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BCMB3904 > Protein analysis > Flashcards

Protein analysis Flashcards

1
Q

Why is it important to study protein-protein interactions?

A

• Reason for studying protein-protein interactions
o Many proteins need to interact with other proteins, ligands, nucleic acids to function
o The regulation of cell function is balanced by the relative affinities of the various protein partners contained within such complexes
o Where two or more proteins interact with each other they may temporarily or permanently 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

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2
Q

How do proteins interact?

A

o How do proteins interact-
 PTMs and charge/charge interactions
 Covalent interactions e.g. intermolecular disulfide bonds
 Non-covalent interactions

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3
Q

What is the best known protein complex?

A

o The best known protein complex is the ribosome

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4
Q

How many proteins are ribosomes made of?

A

 Comprises of more than 50 proteins, some which provide structural support and others which undertake the function

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5
Q

What is the E.Coli 70S ribosomal subunit made of and what is its function?

A

 The E.coli 70S ribosomal subunit
• 29 individual proteins make up the 70S ribosomal subunit in E.coli
• Proteins may be involved in binding rRNA, providing structural integrity, or in translation processes
o Some proteins are physically separated from each other/don’t physically interact whilst some do- binary level of interactions

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6
Q

What is the largest protein complex in S.cerevisiae?

A

The ribosome

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7
Q

What is the mitochondrial protein complex?

A

o The mitochondrial NADH complex
 45 individual proteins make up the complex I of the electron transport chain
• Not all proteins interact with each other
 Proteins may be involved in complex assembly, providing structural integrity, with 7 encoded by mitochondrial DNA

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8
Q

On what chemical basis do proteins interact with each other?

A

• Protein-protein interactions-functional implications

o Proteins interact based on charge-charge basis, dissulfide bonds, structural folds, etc.

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9
Q

What are the biological implications of protein-protein interactions/what do they form?

A

o Receptors
o Interactions form chains as well as complexes
o Chaperones

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10
Q

What are chaperones and are they specific in their interactions?

A

o Chaperones- aid in maintaining correct folding, chaperon proteins to correct sub-cellular location
 Tend to be promiscuous in terms of interactions
o Expect very main interactions

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11
Q

Describe GroEL in E.Coli:

  • What it is
  • What interactions it makes
  • What information can be inferred from it and how this information can be obtained
A

 E.g. GroEL in E.Coli
• GroEL is a chaperone-proteins that assist other proteins to fold-expect a lot of interactions
• Numerous diverse interactions, especially during stress
o Amino acid biosynthesis
o RNA polymerase
o Heat shock proteins
• PPI can provide information regarding protein function
• Pulls down more proteins when there’s been stress
o Stress causes proteins to unfold
• Looked at this using immunoprecipitation

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12
Q

What are methods for looking at protein-protein interactions/complexes? What do they specifically look at?

A 
•	Methods for looking at protein-protein interactions/complexes
o	Yeast-2-hybrid (interactions)
o	Immunoprecipitation (IP)/affinity chromatography (interactions and/or complexes)
o	Native PAGE (BN-PAGE) (complexes)
o	Chemical cross-linking (interactions)
o	Tandem Affinity Purification (TAP) (interactions and/or complexes)
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13
Q

What is the construction and process of yeast-2-hybrid to study protein-protein interactions?

A

o Yeast-2-hybrid (interactions)
 Uses a report gene (e.g. gal1-lacZ- beta galactosidase) that is transcriptionally activated when two proteins interact
• Bait protein and prey protein
 The transcriptional activator has 2 domains:
• A DNA-binding domain
• An activating domain that activates reporter gene
 Find proteins (prey) that interact with a single bait on a genome-wide bases- binary interactions
• Limitation- a lot of work
 Bait is fused to the DNA-binding domain; preys fuse to the activating domain
 Activity is achieved when bait and prey interact- reporter gene is then transcribed

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14
Q

What are the advantages of yeast-2-hybrid?

A
  • Rapid, high throughput

- Can test many 1:1 interactions- good first pass

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15
Q

What are the disadvantages of yeast-2-hybrids?

A
  • False positives if the bait is a transcriptional activator as everything gets switched on
  • The fusion proteins may not have in vivo conformation and therefore not bind with in vivo specificity
  • Bait and prey may not fold correctly in yeast
  • Fusions may be toxic to yeast host
  • Weak interactions are amplified- hence increased number of false positives
  • Gives no information about bigger complexes
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16
Q

What is the most commmonly used method to look at interactions?

A

o Immunoprecipitation (IP)/affinity chromatography (interactions and/or complexes)

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17
Q

How is immunoprecipitation/affinity chromatography used to study protein-protein interactions and/or complexes/ how does it work?

A

 Uses an antibody against a protein-of-interest under non-denaturing/native conditions
 Antibody binds the protein and any interacting partners as a complex (that is X binds to Y binds to X: XYZ will be captured)
 Requires native and gentle conditions
 The complex is then captured and subjected to SDS-PAGE or directly digested with trypsin and subjected to LC-MS/MS
 Proteins within a complex, but not absolute interactions
• Not getting binary interactions

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18
Q

What is the immunoprecipitation workflow?

A

 Immunoprecipitation- workflow
• Make antibody against protein-> do immune precipitation-> do a proteolytic digest or SDS-PAGE
o SDS-PAGE-> excise band-> digest-> MS/MS and identify
o Proteolytic digest-> LC-> MS/MS and identify

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19
Q

What is a disadvantage of immunoprecipitation?

A

 Disadvantage-
• Non-specific binding to immunoglobin chain (will be pulled down because bound to immunoglobin instead of because bound to protein)-> lots of false positives

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20
Q

What is a variation of immunoprecipitation that overcomes its disadvantages of false positives?

A

 Variation- no antibodies/use the protein-of-interest (affinity chromatography)

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21
Q

How is immunoprecipitation performed when the protein-of-interest is used instead of antibodies? How is it designed? What is the disadvantage of this?

A

• The protein of interest (in this case S14) is expressed as a fusion with a cleavable affinity tag
• S14 protein is immobilised onto agarose beads via a tag. Nuclear cell extracts are incubated with the beads and washed. Tag-specific proteases are used to cleave the tag, which results in elution of all proteins that are specifically bound to S14. The eluted proteins are resolved by 1D or 2DE. The bands are excised and analysed by MS
o The advantage is that proteins non-specifically bound to the matrix or the tag are not eluted

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22
Q

What are the types of tags that can be used with immunoprecipitation and what are their respective proteases?

A

o Types of tags and appropriate proteases
 FLAG-tag- can be removed with enteropeptidase
 His-tag- can be removed with endo/exopeptidase
 GST-fusion- can be removed with thrombin

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23
Q

How can false positives be reduced with iDIRT/how does this immunoprecipitation technique work?

A

 Reducing false positives- iDIRT
• 1:1 mix of light (tagged-protein) and heavy (wild-type) sample-> immunoisolation-> mass spectrometry
• If the peak is seen in both the light and heavy sample, means that it binds to the affinity support or tag, it will be seen in both the light and heavy samples (non-specific binding)

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24
Q

How does native PAGE work for analyses of protein complexes? What is it used for?

A

 Non-reducing PAGE (non-denaturing)
 Proteins are retained in complex
• Can resolve complexes spanning 15kDa to approximately 10 mDa
• Will migrate through the gel based on complex size, shape and charge
 Coomassie G-250 acts as a charge shift molecule
• Imparts a negative charge that alters electrophoretic mobility
• Contained within cathode buffer and sample loading buffer
• Blue native PAGE
 Can perform a secondary SDS-PAGE or directly digest complexes with trypsin and subject to LC-MS/MS
• Would take blue native PAGE gel ->then separate on a secondary SDS-PAGE
 Resolves proteins within a complex, but not absolute interactions
 Workflow- 2-dimensional BN/SDS-PAGE
• Preparation of total cellular lysate-> dialysis of the lysate against BN-bugger-> 1st dimension BN-PAGE-> 2nd dimension SDS-PAGE-> Immuno-blotting or silver staining

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25
Q

What are the uses/advantages of Native PAGE to study protein complexes?

A

o Complexes retain activity
 Facilitates experiments for catalytic activity
o Can use the second dimension as denaturing to identify individual components within the complex

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26
Q

In what conditions can chemical cross-linking be performed?

A

o Chemical cross-linking (interactions)
 Chemical cross-linking and mass spectrometry: to identify interactions at a very global scale
• In vitro or in vivo

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27
Q

What are chemical cross-linkers and what is their purpose?

A

• Two reactive groups that are covalently connected by a spacer arm
o Cross-linkers stabilise close interactions
o Chemical cross-linking: chemically joining two or more functional groups of adjacent proteins by a covalent bond
• Covalently links residues that are in close spatial proximity
o Uses chemical that can maintain interaction between two residues by binding to these residues
o Use:
 Primary amines
 Sulfhydryls

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28
Q

In chemical cross-linking, what is the limiting factor for the distance between proximal residues?

A

• The spacer arm length is the limiting factor for the distance between proximal residues

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29
Q

What is the relationship, in chemical cross-linking, between spacer arm length and interaction specificity?

A

o The bigger the spacer arm, the less specific the interaction must be
o The smaller the spacer arm, the more specific the interaction must be

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30
Q

What are the two functional types of cross linkers?

A

• Cross-linkers can be either homobiofunctional or heterobiofunctional

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31
Q

What are homobiofunctional cross linkers?

A

o Homobiofunctional- if they both bind to the same type of site

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32
Q

What are heterobiofunctional cross linkers?

A

o Heterobiofunctional-if they bind to different types of sites

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33
Q

What are examples of MS cleavable cross linkers and what are they? What is their purpose and how are they made?

A

• MS cleavable cross-linkers are available: cross-linker MS (XL-MS)
o Disuccinimidyl sulfoxide (DSSO)
o MS cleavable cross-linkers-cross linkers that when you do tandem mass spectrometry and add fragmentation energy will cleave and leave behind diagnostic
o MS/MS cleavable cross-linkers contain gas-phase cleavable sites in the linker arm:
 MS2 cleavage of cross-linker
• Use MS2 to look for loss of distinctive mass (cleaved cross-linker)
 MS3 identification of peptide
• From the MS2 parent ion that had the loss of the cross-linker
• Process take (MS) time

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34
Q

What are MS cleavable cross-linkers problems?

A

cleavable cross-linkers problems:
 Increased peptide m/z when two peptides are cross-linked
 Increased complexity of MS/MS
 Increased search space to identify two peptides crossed linked (n-square problem)

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35
Q

What are the applications of XL-MS?

A

o XL-MS has many applications in determining protein interactions:
 Protein-complex topology
 De novo modelling with HD-CLMS
 Conformational change by QCLMS
 Native-protein topologies by in situ CLMS

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36
Q

How can XL-MS be applied for protein complex topology?

A

 Protein-complex topology: known complex with unknown topology-> distance restraints to map protein topology-> cross links guide modelling of protein-complex topology (integration of X-ray structures, SAXS data and electron microscopy data)
• Can validate molecular models with cross-linking

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37
Q

How can XL-MS be applied for de novo modelling with HD-CLMS?

A

 De novo modelling with HD-CLMS: protein with unknown tertiary structure-> high density of distance constraints with promiscuous cross-linker-> cross-links guide tertiary structure modelling

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38
Q

How can XL-MS be applied for conformational change by QCLMS?

A

 Conformational change by QCLMS: proteins/complexes with unknown conformations-> comparison of abundance of cross-linked residue pairs-> cross-links guide modelling of conformational changes

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39
Q

How can XL-MS be applied for native protein topologies by in situ CLMS?

A

 Native-protein topologies by in situ CLMS: proteins/complexes with unknown native conformations and topologies-> CLMS-based protein interaction network-> cross-links are used to assign topology and conformation of native complexes including transient binders

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40
Q

What is the workflow for chemical cross-linking?

A

o Select a crosslinker-> react that cross-linker with proteins in the native (in vitro/in vivo) state (XLs form within proteins [tertiary structure] and between proteins)-> Trypsin digest-> (optional) enrichment of cross-linked peptides-> MS analysis (which is complex)

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41
Q

What are properties of a chemical cross-linkers that need to be considered for chemical cross-linking identification of protein-protein identifications?

A

 Properties of the cross-linker to be considered:
• Reactivity of the reactive group
• Spacer length
o Want it to be just right for the space between the two sites of interest
o Normally 10 armstrongs for good spacer length which can search up to 30 armstrong distance
• Enrichment
• Quantitation (isotope label)
• Cleavability in MS/MS

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42
Q

Describe the benefit of tandem affinity purifications for identification of protein-protein interactions and/or complexes?

A

 Reduces false positives arising from non-specific binding to affinity supports

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43
Q

Describe the design/components of tandem affinity purification for identification of protein-protein interactions and/or complexes

A

 Uses a two-step affinity purification
 Uses a molecular fusion of the protein-of-interest with a TEV (Tobacco Etch Virus) protease site flanked by calmodulin-binding peptide (CBP) and protein A
• Protein of interest, protease site, calmodulin-binding peptide and protein A all fused to each other
• Protein A binds to IgG coated beads
• TEV protease added to remove complex
• CBP binds to calmodulin for purification

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44
Q

Describe the workflow of tandem affinity purification for identification of protein-protein interactions and/or complexes

A

 Workflow:
 Uses a two-step affinity purification
• Target protein synthesised and fused to calmodulin binding peptide, have protease cleavage site and protein A that enables binding to IgG beads
• Two rounds of purification-
o First round of purification
 Protein A binds to IgG
 Add TEV protease and it cleaves region so that anything that does not bind to the IgG beads are left behind
o Second round of purification
 Calmodulin binding peptide binds to calmodulin beads, elute with EDTA and have proteins
o Use of other members of a complex acts as a control
 Using, as the protein of interest, different components of the originally identified protein complex
 Each time, should see same banding pattern/same result as targeting other proteins in the protein complex

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45
Q

What are the requirements of diagnostics?

A 
o	Diagnostics need to be:
	Rapid 
	Minimally invasive (not biopsy)
	Found at a stage where therapy is possible (early)
•	Biggest current problem for research
	Reliable (specific)
46
Q

What tissues are the most examined for biomarkers?

A

o Proteins released into blood from injured tissues may represent markers of a particular disease
o Whilst plasma/serum most commonly examined other body fluids (CSF, urine, tears etc.) may also carry biomarkers

47
Q

What is serum and what is the process of serum production?

A

o Serum is the protein solution remaining after plasma (or whole blood) is allowed to clot
 Both serum and plasma are what remains through removal of red blood cells
 Prothrombin is cleaved to thrombin, fibrinogen is removed (to form the clot) and other protein changes (proteolytic cleavages) occur

48
Q

Is research using plasma instead of serum more or less accurate to real life clinical blood tests? Why?

A

o Most of the time, the tests that are actually performed are performed in serum, BUT most of the research is being performed on plasma
 The test therefore needs to be valid in both serum and plasma

49
Q

Why is studying serum technically challenging? Describe

A

o But technically challenging- dynamic range of abundances
 60% of the total protein is made up of serum albumin, and about 80% is comprised of 6 proteins: immune-globulin chains, albumin, serotransferrin and a couple of others
• Hard to find low abundance tissue proteins
o Markers released in very low amounts compared to blood proteins like serum albumin (55% of total protein in plasma)

50
Q

What is a way of studying low abundance proteins in serum, and what is a consideration that must be taken account of when doing so?

A

• To fix this problem, can remove albumin and other abundant proteins-
o Anti-albumin antibodies- remember albumin also complexes some (possibly important) proteins
 Can buy anti-protein antibodies (but quite expensive)
o 3rd dimension- prior fractionation using liquid chromatography
o Isoelectric focusing prior to 2-DE
o Gel filtration
• The problem with removing abundant proteins such as albumin:
o Albumin has some chaperone activity
o Chaperones are some of the most abundant proteins
o By removing chaperones and, by some extent, albumin, completely, would be removing the low-abundant and possibly important proteins that interact with it
• Hence, not only need to remove albumin and high abundance proteins, but DO NOT THROW THEM AWAY, look at what binds to them too

51
Q

What is the albuminome?

A

o Albuminome- what binds to albumin

52
Q

What is C-reactive protein a marker of? Describe

A

• C-reactive protein as a marker of myocardial infarction
o CRP alone is not necessarily specific-it is an acute phase reactant and a marker of inflammation
 They can be detected following a cold
o However, elevated plasma CRP combined with other factors (e.g. elevated LDL cholesterol) is an excellent marker of the risk of CVD

53
Q

Is a single marker enough for indication of a disease?

A

• One single marker SHOULD NOT be indicative of a disease
o Groups of markers should be used to diagnose disease, never just a single marker
 Multiple tests should also be used

54
Q

What is a SELDI-TOF MS and how is it different from MALDI-TOF?

A

• Surface-enhanced laser desorption-TOF MS (SELDI-TOF MS)
• Principle is the same as MALDI-TOF, but the difference is that the metal targets have a surface chemistry that can either bind or not bind proteins and peptides
o Just take blood and put it on SELDI-TOF MS

55
Q

How does SELDI-TOF MS work and what are its components?

A

• Uses chips/targets with different chemistries
o The array/chips have different surface chemistries
 Could be HILIC, Reverse phase, SCX, antibody…
o Sample is applied and only a subset will bind (based on hydrophobicity, affinity etc.) rest are washed away
o Bound sample acidified (allowing ionization), and subjected to MS
o Small ions travel faster (MS)-m/z ratio
o Better MS better resolution= number of ions
o Low resolution MS leads to coalescing peaks
• Diagnostic profiling using SELDI-TOF MS
o Sample (multiple)-> Wash Remove unbound proteins, salt and other contaminants)-> EAM (Matrix: sinupynic acid-> SELDI Reader-> Raw spectra-> Grey scale view of pseudo 2D gel with the most abundant peaks shown as darker bands on the gel

56
Q

What are the potential properties of SELDI-TOF chip arrays?

A 
	Select Chip Array
•	Hydrophobic
•	Anionic
•	Cationic
•	Metal Binding 
•	Antibody
57
Q

What are the potential SELDI-TOF matrices and what are they for?

A

 Matrices-
• Sinapinic acid (for larger biomolecules)
• Alpha-cyano-4-hydroxy cinnamid acid

58
Q

Describe the story of the SELDI-TOF at hospitals, hopes, mistakes made and final outcome and the lesson that must be learned from this

A

• Cytogen would sell SELDI at hospitals who would use this technology to diagnose disease
o Wanted to keep the cost down-> decided to use low resolution mass spectrometer
o Would have big database- would be useful to compare found mass spectrum with mass spectrum of other patients
 Diagnosis would be based on pattern, not individual markers
o This seems like a great idea, but:
 In the original paper that had published this, it was discovered that they had fundamentally failed in experimental design
• They ran all the controls at a different time to running all the tests
o In clinical tests, need to randomise running of controls and tests to overcome technical changes in the instrument across time
 Some of the time changes they’d found was due to this time displacement
o Was difficult to get into hospitals- people did not want to change: high resistance
o The company started to become bankrupt, and sold SELDI-TOF as a research tool
 Although as they tried to sell it as a research tool, due to their use of low resolution, they couldn’t sell it as a research tool
o BioRad bought cytogeny and still couldn’t sell them, so it is not sold anymore
o However, it is still important to acknowledge that pattern-based diagnosis is extremely powerful

59
Q

What are the advantages of SELDI-TOF MS?

A

• SELDI-TOF MS:
o Advantages-
 Rapid, not antibody-based: pattern-based
• Patterns are extremely specific- very powerful
o Very interesting
• Based on patterns rather than single markers
• Multiple markers better than a single marker (this makes sense- checks and balances)
 Does not even matter what the individual molecules are within the pattern
• No need for protein/peptide identification
• Not protein-based: markers may be the result of fragmentation of a larger protein, hence antibodies would not be specific
 Viable in serum/plasma and other body fluids
 Hardware expensive, but individual assays cheap
 Mass spectrometer can be used as a diagnostic device

60
Q

What are the disadvantages of SELDI-TOF MS?

A

o Disadvantages-
 Patient-to-patient variance always an issue-need great statistics and clinical trials
 Day-to-day, lot-to-lot, and machine-to-machine variances introduced by sample handling, storage, and shipping conditions needs to be evaluated, as well as the mass spectrometer itself

61
Q

How is mass spectral tissue profiling/imaging performed and what is its use?

A

• Tissue imaging and profiling provides a histological scale molecular map of disease
• Mass spectrometry of tissue sections for discovery of diagnostic biomarkers
• Thin tissue sections- imaged by microscopy
• 50-150um deposits of MALDI matrix deposited onto tissue section at short intervals (50um) or sprayed onto the surface (imaging)
• Laser fired across each deposit
• Spectra acquired and then processed using bioinformatics to create a tissue image of spectral signals within the tissue space
• Workflow-
o Slice (frozen tissue) on cryostat (about 12 um thick)-thaw slice onto MALDI plate-> apply matrix-> Acquire mass spectra

62
Q

What is the use and process of mass spectral tissue profiling?

A

• Mass spectral tissue profiling-
o Robotic spotting of matrix deposits at regular intervals
o Signals from each spot are processed by MS and stored by informatics
o Profiling- many samples (replicates) of several types, goal is to discover patterns in the profiles that can classify each sample based on biological state (e.g. tumour vs normal) and that can predict biological outcomes (e.g. the prognosis of a patient)
o Used in forensics
o Uses pattern rather than single markers for diagnosis, which is a lot better

63
Q

What is the process of mass spectral tissue imaging and what is its purpose/use?

A

• Mass spectral tissue imaging
o Take tissue section->apply the matrix-> MS surveys across backwards and forwards
o Get one mass spectrum-> shows you where different peaks are distributed in the tissue
o Performed on a small number of samples where the goal is to obtain a relatively highresolution imaging showing the distribution of various proteins in the tissue section. Mass spectra are acquired in a raster over the tissue surface
o Can get ion density map
o MALDI-TOF_TOF: can use first time of flight as a fragmentation chamber and then measure the fragments
o Very powerful technology for looking at the distribution of biomolecules within tissue
o Application of this technology- monitoring drug targets
 Look whether drug is going to drug target
o Can do whole animal cross-section mass spectral tissue

64
Q

What is a big use of CESTA and thermal proteomics?

A

• CETSA and thermal proteomics provide a pathway for linking drug libraries with protein targets

65
Q

What is the basis of thermal proteomics?

A

• Thermal proteomics (drug-protein interactions)

o Heating proteins causes them to denature and precipitate out of solution

66
Q

What is the basis of CETSA for drug discovery?

A

• Cellular thermal shift assay (CETSA) thermal proteomics for drug discovery
o Both CETSA and thermal proteomics work on the principle that proteins denature (unfold or melt) at a given temperature: however, interaction with a binding partner (small molecule drugs for example) stabilize the protein and increase the melting point of the protein
o Can be used to determine molecular targets of commercial drugs and to screen unknown compounds

67
Q

What is the workflow of CETSA and thermal proteomics for drug discovery? What needs to be done/components that need to be considered for this to work?

A

 Workflow: Preparation of cells (cell lysate AND intact cell)-> drug treatments and testing concentration range-> heating procedure-> extraction of soluble proteins-> protein digestion and protein labelling-> mass spectrometry-> data processing
 Determine melting curve with/without a drug
• Different proteins have different melting points-> need to test protein at temperature ranges to work it out
 Also test range of drug concentrations [increased drug= increased stability]
• See if there is limit to the binding
 Extracts and intact cells are used to ensure drug can reach intended target
• Intact cell-> informs us if drug is able to cross the membrane and find its target in a living system
 Large-scale proteomics enables melting curves to be simultaneously generated for about 10000 proteins
 A temperature vs drug concentration heat map (lower right) reveals proteins stabilized by drug at higher temperatures
 Real targets are shown by overlapping the living cells (where you might see flow-on effects) to lysate (where there might be non-specific binding) that are available to the drug because the cell has been lysed

68
Q

What are the disadvantages of using CETSA for drug discovery?

A

 Caveats/Disadvantage: some drugs destabilise proteins
• Drug might cause the protein to unfold and thus be targeted by protease
o Some drug will bind to their protein and cause it to be degraded

69
Q

What is staurosporine and its function?

A

• Staurosporine (St)is a cell permeable alkaloid exhibiting anti-cancer activity and is considered a potent, non-selective inhibitor of protein kinases
o Staurosporine is fundamentally inhibiting the ability of the co-factor to bind to the catalytic and regulatory subunits

70
Q

What is ampicillin and what does it do?

A

• Ampicillin as expected targets penicillin-binding proteins (sig. stabilization, especially DacB), but also impacts many other proteins, especially in translation and tRNA ligase activity

71
Q

What is ciprofloxacin and what does it do?

A

• Ciprofloxacin (DNA repair/SOS response) in E.coli GyrB is a known target. The degradation of LexA and stabilisation of RecA are known SOS responses and results in the upregulation of YebG

72
Q

What is the iKnife, who was it created by and what does it do?

A

o Rely on real-time acquisition of pattern-based spectral data through rapid evaporative ionization MS (Rapid Evaporative Ionisation Mass Spectrometry- REIMS) which is inhaled into the MS
 Don’t need matrix
o Created by Zoltan Takats [imperial college]
o The iKnife coupled to rapid evaporative ionization mass spectrometry [REMIS] can be used for real-time cancer diagnosis in clinical surgery

73
Q

What is the workflow of the iKnife?

A

o iKnife workflow:
 Electricity heats the tip of knife-> the hot blade causes cells in the tissue to explore, releasing molecules in the smoke-> the smoke is sucked up into a tube and fed into a very accurate molecular weighing scale (mass spectrometer)-> the mass spectrometer analyses the molecules and creates a fingerprint-> the fingerprint tells scientist the type of tissue being cut

74
Q

What is the use of the MassSpecPen, who was it created by and how does it work?

A

• MassSpecPen
o Rely on real-time acquisition of pattern-based spectral data through deposits of water droplets that are then taken into the instrument via suction
o Workflow
 Tip and surgical instrument go together-> 3 vessels at the end of the mass spec pen send water on tissue surface as the tissue is being cut-> water makes droplet that picks up molecules from tissue-> gas coming down from central pipe puts the molecules, ionises molecules into gas phase-> go into mass spectrometer-> real time feedback of what tissue is being cut (due to pattern-based recognition
o Created by Livia Eberlin [UT]

75
Q

What is the use of both the iKnife and the MassSpecPen?

A

• The iKnife and MassSpecPen both enable real-time diagnosis during surgery
o Find where healthy tissue begins and where the cancer tissue ends-> enables surgeons to find margins between healthy and cancerous tissue and hence minimise the chance of recurrence in people

76
Q

Can the mass spectrometer be used as a diagnostic device?

A

• The mass spectrometer can hence be used as a diagnostic device with no need for molecule identification
o New era of biomarkers and pattern-based diagnosis
• Mass spectrometers now a valuable in clinic device

77
Q

How can proteomic data be validated?

A
  • Western blotting
  • RT-PCR
  • Immunohistochemistry
  • Selected reaction monitoring (SRM/MRM) validation
  • Metabolomics-validation
78
Q

What is the process of western blotting and its principle?

A

• Western blotting
o Based on the simple principle of antibody-antigen reactivity where your protein-of-interest is the antigen
o One assay per gel/per blot
o Commercial primary antibody, or make your own polyclonal/monoclonal
o Process-
 Proteins separated by SDS-PAGE or 2-DE
 Proteins electro-transferred from gel to membrane (typically nitrocellulose or polyvinylidene fluoride (PVDF))
 To reduce interactions between the membrane and the primary (or secondary) antibody (remember the membrane has a high affinity for all proteins), it is blocked using BSA or skim milk powder (protein-rich)- reduce false positives
 Incubation with primary antibody- the antibody specific for your protein-of-interest
 Incubation with secondary antibody normally ligated to a reporter region- the antibody specific for a region of the primary antibody. Secondary is also linked to a reporter (e.g. biotin, alkaline phosphatase or horseradish peroxidase). HRP cleaves a chemiluminescent agent to produce luminescence proportional to the amount of protein

79
Q

What is a loading control and what is the best loading control?

A

 Loading control-> to prove that more sample isn’t simply being loaded (no cheating)
• DJ-1 is best for a loading control

80
Q

What two test cohorts are needed in clinical experiments for biomarker identification? Why?

A

o Good to have a discovery cohort and a validation cohort
 Discovery cohort samples run with Tandem Mass Spec
• Expensive and takes a lot of time
 Validation cohort-after finding something with the discovery cohort, the validation cohort can be used to validate findings at much lower cost
• With less expensive method and less time

81
Q

How can PTMs be validated?

A

 Western blots
• Antibodies are available for global PTM analysis
 PTM vs protein abundance
• When using a PTM antibody, also use a total protein antibody to prove that there is no change in the abundance of the proteins

82
Q

What are the disadvantages of using western blots for PTM?

A

• Antibodies have varying degrees of reliability- possible false positives
o Cross-reactivity can occur: some of them are not very good
• If we use antibody detection, biologists can find only what they look for
o As a result, the cell is not seen as a whole system, so biologists tread common ground over and over again
o Proteomics can solve this problem, so proteomics can also be used as a validation tool
• But don’t have an antibody for every PTM: validating with what is already known, not validating anything new that data may have discovered

83
Q

What are the advantages of using Western blotting for protein validation?

A 
One-one specificity
Can examine individual variants
Multiple parallel assays
Visual tool-data looks compelling
Comfort zone of many biologists
84
Q

What are the disadvantages of using Western blotting for protein validation?

A

Only proteins for which antibodies are available ($$$ to make new monoclonals)
Subtle differences missed
Not looking at whole picture
Looking at what is known (e.g. no novel proteins or unknown modification sites)

85
Q

What is the use of RT-PCR for protein validation?

A

• RT-PCR
o Transcript levels-RT PCR
 Real time PCR or quantitative PCR to check if there’s increased transcript levels

86
Q

What is the principle of using immunohistochemistry for protein validation?

A

o Uses antibodies to prove distribution of protein of interest in cells or tissues of interest
 Antibodies proven for immunohistochemical applications

87
Q

What is the disadvantage of using immunohistochemistry for protein validation?

A

o Some biological systems are unsuitable for immunohistochemistry

88
Q

What is the human protein atlas, when was it finished, what was its aim and what did it achieve/what did not achieve?

A

o The human protein atlas (published in 2015- was done in about 8-10 years with probably $100,000 spent on this project)
 Ultimate aim to make monoclonal (specific) antibodies against every protein and protein-PTM in the human proteome and determine the localization of these in healthy and diseased cells and tissues
 Originated in Sweden
 This is complete- antibody has been made against every protein in the human proteome
 Protein-PTM side have backed away from: too many protein-PTMs to make an antibody against all of them
• Not economically and functionally viable
• May not be informative
 Tissue based map of the human proteome based on making a high quality monoclonal antibody against every single expressed protein in the human proteome
 Found:
• Secreted proteins tend to be very abundant
• Tissue that contains the most unique proteins are male tissue/testes
o Only expressed in the male tissue
• Brain has the second most unique proteins

89
Q

Describe what is on the website of the human protein atlas

A 
	On website, have-
•	Links to antibodies
•	Additional information:
o	IH-immunohistochemistry
o	IF-immunofluorescence
o	PA-protein arrays
o	WB-western blots 
•	Colour highlights quality of existing data for that antibody
•	Protein information
•	Chromosomal location 
•	Pp-plasma protein or Cc-CVD associated
	Line is where the tissue is expressed
	Red oval- most staining, white oval- no staining
90
Q

What was the human proteome project, what was its aim and what did it find?

A

o The human proteome project- aims to determine whether every protein in the human proteome is ever expressed and where it is expressed
 Has been done as of this year (less than 80 genes that we have yet to see as a protein-most likely they are found in extremely specialised cell types)

91
Q

How does selected reaction monitoring work for validation?

A

o In SRM, the 1st and 3rd quadrupoles act as mass filters for specifically selected predefined m/z values corresponding to the precursor ion (Q1) and a specific, known fragment ion (Q3)
 Tell the mass spectrometer to completely disregard any ions that we are not interested in- focus on masses of interest (assays are therefore extremely quick)
o Each precursor/fragment pair is known as a transition- use several fragments for each precursor- multiple transitions per peptide
o The signal intensity of each transition can be compared for multiple samples to achieve relative quantification
o Helps analysis if the LC retention time is known and reproducible for each peptide
 Tell MS to scan at a particular time frame according to LC retention time

92
Q

What is a transition in selected reaction monitoring and what is their significance?

A

 Transition- parent to fragment ion intensity measured via retention time on the chromatography system to the intensity of that transition
 More transitions you have, the more confident you are in your subsequent data

93
Q

What is needed to be done for absolute quantification each protein of interest in selected reaction monitoring for the validation to work?

A

o For each protein of interest (generally one that has been identified in a shotgun experiment) generate a list of proteotypic peptides (unique, non-modified (e.g. no Met), generate quality fragment ions etc.)
 If you have a standard chemically identical to molecule of interest (make a synthetic peptide and spike it into the sample at a known concentration), you can then do a concentration curve and get absolute quantification of protein/peptide of interest

94
Q

On what basis is a proteotypic peptide chosen for selected reaction monitoring

A

 Proteotypic peptide choice-
• MS properties- based on previous observations in discovery-based experiments (want peptides that yield a few predominant fragments- select transitions based on the most intense fragments- e.g. Proline [P], aspartic acid [D])
• Uniqueness- must be unique to the protein-of-interest (be careful with repeat regions of short length e.g. dipeptides)
• PTM status- modified peptides not detected (since change precursor mass) unless specifically targeted
o Don’t want to use modified peptides unless using this technique to monitor the relative abundance of a PTM
• Chemical modifications- e.g. methionine oxidation, or asparagine deamidation (don’t want these)
• Avoid poor/unpredictable cleavages- e.g. KR, KP, RP etc.

95
Q

What is the SRM/MRM atlas?

A

o SRM/MRM atlas

 To create synthetic prototypic peptides for every protein encoded by the human genome

96
Q

What is the application of Selective reaction monitoring validation analyses?

A

o Applications of SRM analyses
 Validation of discovery proteomics data
 Searching for specific proteins in a complex mixture (e.g. a biomarker amongst patient plasma samples)
 PTM site occupancy (modified vs non-modified versions of a peptide)
• How much of a protein is modified at a given site at a given time
 Pathway analysis- quantify amounts of proteins present in a biochemical pathway
• Develop an assay for the proteins that were not seen in the pathway to see if they are also changed as per the identified proteins

97
Q

How can metabolomics be used to validate proteomic data?

A

o Protein abundance changes- does this result in a relative change in the substrate of this protein or the end-product of protein function
o Metabolomics measures these
o Alternatively, established functional assays can be used to monitor substrate/products
 Enzyme assays can be important for this
o Metabolomics can be used to validate proteomics data
 When have enzymes with known catalytic site, can use functional assay to see if modification influences functionality of protein

98
Q

What is metabolomics and why are they more easily correlated with phenotypes than other -omics?

A

• Metabolomics- small molecules that are chemically transformed during metabolism and, as such, they provide a functional readout of cellular state.
o Unlike genes and proteins, the functions of which are subject to epigenetic regulation and post-translational modifications, respectively, metabolites serve as direct signatures of biochemical activity and are therefore easier to correlated with phenotype

99
Q

What is metabolic targeting?

A

• Metabolic targeting- quantification of a specific metabolite

100
Q

What is metabolomic profiling?

A

• Profiling- quantification of a group of related compounds (e.g. lipidomics or glycomics) or those found in a single biochemical pathway

101
Q

Are metabolomics more or less complex than other -omics?

A

• Metabolomics is substantially more complex analytically than other -omics:
o Lots of compounds share the same mass

102
Q

What is the use of targeted metabolomics, its sample use and its equipment use?

A

• Targeted metabolomics-
o Validate changes at the proteome level (enzyme substrate/product ratio)
o Survey patient blood (plasma/serum) for known metabolites
o Any known biomolecule can be assayed (sugars, glycans, lipids (e.g. fatty acids), amino acids, peptides)
o Relies on SRM-MS

103
Q

What is the workflow of targeted metabolomics?

A

o Workflow-What are the levels of a specific metabolite in a sample
 Standard metabolites-> LC/MS of standard metabolites-> Selected reaction monitoring-> optimization and standard curve for quantification-> Samples (tissue lysates, cells, blood and other biofluids)-> LC/MS of metabolite extracts-> data analysis by comparison of sample groups and/or standards-> quantification of specific metabolites in biological samples

104
Q

How can transitions be used for relative or absolute quantitation of samples by LC-SRM?

A

o The definition of a parent ion in MS is any analyte that can be fragmented to produce product ions
o Even very simple chemical species can be fragmented to produce product ions
o Therefore, can use parent: product ion transitions for relative quantitation by LC-SRM
 Theoretical fragments produced should be able to me mapped to chemical structure of the compound
o Furthermore, if we employ a standard of known quantity we can perform absolute quantitation by comparing a standard curve against test samples
 More easily than can be done with peptides

105
Q

What is the process of selected reaction monitoring for metabolites?

A

o Selected reaction monitoring (SRM/MRM) for small molecules
 In SRM, the 1st and 3rd quadrupoles act as mass filters for specifically selected predefined m/z values corresponding to the precursor ion (Q1) and a specific known fragment ion (Q3)
• Q1- measure the mass of the precursor ion
• Q2- fragment wanted precursor ion
• Q3- measure masses of fragment ions
 Each precursor/fragment pair is known as a transition- use several fragments for each precursor- multiple transitions per metabolite
 The signal intensity of each transition can be compared for multiple samples to achieve relative quantification
 For each metabolite generate a list transitions

106
Q

Are there fewer transitions for peptides than metabolites in selected reaction monitoring?

A

• Generally have fewer transitions for metabolites than peptides

107
Q

What are the known parameters/optimization for targeted LC-SRM?

A

 Targeted LC-SRM: known parameters/optimization
• Q1- parent mass
• Q3- surveyed fragment mass (Q1:Q3= 1 transition)
• RT, LC -retention time
• DP-declustering potential
• CE-collision energy

108
Q

What is the use of untargeted metabolomics and what technology does it rely on?

A

o Identification of unknown compounds
o Relative quantification of both known and unknown compounds under different conditions (akin to shotgun proteomics)
o Relies on LC-MS/MS (and NMR- metabonomics)
 Structural characterisation of small molecules

109
Q

What is the workflow for determining the global metabolic profile of a sample using untargeted metabolomics?

A

o Workflow-What is the global metabolic profile of a sample
 Sample (Tissue lysate, cells, blood and other biofluids)-> LC/MS of metabolite extracts-> overlayed extracted ion chromatograms-> data alignment and analysis-> validation with MS/MS from standards-> global metabolic profile of biological samples

110
Q

What is the workflow for determining the molecular structure of a compound detected by untargeted profiling?

A

o Workflow- what is the molecular structure of a compound detected by untargeted profiling
 Untargeted metabolomic output-> extract m/z values of individual peaks-> search metabolite databases for m/z values of interest->
• No database hit for m/z of interest: more data needed for characterisation
• Obtain data from a standard compound-> comparison of retention times-> comparison of MS/MS spectrum-> characterisation of metabolite(s) as potential diagnostic or therapeutic target
o If the peak of interest doesn’t have the same retention time as any known standards, then don’t have a hit
o Large-scale identification and quantitation of known metabolites Based on m/z values for known compounds and then comparison based on LC retention time and MS/MS
o Compound-of-interest has no m/z hit [or LC retention/ MS/MS do not match known compounds
 Full molecular characterisation based on MS/MS fragments, compound isolation, NMR spectroscopy

BCMB3904 (6 decks)

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