lecture 6 comparative analysis oct 2011 sdmbt
DESCRIPTION
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 SDMBTTRANSCRIPT
Oct 2011 SDMBT 1
Lecture 6Comparative analysis
2
General workflow for proteomic analysis
SampleSample preparation
Protein mixture Sample separation and visualisation
Comparative analysis
DigestionPeptides
Mass spectrometry
MS dataDatabase search
Protein identification
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Data analysis
Sequence of events for comparative analysis
Scanning of image
Image processing
Spot Detection
Gel Matching
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Scanning of imageConvert ‘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
(Biorad, Biosurplus.com, Institute of Arctic Biology)Oct 2011 SDMBT
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(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 )
Densitometry
(UIC)
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Densitometry
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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
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Smoothing Gaussian curves
(BARS,Statlib)
Raw data
curve #1
curve #2
(CBU Imaging Wiki)Oct 2011 SDMBT
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Contrast enhancement
(brneurosci.org)
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Background subtraction
(NIH Image) Oct 2011 SDMBT
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Image Processing
(Olympus) Oct 2011 SDMBT
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Contrast enhancement
(Olympus) Oct 2011 SDMBT
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Smoothing
(Olympus) Oct 2011 SDMBT
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Background subtraction
(Olympus) Oct 2011 SDMBT
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Spot detectionAutomatic detection aided by manual input
Need to adjust sensitivity
Too little sensitivity = missed spots
Too much sensitivity = false positives
(Biorad) Oct 2011 SDMBT
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Spot detection
Overlapping spots
Streaks
(Biorad) Oct 2011 SDMBT
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Gel Matching
(Proteomics – from protein sequence to function, Pennington & Dunn [editors])
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 stepOct 2011 SDMBT
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Gel Matching
(Biorad)Oct 2011 SDMBT
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Manual spot matching Matched
Unmatched
(Biorad)
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(Proteomics – from protein sequence to function, Pennington & Dunn [editors])
Data AnalysisAfter 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
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Data Analysis
(Biorad)
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e.g. with CyDye (GE Bioscience)
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Internal standard to make sure that abundance is normalised and variationIs due to biological variation rather than gel-to-gel variation
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Lecture 7In-gel digestion
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General workflow for proteomic analysis
SampleSample preparation
Protein mixture Sample separation and visualisation
Comparative analysis
DigestionPeptides
Mass spectrometry
MS dataDatabase search
Protein identification
Oct 2011 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
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Trypsin
(ExPasy PeptideCutter)
Arginine or Lysine Proline
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Chymotrypsin
(ExPasy PeptideCutter)
Tryptophan, Tyrosine and Phenylalanine (major)
Proline
Leucine, Methionine and Histidine (minor)
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Peptide masses from tryptic digest
(Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels, Shevchenko et al., Anal. Chem. 1996, 68, 850-858)
Peptide Cutter
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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
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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)