation signalling
o Signaling via phosphorylation allows a rapid response in a time-frame shorter than possible via gene expression alone
Describe the cell response to phosphorylation
o Cell response is specific and amplified via multiple pathways
Describe the architecture of histidine kinases
Sensing domain Conserved transmembrane helices (2,3, or 4) Linker domain catalytic domain Histidine phosphorylation domain • It is autophosphorylated
What does the architecture of histidine kinases allow?
o Allows signal to be transferred from outside to inside the cell
What parts of the histidine kinases are highly conserved?
o Parts of HK are highly conserved (the TMR, His phosphorylation (Dhp[dimerization and His phosphotransfer) and ATP-binding domains)
What parts of the histidine kinases are variable?
o Others are variable (e.g. sensor domain- specific for the ligand: signal transducing domain-specific for second messengers and activities)
What are G protein coupled receptors?
o G-protein coupled receptors (GPCRs) are alpha-helical integral membrane proteins that bind G proteins
o G-proteins are heretotrimeric (alphabetagamma) membrane associated proteins that bind guanosine nucleotides (e.g. GTP) (hence G proteins)
What is the role of G protein coupled receptors?
o G-proteins mediate signal transduction from GPCRs to target proteins- they are inactive when GDP is bound and active when GTP is bound
o The third component is an effector enzyme that is regulated by the active G-protein
o Important in a number of biological/biomedical situations-
G protein mutations found in cancer subtypes
Many drug compounds target G-coupled receptors: easier to design a drug against
o In GPCR signaling, a second messenger (cAMP) activates protein kinases (PKA) that amplifies the signal without altering protein expression
What are enzymes regulated by cAMP dependent phosphorylation
- Glycogen synthase
- Phosphorylase b kinase: alpha subunit and beta subunit
- Pyruvate kinase (rat liver)
- Pyruvate dehydrogenase complex (type L)
- Hormone sensitive lipase
- Phosphofructokinase-2/fructose 2,6- bisphosphatase
- Tyrosine hydroxylase
- Histone H1
- Histone H2B
- Cardiac phospholamban (cardiac pump regulator)
- Protein phosphatase-1 inhibitor-1
- PKA consensus sequence
What sequence is phosphorylated by glycogen synthase and what pathway/process is regulated?
RASCTSSS
Glycogen synthesis
What sequence is phosphorylated by Phosphorylase b kinase
alpha subunit
beta subunit
and what pathway/process is regulated?
Alpha subunit: VEFRRLSI
Beta subunit: RTKRSGSV
Glycogen breakdown
What sequence is phosphorylated by pyruvate kinase and what pathway/process is regulated?
GVLRRASVAL Glycolysis
What sequence is phosphorylated by pyruvate dehydrogenase complex and what pathway/process is regulated?
GYLRRASV
Pyruvate to acetyl-CoA
What sequence is phosphorylated by hormone sensitive lipase and what pathway/process is regulated?
PMRRSV
Triacylglycerol mobilisation and fatty acid oxidation
What sequence is phosphorylated by Phosphofructokinase-2/fructose 2,6- bisphosphatase and what pathway/process is regulated?
LQRRRGSSIPQ Glycolysis/gluconeogenesis
What sequence is phosphorylated by tyrosine hydroxylase and what pathway/process is regulated?
FIGRRQSL
Synthesis of L-dopa, dopamine, norepinephrine, and epinephrine
What sequence is phosphorylated by histone H1 and what pathway/process is regulated?
AKRKASGPPVS
DNA condensation
What sequence is phosphorylated by histone H2B and what pathway/process is regulated?
KKAKASRKESYSVYVYK DNA condensation
What sequence is phosphorylated by cardiac phospholamban and what pathway/process is regulated?
AIRRAST
Intracellular [Ca2+]
What sequence is phosphorylated by protein phosphatase-1 inhibitor-1 and what pathway/process is regulated?
IRRRRPTP
Protein dephosphorylation
What sequence is phosphorylated by PKA consensus sequence and what pathway/process is regulated?
xR[RK]x[ST]B
Many
How do phosphoprotein phosphatases work?
o Remove PO4 (phosphorylation) from target proteins in response to cellular stimulus (or removal of competing stimulus)
o May need more than one phosphatase to remove all phosphates
o Tightly regulated and sometimes works in feedback mechanisms with protein kinases themselves
Why are phosphoprotein phosphatases more promiscuous than kinases?
o More promiscuous than kinases as less of them coded in the genome
What are receptor tyrosine kinases?
• Receptor Tyrosine Kinases (RTKs)
o A family of around 60 plasma membrane receptors that transduce extracellular signals via autophosphorylation
o Various RTKs act as specific receptor for growth factors (e.g. epidermal growth factor; EGF) and insulin
Describe the structure of receptor tyrosine kinases
o RTKs have an extracellular ligand-binding domain, a single transmembrane alpha helix, an activation loop and an enzymatically active kinase domain in the cytoplasm
o The kinase domain is a tyrosine kinase
What are the general mechanisms of receptor tyrosine kinase structure and activation?
RTK cytosolic domain contains a protein tyrosine kinase that is poorly catalytic as a monomer
Two ligands binding to 2 RTK extracellular domains homodimerizes the receptors, bringing together two poorly active cytosolic domain kinases, which phosphorylate each other on an activation loop tyrosine residue. The phosphorylated loop moves out of the kinase catalytic site, increasing ATP and/or protein substrate binding
The activated kinase phosphorylates additional Tyrosine residues in the cytosolic domain, which provides docking sites for binding domains on downstream signal transduction proteins
What type of signalling is the fight or flight response mediated by?
• The flight or fight response is mediated by GPCR signalling
What is adrenaline made by and why?
o Mediates stress response: mobilization of energy-generating machinery (in e.g muscle) in response to stress
What effects does adrenaline binding to GPCRs produce in different locations?
o Binding to GPCRs (Beta-adrenergic receptors) in muscle or liver cells induces breakdown of glycogen
o Binding to GPCRs in adipocytes induces lipid hydrolysis
o Binding to GPCRs in myocytes increases heart rate
What is the name of the G-protein, effect enzyme and second messenger in the adrenaline signalling pathway?
o The G-protein is an activator and is hence referred to as a stimulatory G-protein or GS
o The effector enzyme is adenyl cyclase and the second messenger is cAMP
Describe the process of epinephrine-induced beta-adrenergic signalling
• Epinephrine-induced beta-adrenergic signaling leads to production of cAMP and activation of protein kinase A
o Process-
Epinephrine binds to its specific receptor (Beta-adrenergic receptor)
Hormone-receptor complex causes the GDP bound to GSalpha to be replaced by GTP, activating GSalpha
Activated GSalpha separates GSbetagamma moves to adenylyl cyclase and activates it. Many GSalpha subunits may be activated by one occupied receptor
Adenylyl cyclase catalyzes the formation of cAMP
cAMP activates Protein Kinase A
Phosphorylation of cellular proteins by PKA causes the cellular response to epinephrine
cAMP is degraded, reversing the activation of PKA
How does PKA transmit the adrenergic signal?
• PKA transmits the adrenergic signal via kinase/phosphatase-type phosphorylation
Describe the properties of Ser/Thr and Tyr phosphorylation in terms of:
- Speed
- Coordination
- Permanence
- Strength
- Role
o Ser/Thr and Tyr phosphorylation is:
Rapid (can occur in less than 1 second)
Co-ordinated
• Only targets of PKA can be phosphorylated at that point
Easily reversible (therefore transient)
Amplification of signal (activation of a kinase can lead to the phosphorylation of many proteins)
Acts as a molecular switch (activate or deactivate)
• Without reference to the genome
What is the consequence of production of cAMP in beta-adrenergic signalling?
o In beta-adrenergic signaling, production of the second messenger cAMP activates cAMP-dependent protein kinase (protein kinase A [PKA]),which in turn phosphorylates many target proteins including activation of glycogen phosphorylase which converts glycogen stores to glucose
How does PKA induce gene transcription in beta-adrenergic signalling? Outline
• PKA also induces gene transcription via nuclear translocation and phosphorylation/activation of CREB (cAMP response element binding protein)
o Activated GPCR
o Activation of adenyl cyclase by Gs and production of cAMP
o cAMP activation of PKA, disassociation of the catalytic subunits of PKA and translocation to the nucleus
o PKA phosphorylates CREB
o Activated CREB forms a complex with CBP/P300 enabling binding to CRE (cAMP response elements) in promoters of cAMP-regulated genes promoting their transcription
What is the consequence of increased cAMP on glycogen synthesis and glycogen breakdown and why?
• Increased cAMP-> stimulates Protein Kinase A-> phosphorylates glycogen synthase (becomes inactive) and phosphorylates inhibitor of phosphoprotein phosphatase, which inhibits phosphatase activity (for decreased glycogen synthesis) AND PKA phosphorylates second kinase, which phosphorylates GP (which is responsible for converting glycogen to glucose-1-phosphate) (increase glycogen breakdown)
o Increased cAMP-> Increased glycogen breakdown and decreased glycogen synthesis
What is the consequence of decreased cAMP on glycogen synthesis and glycogen breakdown and why?
• Decreased cAMP-> activation of phosphoprotein phosphatase (as inhibitor of phosphoprotein phosphatase cannot be activated by PKA)-> dephosphorylates glycogen synthase (active glycogen synthase starts to convert glucose to glycogen) (stimulates glycogen synthesis) AND dephosphorylates GPK and GP (prevents glycogen breakdown)
o Decreased cAMP-> decreased glycogen breakdown and increased glycogen synthesis
What is the structure of insulin, what is its purpose and how is it produced?
• Insulin interacts with the cell via a receptor tyrosine kinase (RTK)
o Insulin is comprised of 51 amino acids, just under 6 kDa in size
o Insulin is a peptide hormone that is produced by the beta-cells of islets of Langerhans in the pancreas
Produced in response to high blood glucose
o Insulin reaches target cells, such as liver, muscle, or fat tissue cells via bloodstream
Does insulin signalling affect only one kinase pathhway?
• In insulin signaling, several kinase pathways are activated (e.g. MAPK/AKT)
What initiates insulin signalling and what is the consequence of this?
o Binding of insulin to the insulin receptor (INSR) initiates signal transduction pathways (insulin signalling) that leads to increased glucose uptake and metabolism
What causes diabetes?
o Inability to make (type I diabetics or sense insulin (type II diabetes)-> diabetes
How does activated insulin signalling lead to GLUT4 translocation to the cell surface/movement?
• Activated insulin signalling leads to GLUT4 translocation to the cell surface
o Glucose transporters are stored within the cell in membrane vesicles
o When insulin interacts with its receptor, vesicles move to the surface and fuse with the plasma membrane, increasing the number of glucose transporters in the plasma membrane
o When insulin level drops, glucose transporters are removed from the plasma membrane by endocytosis, forming small vesicles
o The smaller vesicles fuse with a larger endosome
o Patches of the endosome enriched with glucose transporters bud off to become small vesicles, ready to return to the surface when insulin levels rise again
What is the role of INSR in Ras/Raf and MAPK signalling?
INSR phosphorylates insulin receptor substrate-1 (IRS-1) which initiates a series of protein phosphorylations via binding to Grb2, Sos and Ras
What is the role of Ras in Ras/Raf and MAPK signalling?
Ras GTP-binding activates Ras, which in turn activates Raf and initiates mitogen-activated protein kinase (MAPK) signalling (MEK)
What is the role of ERK in Ras/Raf and MAPK signalling?
Phosphorylated extracellular signal-regulated kinase (ERK; a MAPK) enters the nucleus
What is the role of Elk1 in Ras/Raf and MAPK signalling?
A transcription factor (Elk1) becomes phosphorylated and stimulates the expression of specific genes (e.g. glucose transporters)
What is the process of insulin signalling Ras/Raf and MAPK signalling
o Process:
Insulin receptor binds insulin and undergoes autophosphorylation on its carboxyl-terminal Tyr residues
Insulin receptor phosphorylates IRS-1 on its Tyr residues
SH2 domain of Grb2 binds to phosphor-Tyr of IRS-1. Sos binds to Grb2, then to Ras, causing GDP release and GTP binding to Ras
Activated Ras binds and activates Raf-1
Raf-1 phosphorylates MEK on two Ser residues, activating it. MEK phosphorylates ERK on Thr and a Tyr residue, activating it
ERK moves into the nucleus and phosphorylates nuclear transcription factors such as Elk1, activating them
Phosphorylated Elk1 joins SRF to stimulate the transcription and translation of a set of genes needed for cell division
What is the role of AKT phosphorylations in insulin signalling/what does AKT effect? What do they do (downstream applications)
• Insulin Signalling-PI3K and Akt (Protein Kinase B) signalling
o Insulin signalling involves several kinases in addition to MAPKK/MEK and MAPK/ERK, including AKT
o Most AKT phosphorylations are inhibitory to its downstream proteins
Inhibiting TBC1D4 can increase glucose uptake
Inhibiting GSK3Beta increases glycogen synthesis
Inhibiting TSC2 increases mTORC1 activity, which increases SREBP1c activity for lipid synthesis and S6K activity for protein synthesis
o This results in many cellular and metabolism changes, many of which are tissue specific
Multiple signal pathways are activated by insulin signalling
So few receptor tyrosine kinases because there are very few compounds that require such a global response
What is the advantage of large scale phosphoproteomics in studying insulin signalling?
• Large-scale phosphoproteomics provides a temporal and global view of insulin signalling
o Large-scale phosphoproteomics can help decipher the temporal regulation of signal events and identify new targets
What are 4 ways in which 2-DE gels can be used to analyse phosphoproteins/peptides
- 2-D Electrophoresis
- 2-DE combined with stains (PO4-specific)
- 2-DE combined with electroblotting and anti-phosphoantibodies
- Affinity chromatography followed by 2-DE
Why can phosphate modifications be seen on normal 2-DE electrophoresis
- 2-D Electrophoresis
a. Takes advantage of charge states-addition of PO4 results in approximately 0.4 pH unit shift (acidic-HP3O4))
b. Visible as pI isoforms or variants across a 2-DE gel (x-axis)
c. However, many different PTMs modify charge so more is needed
How can stains be combined with 2-DE to detect phosphate modification, give an example and a limitation
- 2-DE combined with stains (PO4-specific)
a. Dual-channel imaging
i. By giving faux colour to different stains and overlapping gel images (the same gel but stained for different features) - Can overlap gel with total protein staining and one with phosphoprotein specific stain- can make the stains on the same gel
ii. The stains can give different information about protein abundance and post-translational modifications
b. Pro-Q diamond
c. Not as specific as desired
How can 2-DE be combined with anti-phosphoantibodies to visualise phosphate modifications? Do all of these anti-phosphoantibodies work equally?
DE combined with electroblotting and anti-phosphoantibodies
a. Anti-phosphotyrosine antibodies work pretty well, but anti-phosphoserine and anti-phosphothreonine antibodies do not work as well
b. Bind samples to anti-phospho antibody column
c. Alternative: Take bound fractions, digest with trypsin and subject to 2nd round of affinity enrichment, then take the bound fractions straight to MS
How can characterisation of phosphoproteins/phosphopeptides by mass spectrometry occur?
• Isolation and identification (peptide mass fingerprinting or Tandem-MS)
• Selective enrichment of phosphopeptides
o Affinity chromatography
o Immobilised metal affinity chromatography
o Titanium dioxide chromatography
• Precursor ion scanning
• On-site phosphatase treatment
What are the possible steps of characterization of phosphoproteins/phosphopeptides by mass spectrometry
o Identification and database prediction
o Comparative MALDI-MS
o Enzymatic treatment (on-target phosphatase)
o Affinity enrichment + MALDI-MS
o Chemical enrichment and large-scale tandem-MS for site discovery
How can identification and database prediction be used to characterize phosphoproteins/ phosphopeptides by mass spectrometry? What are the limitations of this and what can be done for this limitation?
Standard PMF using MALDI-TOF MS-list of matching peptides+list of non-matching peptides
Non-matching could be trypsin, keratin, non-specific cleavages or PTM-peptides
Fraught with danger
• Most PO4 peptides don’t fly in MALDI-MS due to ion suppression caused by signal strength of other peptides-> enrichment is needed
Needs independent verification or comparative MS
Describe phosphopeptides behaviour in MALDI-MS and why
• Most PO4 peptides don’t fly in MALDI-MS due to ion suppression caused by signal strength of other peptides-> enrichment is needed
o Phosphopeptides don’t ionise very well in MALDI-MS spectrometry when other peaks are present
If part of the protein is phosphorylated, one peptide could be in two forms (one phosphorylated and one not phosphorylated)
• Phosphate cannot be seen
Phosphate is labile
• In-source decay
How can comparative MALDI-MS be used to characterize phosphoproteins/ phosphopeptides by mass spectrometry?
o Comparative MALDI-MS
Compare spectra from multiple pI variants (or mass variants) of the same protein as shown on 2-D gels
Examine for both missing peaks and new peaks (PO4 is seen as approximately 80Da (80m/z) shift
Quantity is not so important
Describe how enzymatic treatment can be used to characterize phosphoproteins/ phosphopeptides by mass spectrometry? What phosphatases are used to develop such treatment?
Phosphatase treatment uses commercially available phosphatases to treat peptide mix either in solution or directly on MALDI target plate (incubate for ½ an hour, let it dry, put more MALDI on and take another spectrum)
• Compare first mass spectrum (with phosphates to second mass spectrum (without phosphates due to phosphatase treatment)
B-casein phosphatase treatment method development protein because of its phosphorylation site
Describe the process of how enzymatic treatment can be used to characterize phosphoproteins/ phosphopeptides by mass spectrometry?
- Phosphopeptides are barely visible in spectrum due to ion suppression effects of large non-modified peptides
- Removal of PO4 groups results in large signal intensity of non-phosphorylated forms of those peptides
How specific is affinity enrichment+ MALDI-MS in characterizing phosphoproteins/ phosphopeptides by mass spectrometry?
Specific analysis of phosphopeptides alone- highly selective
What are three chromatographic steps prior to MS that can be performed when characterizing phosphoproteins/ phosphopeptides by mass spectrometry?
- Anti-phospho antibodies
- Immobilized metal affinity (IMAC)
- Titanium dioxide (TiOx)- method of choice for large scale proteomics
Describe how immobilised metal affinity (IMAC) works in selecting for phosphopeptides only, its limitations and its possible adjustments
o Binding occurs due to the formation of a reversible complex between the PO4 group (-ve) and the metal ion (+ve) (under acidic conditions)
Charged based interaction
o Bond is broken at alkaline pH
Elution step
o Carboxylic acid moieties of Glu and Asp may also bind (as they are negatively charged)-need repeated washes
o Some singly (mono-) PO4lyted peptides may not bind if PO4 is inaccessible
IMAC is better at multiply-phosphorylated peptides
If there is folding of peptide, phosphate may become inaccessible
o Choice of different metal ions (copper, gallium, iron etc.) with different properties
Immobilised metal affinity chromatography
How is IMAC performed for phospho-proteins
Control cell vs treated cell
Get proteins and put them through IMAC column
• Once that are phosphorylated stick to column by charged based interactions
• The others flow through
Look for differences between bound gel and unbound 2-DE gel
Do MS for identification and identify
• Not site-specific information
How is IMAC performed for phospho-peptides
Take spot of interest Alkalation step- alkate free thiols Do trypsin digest Desalt (using C18) DO IMAC column Identify by LC-MS/MS • Site specific information
How does a titanium dioxide column select for phosphopeptides? What are its advantages?
o Phosphopeptides interact with TiO2 based on bidendate binding
Partly based on charge interactions and other types of interactions (hydrogen bonds…)
o Similar properties to IMAC but:
Simpler sample handling (no metal ions)
Fewer false negatives and false positives
Stronger binding- multi-phosphopeptides hard to elute
o Use very high pH to elute bound peptides
Straight ammonia solution (pH 11-13)
o Current method of choice for both single protein analysis and large-scale pphosphoproteomics
o Comparative MS of both bound and unbound fractions
o Compatible with label-based quantitation
What is the advantage and disadvantage of using affinity enrichment + MALDI-MS to characterize phosphoproteins/ phosphopeptides by mass spectrometry?
False positives and false negatives
Large-scale analysis by MS/MS
How are phosphopeptides purified using titanium oxide
Purification of phosphopeptides using TiO2
• Mixture containing phosphopeptides->phosphopeptides binding-> washing (5x)-> phosphopeptides attached on TiO2 microspheres-> elution (1% ammonia solution)-> MS
o Peptide mixtures are incubated with TiO2 beads, beads are washed away and collected (non-phosphopeptides are washed away)
o Phosphopeptides are eluted using highly alkaline ammonia solution and analysed by MS
Describe the process of titanium dioxide micro-purification
Titanium dioxide micro-purification
• Gel-separated proteins (2-DE)
• Digest with trypsin and get:
o Peptide mix containing phosphorylated and non-phosphorylated peptides
o Peptide mix passed through GeLoader tip packed with TiOx using air pressure and column washed
Unbound fraction (containing DHB matrix) spotted directly onto MALDI target plate
Bound peptides eluted with high pH onto MALDI target, DHB added
Why can phosphopeptides cause difficulties in MS/MS
Phosphopeptides can cause difficulties in MS/MS
• The phosphate group attached to an amino acid is extremely labile
o If energy is used to break peptide bonds between amino acids, the phosphate group falls off-> because this bond is much weaker than the bonds between the amino acids
• The energy required to remove the phosphate group is much lower than required to break peptide bonds between amino acids
• Sometimes in MS/MS of phosphopeptides, the only fragment ion observed is the loss of phosphate
o When this occurs no sequence data are generated
What happens when phosphopeptides undergo MS/MS? What is the consequence of this?
MS/MS of phosphopeptides
• The phosphate attachment is extremely labile and collision breaks the bond readily- hence both the loss of phosphate and the loss of each amino acid are seen in the MS/MS spectrum; this can be exploited to identify the modification site
Why is MS/MS/MS performed for phosphopeptides for sequence identification instead of MS/MS? What is a limitation of this and what can be done to fix this?
MS/MS/MS
• Like many PTMs, the bond between the phosphate group and the modified amino acid is much more labile than the peptide bonds (linking the amino acids in a chain)
• Therefore in MS/MS (tandem-MS or MS2), fragment ions resulting from the loss of phosphate are generally much more intense than those resulting from the amino acid sequence
o This makes assigning the y- and b-ion peptide series difficult and results in reduced peptide identifications
• MS3 takes advantage of this by performing fragmentation of the (parent ion -98 Da) peak generated in MS/MS
o Enables sequence identification but often results in loss of contextual information about the location of the phosphorylation site
Multi-stage activation retains any positional information
• Overlap MS/MS and MS/MS/MS spectrum
o Do MS to get the mass of the peptide, MS/MS to generate a fragment that loses 98, and do MS/MS/MS on that fragment to get the sequence
What enrichment/purification techniques are available to isolate modified peptides/proteins?
• Enrichment/purification techniques available to isolate modified peptides/proteins
o Hydrophilic interaction liquidd chromotography
o Titanium dioxide
o Immobilised metal affinity chromatography
Describe what basic steps/the manner in which signal pathways during disease should be elucidated. Basic things that should be considered during a phosphopeptide signalling experiment
• Combined with a temporal dimension, quantitative shotgun (iTRAQ, TMTs, SILAC) and PTM-focused proteomics enables elucidation of signal pathways during disease
• Should get a temporal profile of signalling-> signalling is temporally affected
o Phosphorylation can occur really quickly, and phosphoproteomics can direct future experimentation that would never be seen if only look at proteomics level
o Can also do cluster analysis of temporal phosphorylation profile
• Induction of signal transduction can be monitored via Western blotting
o Validation of a single result enables specific hypotheses to be generated
o Use more independent methods of verification
• Need genetic mutagenesis to decipher role of the phosphorylation event
Describe the optimised phosphoproteomics workflow
• Optimised phosphoproteomics workflow
o Ex vivo model-> protein extraction-> digestion-> isobaric tags-> PTM enrichment
Enrichment : Dual enrichment
• IMAC basic elution for multiply phosphorylated peptides->MS
• IMAC acidic elution for primarily single phosphorylated sites-> Titanium dioxide column -> do Hydrophilic interaction chromatography-> MS
