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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
Common technique in simple fractionation
- Chromatography: fractionation by size/charge - Removal of large abundant protein
26
Explain simple enrichment
- Encrichment of protein of particular type protein - Use lectin column to bind glycosylated protein to lectin & remove unbound protein
27
Effects of protein enrichment on protein separation
- Less spots to compare - Simpler analysis - Clearer differences observed
28
Explain specific gel percentage & smaller pH range IPG strip
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
Explain 2Differential Gel Electrophoresis (DIGE)
- Direct comparison of protein in 2 samples - Label using fluorescent dye - Separated together on 1 IPG strip during IEF & same gel
30
Pros of DIGE
- Eliminates problem of spot matching - Quantitative comparison by measuring amount of fluorescene - Overlay of image allow differential expression to be easily detected
31
Dye used in DIGE when overlaid colour is yellow
- Cy3: Green - Cy5: Red
32
DIGE sample preparation
- Protein are evenly label with fluorescent dye - Label either e amino acid group of lysine/ thiol group of cysteine - Easier spot matching
33
Why we need internal standard in DIGE
Each protein can be compared to itself within the internal standard to generate a ratio of relative expression
34
Pros of internal standard
- Reduce effects of variation - Highlight significant differences by including same amount of standard in each gel - Detect differences in abundance of <10%
35
Explain mass spectrometry for protein separation
- 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
Principle of mass spectrometry
- 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
Apart from separation MS allow
- Study PTM - Quantitative analysis using label tag - Identification of protein by peptide mass fingerprinting (PMF)
38
Formula of m/z
Total mass of ion/ Total charge of ion
39
Important parameter in MS
- Resolution: Ability to distinguish peptide - Sensitivity: Ability to detect lowest amount of peptide
40
Resolution in MS defined as
Width of a peak at given height
41
Basic component & functions of MS
- Ion source: Ion formation - Mass analyser: Ion separation - Detector: Detection
42
Sample preparation for MS
- 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
Types of sample ionisation/ formation
- MALDI - SELDI - ESI (Electrospray ionisation)
44
Explain MALDI
- 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
Steps in dried droplet MALDI method
- 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
Steps in SELDI
- 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
Pros & Cons of SELDI
Pros - Robust & automated - High detection limit - Small amount of sample required Cons - Result biased towards peptide & smaller proteins - Sensitivity & resolution fall above 30kDa
48
Define ESI
Tool to study non volatile, thermally labile bio molecular that are not amenable to analysis by other tools
49
Principle of ESI
- 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
Type of mass analyser for ion separation
- TOF Separator - Reflectron - Delayed Extraction Plates - Ion trap - Quadrupole Mass Analyser
51
Principle of TOF
Time taken for ion to reach detector from ion source
52
Explain TOF
- 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
Explain reflectron
- Modification of TOF added with ion mirror to reflect ion to detector - Increase resolution - Able to detect ion that have similar m/z
54
Explain delayed extraction plates
- Modification of TOF added with extraction plates to prepare ion to better separate in vacuum - Placed between source & analyser - Increase resolution
55
Explain iron trap
- Capping electrodes introduced in oscillating electrical field - Allow only specific set of ion to pass through by tuning the oscillating field
56
What is quadrupole mass analyser
- Consist of 4 circular rods - Perfectly parallel to each other
57
Explain quadrupole mass analyser
- 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
Types of detector
- Electron multiplier/ Faraday cups - FTICR
59
Explain Faraday cups
- Series of faraday cups with increasing voltage - Use to amplify ion signal that reach the detector by secondary ion emission
60
Pros of Faradays cups
- Improve ion detection - Spectra quality
61
What is cyclotron in FTICR
Particle accelerator with a strong magnetic field with applied voltage to capture electrons in orbits
62
Explain FTICR
- Cyclotron frequency is related to m/z of ions - Fourier transformation teases out ions that detected simultaneously into individual frequencies
63
2D LC-MS also known as
Shotgun proteomics
64
Explain 2D LC-MS
- 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
Sample type of 2D LC-MS
Peptide in liquid
66
Mass spectrometry output
- 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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