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Chapter 2 Protein Separation
Chapter 2 Protein Separation
Chapter 2 Protein Separation
Protein Separation
Learning Outcomes
http://www.jpp.krakow.pl/journal/archive/1106_s7/articles/05_article.html
Yeast proteome as separated by 2D gel electrophoresis
Saccharomyces cerevisiae
Lower molecular weight Proteome of
Saccharomyces cerevisiae
Proteome of the mouse brain as separated by 2D gel electrophoresis
http://www.proteomefactory.com/vwr/technology/proteomeanalysis/proteomeanalysis.html
Medical Examples of Proteomics Application
Identify and
Proteins as markers for Example: Rice, tomato, corn
determine
plant responses to function of unknown
abiotic/biotic stress proteins in the
proteome
discovery of proteins
Investigate crop growth with beneficial
and quality markers activity for cosmetics,
nutraceutical and
pharmaceutical
To distinguish
• protein profiles/map/expression pattern
• identification and quantification (some sort of)
• interaction, modification
Main approaches
• proteomics analysis- characterisation and
identification of proteins
• expression proteomics- expression profiles
• ‘interactome’- protein-protein interactions and
complexes
Major techniques
• Gel-based- 2D gel electrophoresis
• Mass spectrometry
• Protein arrays
• Interaction arrays
Electrophoresis for Protein separation
2nd dimension
Variable Gel Staining for Visualisation
separation by PAGE
Step 1: Sample preparation
iii.Fluorescent stains
sensitive – almost as good as silver stain – 0.25 to 1ng
best dynamic range for quantitative
compatible with MS
Comparison of protein visualisation
based on various staining methods
Coomassie blue
Silver stain
Fluorescent dye
Qualitative and Quantitative comparison of
Protein spots
Matching spot-to-spot of two different samples e.g.
cancer vs normal cells
Matching intensity of spots concentration of protein
Example: Comparison of a
section of 2D protein profiles
from 9 different samples
Disadvantages
Time consuming
Technically difficult
Difficult to automate
Some proteins e.g. membrane proteins not recovered
Not very useful in protein identification – spot
matching is difficult
Methods to Improve Protein Separation
1. Sample Fractionation
Comunale MA et al. Proteomic Analysis of Serum Associated Fucosylated Glycoproteins in the Development of
Primary Hepatocellular Carcinoma. J. Proteome Res., 2006, 5 (2), pp 308–315
Comunale MA et al. Proteomic Analysis of Serum Associated Fucosylated Glycoproteins in
the Development of Primary Hepatocellular Carcinoma. J. Proteome Res., 2006, 5 (2), pp
308–315
Isoforms in 2D profiles
Cy5
Gel 1
Gel 2
Gel 3
~30,000 genes
at least 10 times as many proteins
may exceed 1,000,000 if include modifications
5% of the proteins make up 80% of cell weight
the dynamic range of the protein concentrations can
vary very greatly to > 106 fold
After 2D gel
Cut protein
spot
Tandem MS/MS
for sequencing
different proteins will give different PMF
If the gene sequence is known, the PMF of a protein can
be derived using software
Important parameters in MS
Automated spot
cutter
2. Protein digestion and extraction
Protocol involves:
-Destaining –removal of dye from gel spot (interference)
* silver stained spots are not favourable
- reduction/alkylation- breaking disulphide bonds
- proteolytic cleavage
- extraction of digested peptides by incubation with extraction buffer
and supernatant collected for next step ie MS
• Binding to the SELDI surface acts as a separation step and the subset of
proteins that bind to the surface are easier to analyze
• Common surfaces include CM10 (weak-positive ion exchange), H50
(hydrophobic surface, similar to C6-C12 RP chromatography, IMAC30 (metal-
binding surface), and Q10 (strong anion exchanger)
• Surfaces can also be functionalized with antibodies, other proteins, or DNA
PRINCIPLE OF SELDI-TOF CHIP ASSAYS
Source: urology.jhu.edu
Advantages of SELDI:
Disadvantages:
• results are biased towards peptides and smaller proteins
(proteins <30 kDa)
• sensitivity and resolution of the TOF analyser falls off
markedly above 30kDa.
3. Electrospray ionisation (ESI)
Taylor
cone
From right to left:
Delivery needle with Taylor cone droplet formation, Coulomb
fission
(droplet explodes) with droplet evaporation gas-phase ion
formation in transfer capillary
Nanoelectrospray
- Efficiency contributed by initial diameter of droplet formed and flow
rate
- Compared to ESI- higher sensitivity (1fmol vs 10fmol)
- smaller sprayer diameter (1-25µm vs 50-200µm)
- flow rate (1-1000nL/min vs 1-500µL/min)
- requires less voltage due to the positioning of the
delivery needle much closer to the transfer capillary
Methods of Ion Separation (Mass Analysers)
1. TOF (Time-of-flight) Separator
COMPLEX PROTEIN
SINGLE MASS SPECTROMETRY
TANDEM MASS
SPECTROMETRY
Exact amino acid
WESTERN sequence, correct
BLOTTING protein ID
No sequence
Specific binding