Proteomics pathway Proteomics pathway Sample Data Analysis Separation Selection of spot(s) G Q R E N K T M E Post-separation analysis ... NRTKGG ... Databases Data Processing Major steps in gel image Major steps in gel image analysis analysis • Data acquisition • Data acquisition • Gel processing / visualization • Gel processing / visualization • Spot detection and • Spot detection and quanti quantit tation ation • Gel matching Gel matching • • Gel comparison • Gel comparison • Data analysis Data analysis • • Gel annotation - building a database • Gel annotation - building a database • Link to databases (Internet) Link to databases (Internet) • 1
MELANIE 2D gel analysis software Swiss Institute of Bioinformatics, Geneva, Switzerland 2-D PAGE Analysis (other software) 2-D PAGE Analysis (other software) Progenesis (Nonlinear Dynamics) Z3 and Z4 (Compugen) Proteomweaver (Definiens) PdQuest (Biorad) ImagepIQ (Proteome Systems) Decyder (Amersham Biosciences) ProteinMine (Scimagix) Delta2D (Decodon ) Phoretix (many names, many distributors ) 2
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Image analysis Visualisation Visualisation 4
Pairwise Comparison Pairwise Comparison Visual comparison By matching automatic or semi-automatic Gel matching Gel matching 5
Transparent mode & aligning Transparent mode & aligning Transparent mode & aligning Transparent mode & aligning Variation of spot Variation of spot intensity intensity Proteins unique to Proteins unique to a particular state a particular state. . 6
Analyzing populations of gels Analyzing populations of gels Progression Based Comparison Progression Based Comparison t = 1 t = 2 t = 3 t = 4 Analyzing populations of gels Analyzing populations of gels Population Based Comparison Population Based Comparison Control Disease 7
Data analysis Data analysis 8
Data management Data management Samples Data types Data Classes Images Analysis Gels Identification Matching Visualisation Query Annotations External links Analysis Experimental Laboratory Exports conditions Data Warehouse Spot Cutter MS Data management / workspace Data management / workspace 9
Proteomic databases databases Proteomic Definition • • Collection of data obtained by 2D gels Collection of data obtained by 2D gels analysis analysis • includes: includes: • – Annotated images of 2D gels – description of identified proteins Proteome analysis analysis Proteome Sample 2D Gel Annotation of 2D image Selection Database of spots update P22323 P54001 Identification 10
Identification of spots Identification of spots • • Comparison with a reference gel Comparison with a reference gel • • Extraction of spots and biochemical Extraction of spots and biochemical analysis analysis • • Search into databases Search into databases Identification Identification 11
Master gel E. E. coli coli Master gel SWISS-2DPAGE SWISS-2DPAGE • • Created in 1993 Created in 1993 • • Collaboration Collaboration – Central Laboratory of Clinical Chemistry, University Hospital of Geneva – Swiss Institute of Bioinformatics 12
Release 17.0, February 2004 Release 17.0, February 2004 • • 36 reference maps 36 reference maps – Plasma (27%), E. coli (18%, 8 maps), Mouse (17%, 6 maps) – Human lymphocytes, Staphyloccocus aeurus Staphylococcus 16% • • 1265 protein entries 1265 protein entries Arabidopsis 4% Human 34% • • 4309 identified spots 4309 identified spots Dictyostelium 1% Yeast 5% Mouse E. coli 14% 26% 13
Annotation examples Annotation examples General information General information 14
2D Annotations 2D Annotations Cross-references Cross-references 15
Query into the databases Query into the databases • • http://www. http://www.expasy expasy.org/ch2d/ .org/ch2d/ • Query by keywords • Query by keywords • • Graphical query Graphical query • • List of identified proteins per master List of identified proteins per master Query by keywords Query by keywords 16
Graphical query Graphical query Query result Query result 17
Other public proteomics Other public proteomics databases databases • 50 2-DE databases 50 2-DE databases • – (fully fed: 20, partially: 22, other: 8) • 270 2-DE map images 270 2-DE map images • • WORLD-2DPAGE (updated list): WORLD-2DPAGE (updated list): • http://www.expasy.org/ch2d/2d-index.html Other proteomic Other proteomic databases databases 35 30 25 20 15 10 2000 5 2001 0 2002 < 5 2003 <10 <15 >15 2-DE 2-DE maps maps / / databases databases 18
Make2ddb Make2ddb • • Objectives: Objectives: – helps to publish your own 2D data on the Web – proposes query tools – proposes a friendly interface • Characteristics: Characteristics: • – implements the rules of federated databases – completely automated 19
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