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BMS661: PROTEOMICS > C3 > Flashcards

C3 Flashcards

1
Q

Define proteome

A

Set of expressed protein in a given type of cell or organisms at given time under defined conditions

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2
Q

Proteome is a collective of all

A

Protein isoforms

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3
Q

Define proteomics

A

Combined techniques applied for the study & analysis of proteome

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4
Q

Example of analysis of proteomics

A
  • Protein identification & measurement
  • Protein sequencing
  • Protein interactions study
  • Protein modelling, structure prediction
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5
Q

What proteomics tools are for

A
  • Large scale analysis of complex sample (urine, cell)
  • Require robust analytical methods (to deal with many sample)
  • To distinguish (protein profile, map, identification)
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6
Q

Main approach of analytical tools in proteomics

A
  • Proteomics analysis: characterisation & identification
  • Expression proteomics: expression profile
  • Interactome: protein-protein interaction & complex
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7
Q

Major techniques in proteomics

A
  • Gel based 2D gel electrophoresis
  • Mass spectrometry
  • Protein arrays
  • Interactions arrays
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8
Q

Why PolyAcrylamide Gel Electrophoresis (PAGE) use for protein separation

A
  • Acrylamide forms linkages polymerise into long chain
  • Result in protein fractionation, separation & isolation
  • Can be prepared with a range of pore size
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9
Q

What are the features that needs attention when observing protein gels

A
  • Shape: band (1D), spots (2D)
  • Separation parameter
  • Intensity of protein band: relate to concentration
  • Individual/ pooled sample
  • 2D: 1 sample per gel
  • 1D: one sample in each lane
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10
Q

Comparison between 1D & 2D page

A

1D
- Separation only size (MW)
- 3 steps, no IEF
- Low resolution
- Poor separation

2D
- Separation size (MW) & isoelectric point (pI)
- 4 steps include IEF
- High resolution
- Good separation

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11
Q

Steps of 2D gel electrophoresis

A
  • Sample preparation: reduction, alkylation
  • Isoelectic focusing (IEF): gel rehydration & focusing 1D
  • Gel electrophoresis: protein on IEF strip (2D)
  • Staining
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12
Q

3 main types of staining

A
  • Coomassie blue: cheap & less sensitive, compatible with MS
  • Silver nitrate: most sensitive, long process, not compatible
  • Fluorescent: sensitive, compatible, best dynamic range
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13
Q

Explain PTM based staining

A
  • Specific fluorescent stain that can detect modified protein
  • Stain bind to specific PTM group on protein
  • Result in less band/spots visualised & simplified analysis
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14
Q

Example of PTM based stain

A
  • Total protein: Sypro Ruby
  • Phosphoproteins: Quercetin, PhosDecor, Sypro Ruby ProQ Diamond
  • Glycoproteins: Sypro Ruby ProQ, Pierce Glycoprotein Staining
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15
Q

Explain Pierce Glycoprotein staining

A
  • Based on periodic acid Schiff (PAS) method
  • 3 reagents: oxidizing, stain & reducing
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16
Q

Pros of Pierce Glycoprotein staining

A
  • Specific for glycoprotein
  • Optimised protocol
  • Colorimetric
  • Qualitative staining
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17
Q

Explain Sypro Ruby ProQ Emerald (Glycoprotein)

A
  • Most advanced technology for detection of glycoproteins in gels & blots
  • Stain react with periodate oxidise carbohydrate group
  • Produce bright green fluorescent signal
  • 3 step: fixation, oxidation & staining
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18
Q

Pros of Sypro Ruby ProQ Emerald Glycoprotein

A
  • Rapid protocol <3 hours
  • Very sensitive
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19
Q

Sypro Ruby ProQ Diamond staining (Phosphoprotein)

A
  • Allow direct in gel detection of phosphate groups attached to tyrosine, serine & threoine
  • 3 steps: fix, stain & destain
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20
Q

Pros of Quercetin staining (Phosphosprotein)

A
  • Specific
  • Cost effective
  • Time saving
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21
Q

Overall advantages of PTM based staining

A
  • Easily highlight protein of interest
  • Confirms the PTM status of protein stain are specific
  • Simplify analysis: less protein to study
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22
Q

Pros & cons of 2D proteomics

A

Pros
- High resolution capacity
- Identification of PTM
- Highly reproducible

Cons
- Time consuming
- Technically difficult
- Difficult to automate

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23
Q

Methods to improve protein separation

A
  • Sample fractionation
  • Sample enrichment
  • Specific gel percentage for PAGE
  • Smaller pH range for IPG strip
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24
Q

Explain sample fractionation

A

To remove unwanted/high abundant protein in order to simplified complex protein profiles & analysis

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25
Q

Common technique in simple fractionation

A
  • Chromatography: fractionation by size/charge
  • Removal of large abundant protein
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26
Q

Explain simple enrichment

A
  • Encrichment of protein of particular type protein
  • Use lectin column to bind glycosylated protein to lectin & remove unbound protein
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27
Q

Effects of protein enrichment on protein separation

A
  • Less spots to compare
  • Simpler analysis
  • Clearer differences observed
28
Q

Explain specific gel percentage & smaller pH range IPG strip

A

Specific gel percentage
- Use certain percentage of gel to separate protein in desired MW range

Smaller pH range
- Use specific range of pI to focus known protein
- Manipulation of various IGP strip available

29
Q

Explain 2Differential Gel Electrophoresis (DIGE)

A
  • Direct comparison of protein in 2 samples
  • Label using fluorescent dye
  • Separated together on 1 IPG strip during IEF & same gel
30
Q

Pros of DIGE

A
  • Eliminates problem of spot matching
  • Quantitative comparison by measuring amount of fluorescene
  • Overlay of image allow differential expression to be easily detected
31
Q

Dye used in DIGE when overlaid colour is yellow

A
  • Cy3: Green
  • Cy5: Red
32
Q

DIGE sample preparation

A
  • Protein are evenly label with fluorescent dye
  • Label either e amino acid group of lysine/ thiol group of cysteine
  • Easier spot matching
33
Q

Why we need internal standard in DIGE

A

Each protein can be compared to itself within the internal standard to generate a ratio of relative expression

34
Q

Pros of internal standard

A
  • Reduce effects of variation
  • Highlight significant differences by including same amount of standard in each gel
  • Detect differences in abundance of <10%
35
Q

Explain mass spectrometry for protein separation

A
  • Molecule or atom ionised, vaporised & introduce into a vacuum to separate before being detected
  • Measure the mass to charge ratio (m/z) of ions
36
Q

Principle of mass spectrometry

A
  • Molecules converted to ions to possess net charge (+/-)
  • Separated & detected based on m/z ratio
  • Involve breaking molecules into fragments making its structure determine
37
Q

Apart from separation MS allow

A
  • Study PTM
  • Quantitative analysis using label tag
  • Identification of protein by peptide mass fingerprinting (PMF)
38
Q

Formula of m/z

A

Total mass of ion/ Total charge of ion

39
Q

Important parameter in MS

A
  • Resolution: Ability to distinguish peptide
  • Sensitivity: Ability to detect lowest amount of peptide
40
Q

Resolution in MS defined as

A

Width of a peak at given height

41
Q

Basic component & functions of MS

A
  • Ion source: Ion formation
  • Mass analyser: Ion separation
  • Detector: Detection
42
Q

Sample preparation for MS

A
  • Undergo protein fragmentation
  • Generate smaller peptide for faster separation
  • Use protease digestion
  • Each enzyme has unique cleavage sure where they cut the protein into peptide
43
Q

Types of sample ionisation/ formation

A
  • MALDI
  • SELDI
  • ESI (Electrospray ionisation)
44
Q

Explain MALDI

A
  • Sample is co crystallised with low molecular mass organic matrix
  • Laser beam use to excite matrix
  • Result in matrix-protein to expand into gas phase
  • Peptide ionised by protonisation (add H+) using energy from laser
45
Q

Steps in dried droplet MALDI method

A
  • Drop of aqueous matrix solution mix with peptide sample
  • Leave to dry before use
  • Bombard with laser, matrix absorb energy while protect peptide
  • Matrix peptide expands into gas phase, enter ion separator of MS
46
Q

Steps in SELDI

A
  • Protein mix spotted on surface
  • Only specific protein bind to surface, other remove by washing
  • Matrix applied to surface to allow crystallise with sample peptide
  • Analysed by TOF-MS
47
Q

Pros & Cons of SELDI

A

Pros
- Robust & automated
- High detection limit
- Small amount of sample required

Cons
- Result biased towards peptide & smaller proteins
- Sensitivity & resolution fall above 30kDa

48
Q

Define ESI

A

Tool to study non volatile, thermally labile bio molecular that are not amenable to analysis by other tools

49
Q

Principle of ESI

A
  • Use high voltage to liquid sample to create aerosol together with temp control & stream of nitrogen gas
  • Produce positively charged ion in gas phase
  • Use nanoelectrospray to contribute initial diameter of droplet formed & flow rate
50
Q

Type of mass analyser for ion separation

A
  • TOF Separator
  • Reflectron
  • Delayed Extraction Plates
  • Ion trap
  • Quadrupole Mass Analyser
51
Q

Principle of TOF

A

Time taken for ion to reach detector from ion source

52
Q

Explain TOF

A
  • Ion accelerated in electrical field & go to detector
  • Ion given same amount of energy
  • Depend on mass
  • m/z ratio can be calculated from PMF generated
53
Q

Explain reflectron

A
  • Modification of TOF added with ion mirror to reflect ion to detector
  • Increase resolution
  • Able to detect ion that have similar m/z
54
Q

Explain delayed extraction plates

A
  • Modification of TOF added with extraction plates to prepare ion to better separate in vacuum
  • Placed between source & analyser
  • Increase resolution
55
Q

Explain iron trap

A
  • Capping electrodes introduced in oscillating electrical field
  • Allow only specific set of ion to pass through by tuning the oscillating field
56
Q

What is quadrupole mass analyser

A
  • Consist of 4 circular rods
  • Perfectly parallel to each other
57
Q

Explain quadrupole mass analyser

A
  • Direct current & radio frequency voltage applied across the rods
  • Only ion of certain m/z travel through the centre of rods at given voltage ratio
  • Other ions has unstable trajectory & collided with rods
58
Q

Types of detector

A
  • Electron multiplier/ Faraday cups
  • FTICR
59
Q

Explain Faraday cups

A
  • Series of faraday cups with increasing voltage
  • Use to amplify ion signal that reach the detector by secondary ion emission
60
Q

Pros of Faradays cups

A
  • Improve ion detection
  • Spectra quality
61
Q

What is cyclotron in FTICR

A

Particle accelerator with a strong magnetic field with applied voltage to capture electrons in orbits

62
Q

Explain FTICR

A
  • Cyclotron frequency is related to m/z of ions
  • Fourier transformation teases out ions that detected simultaneously into individual frequencies
63
Q

2D LC-MS also known as

A

Shotgun proteomics

64
Q

Explain 2D LC-MS

A
  • Mimics the separation principle as 2D gel
  • Use 2 types of column to separate complex protein sample
  • Each fraction fed sequentially into MS
  • Able to analyse protein content of entire tissue samples
65
Q

Sample type of 2D LC-MS

A

Peptide in liquid

66
Q

Mass spectrometry output

A
  • Different protein have different PMF
  • Gene known, PMF protein can derived using software
  • Distribution of cleavage sites determine length & number of peptide fragments

BMS661: PROTEOMICS (6 decks)

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