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Transcript
Lecture 6
Comparative analysis
Oct 2011 SDMBT
1
General workflow for proteomic analysis
Sample
Sample preparation
Protein mixture
Sample separation and visualisation
Comparative analysis
Digestion
Peptides
Mass spectrometry
MS data
Database search
Protein identification
Oct 2011 SDMBT
2
Sequence of events for
comparative analysis
Scanning of image
Image processing
Spot Detection
Gel Matching
Data analysis
Oct 2011 SDMBT
3
Scanning of image
Convert ‘analog’ spots on gel
into digital data
High resolution images on
densitometers/imaging systems
For wet or dried gels that have
been stained, X-ray films and
blots
Oct 2011 SDMBT
(Biorad, Biosurplus.com, Institute of Arctic Biology)
4
Densitometry
(UIC)
(Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and
Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells, Singh et
al., Int J Biol Sci 2007; 3:27-39 )
Oct 2011 SDMBT
5
Densitometry
Oct 2011 SDMBT
6
Image Processing
Digital data converted into Gaussian curves.
Algorithms used to smoothen curve, removing statistical
noise
Contrast enhancement to see better spots
Background subtraction to remove meaningless changes
in the background of the gel
Oct 2011 SDMBT
7
Smoothing Gaussian curves
Raw data
curve #1
curve #2
Oct 2011 SDMBT
(BARS,Statlib)
(CBU Imaging Wiki)
8
Contrast enhancement
(brneurosci.org)
Oct 2011 SDMBT
9
Background subtraction
(NIH Image)
Oct 2011 SDMBT
10
Image Processing
(Olympus)
Oct 2011 SDMBT
11
Contrast enhancement
(Olympus)
Oct 2011 SDMBT
12
Smoothing
(Olympus)
Oct 2011 SDMBT
13
Background subtraction
(Olympus)
Oct 2011 SDMBT
14
Spot detection
Automatic detection
aided by manual input
Need to adjust
sensitivity
Too little sensitivity =
missed spots
Too much sensitivity =
false positives
(Biorad)
Oct 2011 SDMBT
15
Spot detection
Streaks
Overlapping spots
(Biorad)
Oct 2011 SDMBT
16
Gel Matching
Compare identical spots on
different gels
Matching is seldom 100%
due to variations in
experimental techniques
(staining, gel preparation)
Use of landmarks to
improve matching
Most time-consuming step
(Proteomics – from protein sequence to function, Pennington &
Dunn [editors])
Oct 2011 SDMBT
17
Gel Matching
Oct 2011 SDMBT
(Biorad)
18
Manual spot matching
Matched
Unmatched
(Biorad)
Oct 2011 SDMBT
19
Data Analysis
After matching, data are
arranged into a table
Subjected to normalisation to
account for inconsistencies in
staining and gel preparation
Normalise by:
•Total gel intensity
•Total intensity of subset of
spots
(Proteomics – from protein sequence to function, Pennington & Dunn [editors])
Oct 2011 SDMBT
20
Data Analysis
(Biorad)
Oct 2011 SDMBT
21
e.g. with CyDye (GE Bioscience)
Oct 2011 SDMBT
22
Oct 2011 SDMBT
23
Internal standard to make sure that abundance is normalised and variation
Is due to biological variation rather than gel-to-gel variation
Oct 2011 SDMBT
24
Lecture 7
In-gel digestion
Oct 2011 SDMBT
25
General workflow for proteomic analysis
Sample
Sample preparation
Protein mixture
Sample separation and visualisation
Comparative analysis
Digestion
Peptides
Mass spectrometry
MS data
Database search
Protein identification
Oct 2011 SDMBT
26
Rationale for digestion of proteins
Error is proportional to mass of the protein
PTMs further complicate assignments based on mass
Sensitivity of MS measurement increases with the use of
smaller peptides (6-20 amino acids)
Proteases are able to cut at specific amino acid residues
Oct 2011 SDMBT
27
Trypsin
Arginine or Lysine
(ExPasy PeptideCutter)
Proline
Oct 2011 SDMBT
28
Chymotrypsin
Leucine, Methionine and
Histidine (minor)
Tryptophan, Tyrosine and
Phenylalanine (major)
(ExPasy PeptideCutter)
Proline
Oct 2011 SDMBT
29
Peptide masses from tryptic digest
Peptide Cutter
(Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels,
Shevchenko et al., Anal. Chem. 1996, 68, 850-858)
Oct 2011 SDMBT
30
Typical protocol for in-gel digestion
•Excision of Commassie stained spot(s) from gel(s)
•Destaining with NH4HCO3 /acetonitrile
•Reduction with DTT
•Alkylation with IAA
Oct 2011 SDMBT
31
Overview of in-gel digestion
•Absorption of minimal amount of trypsin into gel (on
ice)
•Overnight incubation of trypsin at 37ºC
•Extraction of peptides from gel with 5% formic acid in
NH4HCO3 /acetonitrile, or trifluoroacetic acid
•Clean up by ZipTips (removes ionic salts)
Oct 2011 SDMBT
32