Lecture 6 Comparative analysis

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

Oct 2011 SDMBT

3

Data analysis

Sequence of events for comparative analysis

Scanning of image

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

(Biorad, Biosurplus.com, Institute of Arctic Biology)Oct 2011 SDMBT

5

(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)

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

(BARS,Statlib)

Raw data

curve #1

curve #2

(CBU Imaging Wiki)Oct 2011 SDMBT

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


13

Smoothing


14

Background subtraction


15

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

16

Spot detection

Overlapping spots

Streaks


17

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

18

Gel Matching


19

Manual spot matching Matched

Unmatched

(Biorad)

Oct 2011 SDMBT

20

(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

Oct 2011 SDMBT

21

Data Analysis

(Biorad)

Oct 2011 SDMBT

22

e.g. with CyDye (GE Bioscience)

Oct 2011 SDMBT

23Oct 2011 SDMBT

24

Internal standard to make sure that abundance is normalised and variationIs due to biological variation rather than gel-to-gel variation

Oct 2011 SDMBT

Oct 2011 SDMBT 25

Lecture 7In-gel digestion

Oct 2011 SDMBT 26

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

Oct 2011 SDMBT 28

Trypsin

(ExPasy PeptideCutter)

Arginine or Lysine Proline

Oct 2011 SDMBT 29

Chymotrypsin

(ExPasy PeptideCutter)

Tryptophan, Tyrosine and Phenylalanine (major)

Proline

Leucine, Methionine and Histidine (minor)

Oct 2011 SDMBT 30

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

Oct 2011 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 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)

Lecture 6 Comparative analysis

Documents

Transcript of Lecture 6 Comparative analysis