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BCMB3904 > Practical knowledge > Flashcards

Practical knowledge Flashcards

1
Q

What data can be extracted from a 1D SDS-PAGE?

A

• SDS-Page- separates proteins in the dimension of mass

o The intensity of bands on the gel are linked to the abundance of that protein

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2
Q

Why is 2D gel electrophoresis (2-DGE) preferential to a 1D SDS-PAGE?

A

• 2D gel electrophoresis has higher resolution than SDS-Page

 Can further visualise how many proteins are in the sample

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3
Q

What is the purpose of a 2-DGE gel?

A

• 2-DGE separates individual proteins from a complex mixture in a single experiment

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4
Q

What is the first dimension (x axis) of a 2-DGE gel and what is the purpose of this dimension?

A

• First dimension (x axis) is isoelectric focusing (IEF)

o Separates by charge

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5
Q

Describe what the isoelectric focusing (IEF) dimension of a 2-DGE gel is composed of, how it works and how the results in this dimension are interpreted.

A

o Uses immobilised pH gradients
 Acidic proteins are on the left
 Basic proteins are on the right
o When electric current is added, proteins migrate toward the pH point at which they have no net charge (their pl)
 Proteins are positively charged when at pH values below their pl and negatively charged when at pH values above their pI
 Once proteins have reached their pI, they can no longer move

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6
Q

What is the second dimension (y axis) of a 2-DGE gel and what is the purpose of this dimension?

A

• Second dimension is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
o Separates by mass

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7
Q

How are the results in the second dimension of the 2-DGE gel interpreted?

A

 High mass is at the top

 Low mass is at the bottom

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8
Q

Describe what data can be extracted from a 2-DGE gel

A
  • Protein charge
  • Protein mass
  • Protein abundance (density of spots)
  • Approximation of number of types of protein in sample
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9
Q

Why are 2-DGE gels clinically important?

A

• Proteins are an important tool for clinical diagnosis in samples that do not contain DNA
o E.g. blood, plasma, CSF, urine and ascites

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10
Q

What is the most crucial step for a 2-DGE and why?

A

• Most crucial step for a 2-DGE is sample preparation
o Unlike nucleic acid, protein extraction is sample dependent
o Each sample contains a complex mixture of proteins with different abundances, chemical and physical properties
o Method of extraction of proteins out of cells is sample dependent
o Need a pure sample only containing proteins as any other molecule can interfere with the 2-DGE process

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11
Q

How is a protein sample prepared for a 2-DGE?

A

o Proteins need to be extracted from cells/tissues and solubilised in buffer compatible with isoelectric focusing
o Remove insoluble and interfering material (sugar, lipids, DNA, RNA…)
 Centrifugation steps and DNAse
o Sub-fractionation
 Look at different parts of the proteome

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12
Q

What is contained in the buffer in which proteins extracted from cells/tissues are solubilised in for preparation for a 2-DGE?

A 
	Chaotropes
	Zwitterionic detergents  
	Reducing agents
	Protease inhibitors (optional)
	Carrier ampholytes (pH buffer)
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13
Q

What are chaotropes used for during sample preparation for a 2-DGE and what is an example of chaotropes used for this purpose?

A

 Chaotropes (unfolds proteins by disrupting hydrogen bonds)
• Can be urea (6 Molar)
• Need to unfold tertiary structure so that proteins may be separated according to charge

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14
Q

What are zwitterionic detergents used for during sample preparation for a 2-DGE and what is an example of zwitterionic detergents used for this purpose?

A

 Zwitterionic detergents
• Need detergents with no net charge
• Cannot use SDS due to its negative charge, which will interfere with protein separation during 2-DGE sample preparation
• Examples include CHAPS, sulfur butane-14, sulfur butaine-3 to 10
• Used to solubilise the proteins and protect their native state without altering their charge

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15
Q

What are reducing agents used for during sample preparation for a 2-DGE and what is an example of reducing agent used for this purpose?

A

 Reducing agents
• To remove disulfide bonds to further unfold proteins
• Examples: DTT, TBP

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16
Q

What are protease inhibitors used for during sample preparation for a 2-DGE and what is an example of protein inhibitor used for this purpose?

A

 Protease inhibitors (optional)
• Extracting proteins from cells can instigate death processes in the cell
o Proteases are produced in high quantity in dead/dying cells to degrade/recycle proteins
• However, add strong chaotropes and zwitterionic detergents quickly, death processes are not triggered and this step is not necessary
• Example: PMSF

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17
Q

How is the first dimension of the 2-DGE gel prepared?

A

o Immobilised pH gradients
 Buffering molecules are covalently bonded into the polyacrylamide matrix as it is cast
• Uses well-characterised acrylamido buffers
• Single acidic or basic buffering group linked to an acrylamide monomer and distributed evenly to create a pH gradient

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18
Q

Describe how the structure of the acrylamide matrix allows for mass sorting in 2-DGE gels

A

o Pore size dictates the migration rate of the proteins
 The bigger the pore size, the faster the proteins can move through
o Gels can be fixed percentage or gradients
o Proteins migrate until they cannot pass through the pores of the acrylamide matrix
 Get trapped in the matrix
o Higher mass proteins migrate more slowly and less downwards, the smaller mass proteins migrate more quickly and more downwards

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19
Q

What determines pore size in a polyacrylamide gel?

A

o Pore size determined by the amount of acrylamide (monomer) (%T) present and the amount of bisarylaminde (cross-linker) (%C)

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20
Q

What are the dimensions of a 2-DGE IPG strip?

A

 The IPG strip is approximately 0.5cm thick and 7-24 cm in length

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21
Q

Are IPG strips dehydrated or hydrated when first delivered?

A

dehydrated- they need to be rehydrated

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22
Q

What is the relationship between IPG strip length and resolution?

A

• The shorter the strip, the less resolution

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23
Q

How is the resolution of a 2-DGE gel determined?

A

o Resolution= strip length (cm)/number of pH units

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24
Q

When is a large pH range IPG strip appropriate in a 2-DGE gel?

A

• Large ranges are useful to visualise a large amount of the proteome

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25
Q

When is a narrow pH range IPG strip appropriate in a 2-DGE gel?

A

• Narrow ranges are useful to determine slight changes in post-translational modifications

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26
Q

What are the 4 ways in which sample can be applied to a 2-DGE gel?

A

o In-gel rehydration
 Active vs passive
o Cup-loading
o Paper bridge

27
Q

Describe active in-gel rehydration for sample application in 2-DGE gels

A

• Active- dried IPG strip goes over sample and current is added (A)
o Proteins separated at the same time

28
Q

Describe passive in-gel rehydration for sample application in 2-DGE gels

A

• Passive- (B)
o Sample is added over top of IPG strip and left overnight to hydrate
o IPG strip is added to the isoelectric focusing cell and current is run through it for protein separation

29
Q

Describe cup loading for sample application in 2-DGE gels

A

 Gel rehydrated without sample
• Strip rehydrated in the sample buffer
 Sample applied in a cup (C)
• Useful for basic range IPG strips as it acts to isolate these (pHs 6-11)

30
Q

Describe paper bridge for sample application in 2-DGE gels

A

 Gel rehydrated without sample
• Strip rehydrated in the sample buffer
 Chromatography paper soaked in sample (D)

31
Q

What is the purpose of equilibration during preparation of the 2-DGE gel and how is it done?

A

• Equilibration for mass analysis
o Prepare IPG strips for SDS-PAGE
o Addition of dye front for visualisation
o Strip loaded on top of the second dimension SDS-PAGE gel, agarose poured on it and current run through it to separate proteins based on mass based on pore size of the gel

32
Q

How are IPG strips prepared for SDS-PAGE during the equilibration step for a 2-DGE gel and what is the purpose of each step?

A

 Reduction
• Reduce with DTT
• Prevents reformation of disulfide bonds
 Alkylation
• Prevents oxidation of thiol groups
 Detergent-exchange
• Removal of Zwitterionic detergents and coating of all proteins in SDS
o SDS gives all proteins equal net charge and eliminates tertiary structure

33
Q

What are the requirements for stains used in the visualisation of 2-DGE gels and why?

A

o Sensitivity
 Ability to visualise low abundance proteins
o Linearity
 Wide dynamic range
 Amount of staining seen has to be proportional to protein abundance
o Reproducibility
 Stain properties consistent from gel-to-gel, thus allowing comparative analysis
o Compatibility
 Stain all proteins equally
 Post-separation analysis with as few steps as possible

34
Q

List the steps in performing 2-DGE gel analysis from the beginning

A
  • Sample preparation
  • Preparing the gel
  • Sample application
  • Isoelectric focusing dimension analysis
  • Equilibration for mass analysis
  • Staining requirements
35
Q

How are differences spotted between gels?

A

• Differential protein display
o Visual comparison- spot the difference
o Sophisticated computation is used to compare gels for statistical analysis
o Look for differing spots between two gels and differing abundance
o Easier to break the gel into smaller parts to spot differences between gels: better to start at bottom of the gel

36
Q

What is the workflow of identifying a protein from a protein spot?

A

• Protein identification

o Cut spots, taking the spots, digest them with trypsin, identification with mass spectrometry

37
Q

What protein parameters does a 2-DGE measure and how?

A

DGE measures:
o Depends on the method of staining
o Protein abundance- if using a visual stain (fluorescent or visible), then this measures the amount of each individual protein at that point in time
 Contributed to by expression and degradation rate
o Protein expression- if using radiolabelling then measures the amount of each protein made over a period of time

38
Q

What is the purpose of 2-DIGE?

A

o Difference In-Gel electrophoresis (DIGE)-protein abundance
• DIGE-spectrally distinct dye so can use internal standard giving better quantitation with fewer gels

39
Q

How are DIGE gels made, used, their advantages and disadvantages?

A

o Difference In-Gel electrophoresis (DIGE)-protein abundance
 Fluorescent dyes that label protein sample prior to electrophoresis
• Can label different experiments with different dyes
 Standard used to normalise between gels
 Eliminates the need to run technical replicates of each sample
 Internal standard- mix of standard and test dyes
 But these are expensive
 Look at differences within a single gel
 No variation in spot mobility
 Pre-labelled so no staining step, gels scanned while still in glass plates
 Results in lower gel warping and dust, as well as increases ease of spot matching

40
Q

What types of dyes are used in DIGE and how are they identified as different?

A

 Cy dyes that are size and charge matched
• Different chemical structures means they are spectrally distinct
• Two types:
o Minimal labelling
o Saturation labelling

41
Q

What protein parameter can be visualised with radiolabelling?

A

o Radiolabelling-protein expression

• 2-DE gels only tell you the protein abundance, not expression, unless using radioisotopes

42
Q

How is 2D radiolabelling performed and what are its advantages/disadvantages?

A

o Radiolabelling-protein expression
 Use [35S] Methionine or 14C
 Most sensitive detection method available
 No staining of proteins without methionine (if using [35S]met)
 Only for actively growing samples that can be labelled (not tissue, plasma, etc.)
 Best detection method for analysis of differential protein expression in growing cells under different growth conditions e.g. stress treatment

43
Q

How can protein abundance and expression (2-DIGE and radiolabelling) be simultaneously visualised?

A

o Can combine silver and radioactive staining- expression vs abundance
 Dual-channel imaging
• Combination of silver stain and radioactively labelled spots but each stain had a different colour
• Overlaid images (the stains were in 2 different gels)

44
Q

What are the limitations of 2-DEs?

A

DE’s limitations
o Can’t measure all proteins all of the time
o Limitations due to physical properties of the protein
 High/low mass
• Proteins above 100kDa are not great for 2-DE as they can’t get into the pores
 Acidic or basic
• Highly basic proteins are very difficult to see (above pH 9.5)
 High/low abundance
• Overloading is a problem- abundant proteins will not focus properly
 Hydrophobic
• Hard to get into solution during electrophoresis and in buffers
o Generally, of these the 2-DE is most poor at separating low abundance and/or hydrophobic proteins
 2-DE misses highly hydrophobic proteins and those with more than 3TMR (integral membrane proteins mainly)

45
Q

Describe the workflow of identifying proteins from gel spots

A
  1. 2D gel
    a. Excise spots from 2D gel
  2. In-gel digestion
    a. Using a proteolytic enzyme to break the protein sequence into smaller peptides
    b. Measures masses of peptides-very specific for a sequence in the database
  3. Desalting/concentration of the remaining peptide solution
    a. Better signal using a handheld microcolumn filled with chromatography resin
  4. High mass accuracy MALDI MS analysis
    a. Can use different types of matrices for different types of analytes
    b. MALDI process
  5. Protein identification possible. If not:
  6. Desalting/concentration of remaining peptide solution
  7. Nanoelectrospray tandem MS Sequencing
  8. Database search by peptide sequence tags
  9. Protein identification: if not
  10. Compare sets of Nano ES tandem MS data to obtain amino acid sequences for oligonucleotide probe design and cloning
46
Q

Describe the enzymatic action of endoproteinase Lys-C

A

ii. Endoproteinase Lys-C- cuts C-terminal to K, but not if followed by P

47
Q

Describe the enzymatic action of endoproteinase Asp-N

A

iii. Endoproteinase Asp-N- cuts N-terminal to D

48
Q

Describe the enzymatic action of chymotrypsin

A

iv. Chymotrypsin- cuts C-terminal to aromatic amino acids, but not if followed by P

49
Q

Describe the enzymatic action of endoproteinase

A

v. Endoproteinase Glu-C- cuts C-terminal to E (or D), but not if followed by P

50
Q

What type of matrix is used for peptides in MALDI-MS analysis?

A

i. Alpha-cyano-4-hydroxycinammic acid (used in prac) used for peptides

51
Q

What type of matrix is used for PTMs in MALDI-MS analysis?

A

ii. 2,5-dihydroxybenzoic acid used for post-translational modification

52
Q

What type of matrix is used for larger proteins in MALDI-MS analysis?

A

iii. Sinapinic acid- larger proteins (polypeptides above 5kDa to 40kDa in size)

53
Q

What type of matrix is used for DNA/RNA in MALDI-MS analysis?

A

iv. 3-amino-4-hydroxybenzoic acid- DNA and RNA

54
Q

Describe the MALDI process

A

b. MALDI process
i. Put sample/analytes down on MALDI target plate
ii. Fire a laser pulse at target plate
iii. Matrix molecules and analyte molecules fly off the surface and pass to the gas phase of the mass spectrometer- they are ionized in this process
iv. Pass down time of flight
v. Length of time to reach the end of the TOF detector is proportional to their mass/charge
1. Longer peptides with higher mass take longer to pass down the time of flight tube than smaller peptides

55
Q

What are tips for sample preparation for gel visualisation and extraction

A

• Tips:
o Don’t cut the spots too big (non-stained areas don’t have protein: trypsin can only diffuse into the gel a limited distance
o Gloves, labcoat, hair-ties (all minimize keratin contamination)
o When concentrating and desalting, work slowly but consistently- too fast nothing binds, too slow sample dries
 Try not to get air in there
o Be careful with sample volumes (not too dilute)

56
Q

What are the advantages of using millipore C18 Zip Tip clean-up for MALDI-TOF

A 
•	Millipore C18 Zip Tip clean-up for MALDI-TOF
o	Zip Tip clean-up-Zip Tip advantage
	Improves signal/noise ratio
	Concentrates analytes
	Desalts and removes detergent
	Fast and easy
	Compatible with automation
	Co-elution with matrix
57
Q

What are the two ways of doing mass accuracy calibration? What did we do in the prac?

A 
•	Two ways of doing calibration
o	Internal (in this prac as we have used peptide signals from trypsin/keratin)
o	External calibrant on MALDI target
58
Q

How is internal MS calibration performed?

A

o Internal (in this prac as we have used peptide signals from trypsin/keratin)
 Add two known peptides to the sample
 Use peptide signals from either trypsin auto-digestion or keratin
 Use mass windows
 The calibration is made after the spectrum is obtained
 Mass accuracy 10-50 ppm

59
Q

How is external MS calibration performed?

A

o External calibrant on MALDI target
 Use a peptide mixture with known peptides to calibrate the instrument
 The calibration follow the method file and are used for each measurements
 Mass accuracy 0.1-0.5 Da depending on the target and position on the target

60
Q

What are different strategies to confirm protein match identification/ different factors that should be accounted for during MASCOT identification for protein matches, and how these factors could occur?

A

• Different strategies to confirm identification
o The species from where the protein originates
o Molecular weight and pI value estimated from the gel
o Induced oxidation of methionine residues
 Throughout sample prep process, there is the ability to artificially oxidise methionine residues
• Oxidised form is more intense than non-oxidised form
 Cysteine not much of a problem as these are alkylated
 In-source decay peak- caused by laser and desorption process causing the labile-methyl sulfoxide compound to fall off that peptide
• Not counted/not annotated
o Incomplete digestion- ‘missed cleavages’
 Sometimes, when lysines/arginines are close to each other, trypsin gets lazy and doesn’t cut between the two
o MS/MS or Edman sequence
 If don’t get identification- Tandem Mass spectrometry

61
Q

What are problems associated with the MALDI strategy for protein identification?

A

• Problems associated with the MALDI strategy for protein identification
o Contamination with skin and dust proteins- keratins
o Low concentration of peptides (poorly abundant proteins)
o Concentration of matrix and trypsin autolysis products
o High concentrations of salts- salt adduct formation
o Few peptides e.g. low number of tryptic digestion sites (membrane proteins, small proteins etc.)
o Protein fragments
o Database searching-what is a real match?
• Problems may come across
o Identification of two proteins in one spot
o Protein subunits and fragmentation
o Redundancy in the database (same protein sequence can have 2 entries in the database)

62
Q

What are ways to overcome problems with the MALDI strategy for protein identification?

A

• Ways to overcome problems with the MALDI strategy for protein identification
o Working in flow bench, using gloves, labcoat, etc.- reducing keratin contamination
o Optimized sample handling: Zip-Tips and chromatography microcolumns for desalting and concentration of the peptide solution
o Alternative enzymes for in-gel digestion
 One with different specificity
o 90-95% of proteins should be identified from fully sequenced genomes, drops to <10% for non-sequenced genomes
 Depends upon number of lysin/arginine residues

63
Q

What are protein identification programs online?

A 
•	Protein identification programs on-line
o	Peptident
o	MASCOT
o	PepSea
o	Peptide Search
o	Protein Prospector (MS-fit, MS-tag)
o	Profound
o	PepMAPPER
o	All except peptident are able to identify proteins based on peptide mass maps or MS sequence information

BCMB3904 (6 decks)

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