Proteomics

Question 1

Which is capable of identifying a higher proportion of the proteome accurately? Peptide-centric or protein-centric approaches? Why?

Answer 1

• 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

Question 2

What is the difference between relative and absolute quantitation?

Answer 2

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

Question 3

What is the aim of comparative quantitative shotgun proteomics?

Answer 3

• Measures relative abundance across two different samples (one control and one test)

Question 4

What are 3 methods to perform comparative quantitative shotgun proteomics?

Answer 4

• Method A-metabolic stable isotope labelling
• Method B- isotope tagging by chemical reaction
• Method C- stable-isotope incorporation via enzyme reaction

Question 5

Describe the process of metabolic stable isotope labelling for quantitative shotgun proteomics and when it can be used

Answer 5

• 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)

Question 6

What is an example of a tag/method used for metabolic stable isotope labelling

Answer 6

• Stable isotope labelling by amino acids in cell culture (SILAC)

Question 7

What is the process of stable isotope labelling by amino acids in cell culture (SILAC) and what are its requirements?

Answer 7

• 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

Question 8

Describe an example of proteomics of B cell differentiation using SILAC quantitative proteomics

• Define B cells
• Differentiation of B cells
• Study details

Answer 8

• 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

Question 9

What is cluster analysis?

Answer 9

 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

Question 10

What are the advantages of stable isotope labelling by amino acids in cell culture (SILAC)?

Answer 10

• Very reproducible
• Generally high labelling efficiency
• Multiple peptides per protein- therefore good statistical confidence
• Compatible with PTMs
• Compatible with most cell-based systems

Question 11

What are the disadvantages of stable isotope labelling by amino acids in cell culture (SILAC)?

Answer 11

• 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

Question 12

Describe the SILAC mouse, how it is produced and why it is so useful

Answer 12

• 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

Question 13

What is a disadvantage of the SILAC mouse and how is it overcome? What is a potential problem with the solution?

Answer 13

 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

Question 14

What is the SILAC fly useful for and how is it produced?

Answer 14

• 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

Question 15

What is the SILAC worm useful for and how is it produced?

Answer 15

• 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

Question 16

What is the procedure for isotope tagging by chemical reaction in comparative shotgun proteomics and when can it be used?

Answer 16

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

Question 17

What are isobaric tags in isotope tagging by chemical reaction in comparative shotgun proteomics and why are they useful?

Answer 17

• 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

Question 18

What are tags used in isotope tagging by chemical reaction in comparative shotgun proteomics?

Answer 18

• Tags- Isotope coded affinity tags (ICAT)
• Tags- Isobaric affinity tags (iTRAQ-Isobaric tags for relative and absolute quantitation)
• Tags- Tandem Mass tags

Question 19

Describe the process of isotope coded affinity tags for isotope tagging by chemical reaction in comparative shotgun proteomics and the quantification process

Answer 19

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

Question 20

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

Answer 20

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

Question 21

What are the advantages of ICAT (isotope coded affinity tags) for isotope tagging by chemical reaction in comparative shotgun proteomics

Answer 21

• Reduces complexity
• Easy analysis
• Reproducible

Question 22

What are the disadvantages of ICAT (isotope coded affinity tags) for isotope tagging by chemical reaction in comparative shotgun proteomics

Answer 22

-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

Question 23

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

Answer 23

• 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

Question 24

Describe the components of isobaric affinity tags (iTRAO) for isotope tagging by chemical reaction in comparative shotgun proteomics and their uses

Answer 24

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

Question 25

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

Answer 25

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)

Question 26

What are the advantages of using isobaric affinity tags (iTRAO) for isotope tagging by chemical reaction in comparative shotgun proteomics

Answer 26

- 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

Question 27

What are the disadvantages of using isobaric affinity tags (iTRAO) for isotope tagging by chemical reaction in comparative shotgun proteomics

Answer 27

- 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

Question 28

How do tandem mass tags for isotope tagging by chemical reaction in comparative shotgun proteomics work?

Answer 28

• Tags- Tandem Mass tags | o Work like ITRAQ tags: isobaric tags where quantification is performed at mass spectrometry level

Question 29

Describe how stable-isotope incorporation via enzyme reaction works in comparative shotgun proteomics

Answer 29

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

Question 30

What is the surfaceome and why is it useful to understand?

Answer 30

• 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

Question 31

What is the secretome?

Answer 31

• Secretomes- proteins secreted from a cell into the extracellular space

Question 32

What are organellar proteomics?

Answer 32

• Organellar proteomics- nuclear proteins, ribosomal proteins, etc.

Question 33

How is a good vaccine made/ what are requirements of a good vaccine?

Answer 33

• 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

Question 34

Who pioneered pathogen genomics for reverse vaccinology?

Answer 34

o Pioneered by Dr. Rino Rappuoli

Question 35

What are the phases of pathogen genomics for reverse vaccinology?

Answer 35

 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

Question 36

What are the symptoms of meningitis and sepsis?

Answer 36

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

Question 37

What is neisseria meningitidis?

Answer 37

o Neisseria meningitidis- Gram negative bacterium with five major serotypes (antibody binding): A, B,C,Y and W135

Question 38

Why was serotype B of meningitidis difficult to make a vaccine of?

Answer 38

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

Question 39

How was reverse vaccinology used to make a vaccine for serotype B of meningitidis? Describe the difficulties in the process and the end result

Answer 39

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)

Question 40

What elements are contained in the pan genome of serotype B N.meningitidis? Describe their significance

Answer 40

• 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)

Question 41

What are pathogenicity islands?

Answer 41

o Pathogenicity islands- little bits of DNA acquired from other microorganisms or from the environemtn that are not shared with all the strains

Question 42

What is the process of reverse vaccinology and the time frame?

Answer 42

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

Question 43

What are the essential components for reverse vaccinology to be successful?

Answer 43

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)

Question 44

What is the process of conventional vaccinology and at what scale is it? What is its time frame?

Answer 44

``` 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 ```

Question 45

What is western blotting based on and how can it be used in vaccine development?

Answer 45

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

Question 46

What is pre-fractionation of the proteome and why is it useful? What cells/tissues can be pre-fractionated?

Answer 46

• 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

Question 47

What are the advantages of pre-fractionation of the proteome for study?

Answer 47

- 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

Question 48

What are the disadvantages of pre-fractionation of the proteome for study?

Answer 48

- 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

Question 49

What are the two main modes of study of the surfaceome and how good are they at studying the surfaceome?

Answer 49

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

Question 50

What is the process of using 2-DE gels to study the surfaceome and what is a problem with this approach?

Answer 50

 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

Question 51

What is the process of using shotgun proteomics to study the surfaceome and why does this approach work so well with the surfaceome?

Answer 51

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.)

Question 52

What are epitopes?

Answer 52

o Epitopes- regions of proteins that can trigger a cellular immune response mediated by T or B cells

Question 53

What is a way of studying surfaceome epitopes and why would we do so?

Answer 53

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

Question 54

Describe the process of cell shaving for surfaceome epitope study

Answer 54

• 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

Question 55

What is the secretome?

Answer 55

• Secretome- all the proteins that are released into the extracellular environment

Question 56

Why is it important to look at the secretome and what is the proteomic advantage/disadvantage of looking at this -ome

Answer 56

* 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

Question 57

Describe the process of examining nuclear proteins/how they are collected and visualised

Answer 57

* 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)

Question 58

Why is it important to enrich transcription factors when looking at nuclear proteins?

Answer 58

• 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

Question 59

What types of environments do cells, tissues and organisms respond to and how?

Answer 59

• Cells, tissues and organisms respond to changes in their internal (genetic) and external environment by molecular adaptation

Question 60

What is molecular adaptation?

Answer 60

o Molecular adaptation is a process involving changes in the expression of genes (and thus proteins) needed for survival under those altered environmental conditions

Question 61

What are regulons?

Answer 61

* 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

Question 62

What are stimulons?

Answer 62

* 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)

Question 63

What information is shown on genomic heat maps?

Answer 63

• 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

Question 64

What does transcriptomics measure?

Answer 64

o Transcriptomics measures the relative levels of mRNAs in a given sample

Question 65

Describe and explain the components influencing the correlation between transcript and protein

Answer 65

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

Question 66

What influences the protein abundance?

Answer 66

 Stability of mRNA/protein, protein abundance relates to degradation as much as synthesis [energy required to synthesise a protein]

Question 67

What are problems with examining transcriptomics alone to solve a protein problem?

Answer 67

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

Question 68

How can the regulon/regulome be analysed/studied?

Answer 68

• 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

Question 69

What is the role of regulatory proteins

Answer 69

• 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

Question 70

What is Tup1?

Answer 70

• Responsible for the repression of glucose-repressed genes

Question 71

What is Yap1?

Answer 71

• Activator of environmental stress genes

Question 72

What symbol represents deletions?

Answer 72

• Deletions have a Δ in front of them

Question 73

What symbol represents overexpressions?

Answer 73

• Overexpressions have a +++ in front of them

Question 74

What is sarA in S.aureus and what is its role?

Answer 74

 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

Question 75

How is sarA studied for possible interventional targets and describe the success of this/ the reason for this level of success

Answer 75

• 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

Question 76

Describe hypoxia-inducible factor 1 (HIF-1) under normoxia (normal oxygen conditions)

Answer 76

• 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

Question 77

Describe hypoxia-inducible factor 1 (HIF-1) under hypoxia (not enough oxygen conditions)

Answer 77

• 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)

Question 78

How does HIF-1 stimulate glycolysis in hypoxia and why is this important?

Answer 78

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

Question 79

How does HIF-1 allow tumour cells to survive?

Answer 79

• 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

Question 80

Describe 3 manners of visualising proteomics data and how these were used to visualise the HIF-1 regulome?

Answer 80

• 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

Question 81

How can the stimulome be studied?

Answer 81

• Omics approaches can also be used to study how cells, tissues and organisms respond to a change in the external environment

Question 82

What is the stimulome required for and how does it behave?

Answer 82

• 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

Question 83

Describe the yeast stimulome and how it was found, and why the stimulome is such

Answer 83

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

Question 84

What is the magnitude of the stimuloe response dependent on?

Answer 84

• The greater the shock that is provided to the cell, the greater the magnitude of the genomic response

Question 85

Does the stimulome adapt to environmental conditions? What happens when it does so and what is this change regulated by?

Answer 85

• 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

Question 86

What is the role of peroxiredoxin?

Answer 86

• Peroxiredoxin: uses the free thiols on its cysteines as oxidant mops o Proteins most likely mopping free radicals

Question 87

Does a single gene give rise to only a single protein? Why/why not?

Answer 87

• 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

Question 88

What are two different proteins that can result in the Nox1 gene and how? What are the purposes of each of these variants?

Answer 88

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

Question 89

What are post-translational modifications?

Answer 89

• PTMs are additions or subtractions to translated proteins that can alter the chemical structure of a protein and therefore modify its function

Question 90

What is the function/role of post-translational modifications?

Answer 90

• 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

Question 91

What is the purpose of protein-protein interactions?

Answer 91

 Protein-protein interactions | • Many proteins need to interact with other proteins, ligands, nucleic acids to function

Question 92

What occurs when proteins interact with each other?

Answer 92

• 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

Question 93

What are protein tertiary structure and protein-protein interactions determined by?

Answer 93

• 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

Question 94

What protein-protein interactions are influenced by regulatory PTMs

Answer 94

```  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 ```

Question 95

What are inducible protein-protein interactions influenced by regulatory PTMs?

Answer 95

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

Question 96

What are cooperative protein-protein interactions influenced by regulatory PTMs?

Answer 96

o Multiple domains bind in an obligatory fashion to two phosphorylated residues (both must be modified to proceed)

Question 97

What are multi-site switches protein-protein interactions influenced by regulatory PTMs?

Answer 97

o Phosphorylation required on several sites before interaction can occur

Question 98

What are sequential protein-protein interactions influenced by regulatory PTMs?

Answer 98

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).

Question 99

What are mutually exclusive protein-protein interactions influenced by regulatory PTMs?

Answer 99

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

Question 100

What are antagonist protein-protein interactions influenced by regulatory PTMs?

Answer 100

o Modification by phosphorylation allows binding as per 1 , however this inhibits modification at a second site proximal to the first.

Question 101

What are intramolecular regulation protein-protein interactions influenced by regulatory PTMs?

Answer 101

o Autoinhibition by self-modification and interaction between domains within the protein

Question 102

What are convergent protein-protein interactions influenced by regulatory PTMs?

Answer 102

o A single PTM can be recognized by different types of protein interaction domain.

Question 103

What are the two main ways in which PTMs are produced?

Answer 103

• Enzymatic vs non-enzymatic PTM

Question 104

What are enzymatic PTMS? What PTMs are enzymatic? Describe.

Answer 104

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

Question 105

What PTMs are non-enzymatic? Describe

Answer 105

o Non-enzymatic  Oxidation of proteins in the presence of reactive oxygen species (generally Cys [Cys-SOH, SO2H, SO3H], sometimes Met)  Deamidation

Question 106

What are the major types of PTMs?

Answer 106

``` o Phosphorylation o Acetylation o Glycosylation o Redox (redox biology and protein oxidation) o Methylation o Deamidation o Proteolysis o Ubiquitination (Ubq) ```

Question 107

Describe the phosphorylation PTM in terms of: - Definition - Role - Enrichment methods - Impact on the protein

Answer 107

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

Question 108

Describe the acetylation PTM in terms of: - Definition - Types - -----Definition - ----Role - ----Enrichment methods

Answer 108

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

Question 109

Describe the glycosylation PTM in terms of: - Definition - Abundance - Role - Enrichment methods - Impact on the protein - Study methods - Motif

Answer 109

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

Question 110

Describe the methylation PTM in terms of: - Definition - Role - Enrichment methods

Answer 110

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

Question 111

Describe the deamidation PTM in terms of: - Definition - Role - Impact on the protein

Answer 111

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

Question 112

Describe the proteolysis PTM in terms of: - Definition - Role - Enrichment methods - Impact on the protein

Answer 112

``` 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 ```

Question 113

Describe the ubiquitin PTM in terms of: - Definition - Role - Enrichment methods

Answer 113

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

Question 114

Describe the redox PTM in terms of: - Definition - Role - Enrichment methods

Answer 114

``` 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 ```

Question 115

How is the diversity of protein modifications studied?

Answer 115

• 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

Question 116

How are PTMs characterised? Describe the principle behind this method and any adjustments sometimes required.

Answer 116

• 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

Question 117

What are different modes of PTM crosstalk? Describe them

Answer 117

• 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)

Question 118

Describe how acetyl-K can induce structural flexibility allowing phosphatase activity with the kinase consensus motif KXXS

Answer 118

• 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).

Question 119

What is signalling and what does it involve?

Answer 119

• 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)

Question 120

What are the two main pathways through which signalling can occur?

Answer 120

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

Question 121

What extracellular signals/stimuli do cells respond to?

Answer 121

• Cells respond to extracellular signals/stimuli that activate membrane-associated or cytosolic receptors

Question 122

What receptor categories do membrane receptors include?

Answer 122

o Membrane receptors include G-Protein coupled receptors (GPCRs) and Receptor Tyrosine Kinases (RTKs)

Question 123

About how many G protein coupled receptors are encoded in the genome?

Answer 123

 About 800-900 G protein coupled receptors

Question 124

About how many receptor tyrosine kinases are encoded in the genome?

Answer 124

 About 60-70 Receptor Tyrosine Kinases

Question 125

What are the possible roles of activated receptors?

Answer 125

• Activated receptors can act as direct transcription factors (cytosolic receptors) or regulate downstream pathways via protein phosphorylation and/or intracellular second messengers

Question 126

What does protein phosphorylation/dephosphorylation regulate?

Answer 126

• 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

Question 127

What amino acids do protein kinases target in eukaryotes?

Answer 127

• In eukaryotes, protein kinases target serine, threonine and tyrosine residues

Question 128

Describe what types of receptors an external signal can interact with

Answer 128

1. G protein-coupled receptor 2. Receptor enzyme (tyrosine kinase) 3. Gated ion channel 4. Nuclear receptor

Question 129

How do G-protein coupled receptors work?

Answer 129

1. 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

Question 130

How do receptor tyrosine kinases work?

Answer 130

2. Receptor enzyme (tyrosine kinase)- ligand binding activates tyrosine kinase activity by autophosphorylation o Kinase activates transcription factor (T) altering gene expression

Question 131

How do gated ion channels work?

Answer 131

3. Gated ion channel- Channel opens or closes in response in response to concentration of signal or membrane potential

Question 132

How do nuclear receptors work?

Answer 132

4. Nuclear receptor- Hormone binding allows the receptor to regulate the expression of specific genes

Question 133

How do bacterial two-component regulatory systems work?

Answer 133

• 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

Question 134

What are the components of the bacterial 2 component regulatory system?

Answer 134

• The 2 components are a SENSOR histidine kinase (HK) and a response regulator (RR)

Question 135

Describe the structure of the histidine kinase in the bacterial 2 component regulatory system

Answer 135

o HK is located in the membrane- has external sensor domain, and part of the protein is located in the plasma membrane and cytoplasm

Question 136

Describe the structure of the response regulator in the bacterial 2 component regulatory system and the role of these structures

Answer 136

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

Question 137

What is the purpose of the PhoP-PhoQ system and how does it work?

Answer 137

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)

Question 138

What is a protein kinase

Answer 138

• Protein kinase- protein/enzyme that phosphorylates another protein or itself

Question 139

Describe how a protein kinase works

Answer 139

o When it adds the phosphate group, it uses the phosphate from ATP  ATP dependent process

Question 140

Describe how a protein phosphatase works

Answer 140

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

Question 141

What can activated kinases do?

Answer 141

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

Question 142

Where do activated kinases phosphorylate?

Answer 142

o Phosphorylate at a particular sequence motif

Question 143

Are kinases very specific to a particular site or are they more promiscuous? Discuss

Answer 143

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])

Question 144

Is phosphorylation only stimulatory?

Answer 144

o Phosphorylation can be stimulatory and inhibitory

Question 145

Do phosphorylation effects only affect a single protein?

Answer 145

o Phosphorylation can have cascade effects

Question 146

Describe the time frame of phosphorylation signalling

Answer 146

o Signaling via phosphorylation allows a rapid response in a time-frame shorter than possible via gene expression alone

Question 147

Describe the cell response to phosphorylation

Answer 147

o Cell response is specific and amplified via multiple pathways

Question 148

Describe the architecture of histidine kinases

Answer 148

```  Sensing domain  Conserved transmembrane helices (2,3, or 4)  Linker domain  catalytic domain  Histidine phosphorylation domain • It is autophosphorylated ```

Question 149

What does the architecture of histidine kinases allow?

Answer 149

o Allows signal to be transferred from outside to inside the cell

Question 150

What parts of the histidine kinases are highly conserved?

Answer 150

o Parts of HK are highly conserved (the TMR, His phosphorylation (Dhp[dimerization and His phosphotransfer) and ATP-binding domains)

Question 151

What parts of the histidine kinases are variable?

Answer 151

o Others are variable (e.g. sensor domain- specific for the ligand: signal transducing domain-specific for second messengers and activities)

Question 152

What are G protein coupled receptors?

Answer 152

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)

Question 153

What is the role of G protein coupled receptors?

Answer 153

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

Question 154

What are enzymes regulated by cAMP dependent phosphorylation

Answer 154

- 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

Question 155

What sequence is phosphorylated by glycogen synthase and what pathway/process is regulated?

Answer 155

RASCTSSS | Glycogen synthesis

Question 156

What sequence is phosphorylated by Phosphorylase b kinase alpha subunit beta subunit and what pathway/process is regulated?

Answer 156

Alpha subunit: VEFRRLSI Beta subunit: RTKRSGSV Glycogen breakdown

Question 157

What sequence is phosphorylated by pyruvate kinase and what pathway/process is regulated?

Answer 157

GVLRRASVAL Glycolysis

Question 158

What sequence is phosphorylated by pyruvate dehydrogenase complex and what pathway/process is regulated?

Answer 158

GYLRRASV | Pyruvate to acetyl-CoA

Question 159

What sequence is phosphorylated by hormone sensitive lipase and what pathway/process is regulated?

Answer 159

PMRRSV | Triacylglycerol mobilisation and fatty acid oxidation

Question 160

What sequence is phosphorylated by Phosphofructokinase-2/fructose 2,6- bisphosphatase and what pathway/process is regulated?

Answer 160

LQRRRGSSIPQ Glycolysis/gluconeogenesis

Question 161

What sequence is phosphorylated by tyrosine hydroxylase and what pathway/process is regulated?

Answer 161

FIGRRQSL | Synthesis of L-dopa, dopamine, norepinephrine, and epinephrine

Question 162

What sequence is phosphorylated by histone H1 and what pathway/process is regulated?

Answer 162

AKRKASGPPVS | DNA condensation

Question 163

What sequence is phosphorylated by histone H2B and what pathway/process is regulated?

Answer 163

KKAKASRKESYSVYVYK DNA condensation

Question 164

What sequence is phosphorylated by cardiac phospholamban and what pathway/process is regulated?

Answer 164

AIRRAST | Intracellular [Ca2+]

Question 165

What sequence is phosphorylated by protein phosphatase-1 inhibitor-1 and what pathway/process is regulated?

Answer 165

IRRRRPTP | Protein dephosphorylation

Question 166

What sequence is phosphorylated by PKA consensus sequence and what pathway/process is regulated?

Answer 166

xR[RK]x[ST]B | Many

Question 167

How do phosphoprotein phosphatases work?

Answer 167

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

Question 168

Why are phosphoprotein phosphatases more promiscuous than kinases?

Answer 168

o More promiscuous than kinases as less of them coded in the genome

Question 169

What are receptor tyrosine kinases?

Answer 169

• 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

Question 170

Describe the structure of receptor tyrosine kinases

Answer 170

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

Question 171

What are the general mechanisms of receptor tyrosine kinase structure and activation?

Answer 171

 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

Question 172

What type of signalling is the fight or flight response mediated by?

Answer 172

• The flight or fight response is mediated by GPCR signalling

Question 173

What is adrenaline made by and why?

Answer 173

o Mediates stress response: mobilization of energy-generating machinery (in e.g muscle) in response to stress

Question 174

What effects does adrenaline binding to GPCRs produce in different locations?

Answer 174

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

Question 175

What is the name of the G-protein, effect enzyme and second messenger in the adrenaline signalling pathway?

Answer 175

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

Question 176

Describe the process of epinephrine-induced beta-adrenergic signalling

Answer 176

• 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

Question 177

How does PKA transmit the adrenergic signal?

Answer 177

• PKA transmits the adrenergic signal via kinase/phosphatase-type phosphorylation

Question 178

Describe the properties of Ser/Thr and Tyr phosphorylation in terms of: - Speed - Coordination - Permanence - Strength - Role

Answer 178

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

Question 179

What is the consequence of production of cAMP in beta-adrenergic signalling?

Answer 179

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

Question 180

How does PKA induce gene transcription in beta-adrenergic signalling? Outline

Answer 180

• 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

Question 181

What is the consequence of increased cAMP on glycogen synthesis and glycogen breakdown and why?

Answer 181

• 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

Question 182

What is the consequence of decreased cAMP on glycogen synthesis and glycogen breakdown and why?

Answer 182

• 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

Question 183

What is the structure of insulin, what is its purpose and how is it produced?

Answer 183

• 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

Question 184

Does insulin signalling affect only one kinase pathhway?

Answer 184

• In insulin signaling, several kinase pathways are activated (e.g. MAPK/AKT)

Question 185

What initiates insulin signalling and what is the consequence of this?

Answer 185

o Binding of insulin to the insulin receptor (INSR) initiates signal transduction pathways (insulin signalling) that leads to increased glucose uptake and metabolism

Question 186

What causes diabetes?

Answer 186

o Inability to make (type I diabetics or sense insulin (type II diabetes)-> diabetes

Question 187

How does activated insulin signalling lead to GLUT4 translocation to the cell surface/movement?

Answer 187

• 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

Question 188

What is the role of INSR in Ras/Raf and MAPK signalling?

Answer 188

 INSR phosphorylates insulin receptor substrate-1 (IRS-1) which initiates a series of protein phosphorylations via binding to Grb2, Sos and Ras

Question 189

What is the role of Ras in Ras/Raf and MAPK signalling?

Answer 189

 Ras GTP-binding activates Ras, which in turn activates Raf and initiates mitogen-activated protein kinase (MAPK) signalling (MEK)

Question 190

What is the role of ERK in Ras/Raf and MAPK signalling?

Answer 190

 Phosphorylated extracellular signal-regulated kinase (ERK; a MAPK) enters the nucleus

Question 191

What is the role of Elk1 in Ras/Raf and MAPK signalling?

Answer 191

 A transcription factor (Elk1) becomes phosphorylated and stimulates the expression of specific genes (e.g. glucose transporters)

Question 192

What is the process of insulin signalling Ras/Raf and MAPK signalling

Answer 192

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

Question 193

What is the role of AKT phosphorylations in insulin signalling/what does AKT effect? What do they do (downstream applications)

Answer 193

• 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

Question 194

What is the advantage of large scale phosphoproteomics in studying insulin signalling?

Answer 194

• 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

Question 195

What are 4 ways in which 2-DE gels can be used to analyse phosphoproteins/peptides

Answer 195

1. 2-D Electrophoresis 2. 2-DE combined with stains (PO4-specific) 3. 2-DE combined with electroblotting and anti-phosphoantibodies 4. Affinity chromatography followed by 2-DE

Question 196

Why can phosphate modifications be seen on normal 2-DE electrophoresis

Answer 196

1. 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

Question 197

How can stains be combined with 2-DE to detect phosphate modification, give an example and a limitation

Answer 197

2. 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) 1. 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

Question 198

How can 2-DE be combined with anti-phosphoantibodies to visualise phosphate modifications? Do all of these anti-phosphoantibodies work equally?

Answer 198

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

Question 199

How can characterisation of phosphoproteins/phosphopeptides by mass spectrometry occur?

Answer 199

• 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

Question 200

What are the possible steps of characterization of phosphoproteins/phosphopeptides by mass spectrometry

Answer 200

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

Question 201

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?

Answer 201

 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

Question 202

Describe phosphopeptides behaviour in MALDI-MS and why

Answer 202

• 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

Question 203

How can comparative MALDI-MS be used to characterize phosphoproteins/ phosphopeptides by mass spectrometry?

Answer 203

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

Question 204

Describe how enzymatic treatment can be used to characterize phosphoproteins/ phosphopeptides by mass spectrometry? What phosphatases are used to develop such treatment?

Answer 204

 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

Question 205

Describe the process of how enzymatic treatment can be used to characterize phosphoproteins/ phosphopeptides by mass spectrometry?

Answer 205

* 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

Question 206

How specific is affinity enrichment+ MALDI-MS in characterizing phosphoproteins/ phosphopeptides by mass spectrometry?

Answer 206

 Specific analysis of phosphopeptides alone- highly selective

Question 207

What are three chromatographic steps prior to MS that can be performed when characterizing phosphoproteins/ phosphopeptides by mass spectrometry?

Answer 207

* Anti-phospho antibodies * Immobilized metal affinity (IMAC) * Titanium dioxide (TiOx)- method of choice for large scale proteomics

Question 208

Describe how immobilised metal affinity (IMAC) works in selecting for phosphopeptides only, its limitations and its possible adjustments

Answer 208

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

Question 209

How is IMAC performed for phospho-proteins

Answer 209

 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

Question 210

How is IMAC performed for phospho-peptides

Answer 210

```  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 ```

Question 211

How does a titanium dioxide column select for phosphopeptides? What are its advantages?

Answer 211

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

Question 212

What is the advantage and disadvantage of using affinity enrichment + MALDI-MS to characterize phosphoproteins/ phosphopeptides by mass spectrometry?

Answer 212

 False positives and false negatives |  Large-scale analysis by MS/MS

Question 213

How are phosphopeptides purified using titanium oxide

Answer 213

 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

Question 214

Describe the process of titanium dioxide micro-purification

Answer 214

 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

Question 215

Why can phosphopeptides cause difficulties in MS/MS

Answer 215

 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

Question 216

What happens when phosphopeptides undergo MS/MS? What is the consequence of this?

Answer 216

 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

Question 217

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?

Answer 217

 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

Question 218

What enrichment/purification techniques are available to isolate modified peptides/proteins?

Answer 218

• Enrichment/purification techniques available to isolate modified peptides/proteins o Hydrophilic interaction liquidd chromotography o Titanium dioxide o Immobilised metal affinity chromatography

Question 219

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

Answer 219

• 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

Question 220

Describe the optimised phosphoproteomics workflow

Answer 220

• 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