1. Lecture 6
Comparative analysis
Oct 2011 SDMBT

2. 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

3. Sequence of events for comparative analysis
Scanning of image
Data analysis
Image processing
Spot Detection
Gel Matching
Oct 2011 SDMBT

4. 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)

5. Densitometry
(Proteomic Identification of ζ 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 )
(UIC)
Oct 2011 SDMBT

6. Densitometry
Oct 2011 SDMBT

7. 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

8. Smoothing Gaussian curves
Raw data
curve #1
curve #2
(CBU Imaging Wiki)
Oct 2011 SDMBT
(BARS,Statlib)

9. Contrast enhancement
(brneurosci.org)
Oct 2011 SDMBT

10. Background subtraction
(NIH Image)
Oct 2011 SDMBT

11. Image Processing
(Olympus)
Oct 2011 SDMBT

12. Contrast enhancement
(Olympus)
Oct 2011 SDMBT

13. Smoothing
(Olympus)
Oct 2011 SDMBT

14. Background subtraction
(Olympus)
Oct 2011 SDMBT

15. Spot detection Automatic detection aided by manual input
Need to adjust sensitivity
Too little sensitivity = missed spots
Too much sensitivity = false positives
Oct 2011 SDMBT
(Biorad)

16. Spot detection
Streaks
Overlapping spots
Oct 2011 SDMBT
(Biorad)

17. 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

18. Gel Matching
Oct 2011 SDMBT
(Biorad)

19. Manual spot matching
Matched
Unmatched
(Biorad)
Oct 2011 SDMBT

20. 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

21. Data Analysis
(Biorad)
Oct 2011 SDMBT

22. e.g. with CyDye (GE Bioscience)
Oct 2011 SDMBT

23. Oct 2011 SDMBT

24. Is due to biological variation rather than gel-to-gel variation
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

25. Lecture 7
In-gel digestion
Oct 2011 SDMBT
October 2010 SDMBT

26. 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
October 2010 SDMBT

27. 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
October 2010 SDMBT

28. Trypsin Arginine or Lysine Proline (ExPasy PeptideCutter)
Oct 2011 SDMBT

29. Chymotrypsin Leucine, Methionine and Histidine (minor)
Tryptophan, Tyrosine and Phenylalanine (major)
Proline
(ExPasy PeptideCutter)
Oct 2011 SDMBT

30. Peptide masses from tryptic digest
Peptide Cutter
(Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels, Shevchenko et al., Anal. Chem. 1996, 68, )
Oct 2011 SDMBT
October 2010 SDMBT

31. 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
October 2010 SDMBT

32. 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
October 2010 SDMBT