Lecture 6 Comparative analysis Oct 2011 SDMBT

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

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

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)

Densitometry (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 ) (UIC) Oct 2011 SDMBT

Densitometry Oct 2011 SDMBT

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

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

Contrast enhancement (brneurosci.org) Oct 2011 SDMBT

Background subtraction (NIH Image) Oct 2011 SDMBT

Image Processing (Olympus) Oct 2011 SDMBT

Contrast enhancement (Olympus) Oct 2011 SDMBT

Smoothing (Olympus) Oct 2011 SDMBT

Background subtraction (Olympus) Oct 2011 SDMBT

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)

Spot detection Streaks Overlapping spots Oct 2011 SDMBT (Biorad)

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

Gel Matching Oct 2011 SDMBT (Biorad)

Manual spot matching Matched Unmatched (Biorad) Oct 2011 SDMBT

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

Data Analysis (Biorad) Oct 2011 SDMBT

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

Oct 2011 SDMBT

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

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

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

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

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

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

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 October 2010 SDMBT

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

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