-Presented by:

Peter Oledzki

John Pinney

Ashwin Sivakumar

Proteomics has been said to be the next step from genomics

Proteomics is the sudy of the proteome.

The proteome is the complete complement of proteins found in a complete genome or specific tissue.

Proteomics

Proteomics and genomics are inter-dependent

Genome Sequence

mRNA

Primary Protein products

Functional protein products

Determination of gene

Genomics

Proteomics

Proteomics

Protein Fractionation

2-D Electrophoresis

ProteinIdentification

Post-TranslationalModification

Aims of Proteomics

Detect the different proteins expressed by tissue, cell culture, or organism using 2-Dimensional Gel Electrophoresis

Store those information in a database Compare expression profiles between a

healthy cell vs. a diseased cell The data comparison can then be used for

testing and rational drug design.

Gel Electrophoresis

Motion of charged molecules in an electric field.

Polyacrylamide gel provides a porous matrix– (PAGE – Polyacrylamide Gel Electrophoresis)

Sample is stained with comassie blue to make it visible in the gel.

Sample placed in wells on the gel.

1-D Gel electrophoresis

Separation in only 1 dimension: size.

Smaller molecules travel further through the gel then large molecules, thus separation.

1-D continued

Electric field across gel separates molecules.– Negatively charged molecules travel towards the

positive terminal and vice-versa.– Western blotting(Protein) not to be confused with Southern

blotting (DNA) or Northern blotting (RNA)

Proteins are treated with the denaturing detergent SDS (sodium dodecyl sulfate) which coats the protein with negative charges, hence SDS-PAGE.

2-D – Separation is based on size and charge

First step is to separate based on charge or isoelectric point, called isoelectric focusing.

Then separate based on size (SDS-PAGE).

Isoelectric Focusing

The isoelectric point is the pH at which the net charge of the protein molecule is neutral.

Different proteins have different isoelectric points.

Isoelectric point is found by drawing the sample through a stable pH gradient.

The range of the gradient determines the resolution of the separation.

SDS-PAGE

Second Dimension. Separation by size. Run perpendicular to Isoelectric focusing. The only unresolved proteins after the first

and second dimensions are those proteins with the same size and same charge – rare!

2-D Proteomics Example

2D-PAGE Analysis Software

2D-PAGE technology has been in use for over 20 years, and potentially provides a vast amount of information about a protein sample.

However, due to difficulties with data analysis, it remains only partially exploited.

Analysis problems

It can be very difficult to compare the results of two experiments to yield a differential expression profile:

Can be severe warping of gel due to– uneven coolant flow– voltage leaks– tears in gel

Can be problems with normalisation of– background– spot intensity

Can be differences in sample preparations.

Current state of software

Correct identification and alignment of spots from the two gels has generally been a process with a lot of manual intervention - hence very slow.

The processing power available with today’s PCs means that automated analysis is starting to become possible.

One vendor claims a throughput of 4 gel pairs per hour can be compared and annotated by an experienced user of their package.

Automated gel matching Gel matching, or “registration”, is the process of aligning

two images to compensate for warp. Some packages still require the user to identify

corresponding spots to help with gel matching. The Z3 program from Compugen has a fully-automated

gel matching algorithm:– define set of small, unique rectangles.– compute optimal local transformations for rectangles.– Interpolate to make smooth global transformation.

Note that this makes use of spot shape, streaks, smears and background structure, which other programs discard.

Spot detection

Once the gel images have been matched, the program automatically detects spots. Algorithms are generally based on Gaussian statistics.

Spot Quantitation

The positions of detected spots are calibrated to give a pI / mW pair for each protein.

A value for the expression level of the protein can be calculated from the overall spot intensity.

Some programs do not quantitate each gel separately, but calculate relative intensity pixel by pixel. This may be a more accurate approach.

Differential Expression

The user can set threshold values for the detection of differential expression. This helps reduce the amount of information displayed at once.

In this example, a protein expressed only in the second sample is circled in red. The yellow circles show proteins which are differentially expressed.

Annotation

Some systems allow semi-automatic annotation of spots, based on a database of proteins listing their pI / mW values.

Proteins of interest can also be excised from the gel and sent on to mass spectrometry for definitive identification. The ProteomeWorks system from Bio-rad offers such an integrated solution for 2D-PAGE and MALDI.

Multi-experiment Analysis

One useful feature of modern programs is the ability to collate data from many runs of the same experiment.

Spots which only appear in one gel are likely to be artifacts, and are removed from the analysis.

This is an excellent way to reduce noise and enhance weak signals.

Links

Z3 system (Compugen) - http://www.2dgels.com/ Melanie3 (SIB) - http://us.expasy.org/melanie/ ProteomWeaver (Definiens) - http://

www.proteomweaver.com/ PDQuest (Bio-Rad) - http://www.biorad.com/ Delta2d (Decodon) - http://www.decodon.com/

Introduction to the databases

•With the advent of many 2-D PAGE databases there are a number of protein spots that are already "identified" in a few cell lines. Combined with the aims of the experiment, these databases may give one the opportunity to guess at the identity of a particular protein spot and confirm or deny this by immunoblotting. The approach of obtaining accurate peptide masses from specifically cleaved proteins to search protein sequence databases, known as peptide mass fingerprinting, provides one with another opportunity to identify a previously sequenced protein or (hopefully) confirm that it is indeed novel.

An animated SDS PAGE presentation

•A number of 2-D Gel databases exist.

•Quantitative databases: S.cervisiae and REF52.

•Annotative databases: E.coli and human keratinocytes.

•An annual issue of the journal “Electrophoresis”-Major database for these databases!!!…(I mean has links to many of these).

•A best one would obviously the database which is regularly updated.(Eg: Swiss 2D page).

List of 2-D GEL DATABASES

One can find an extensive list of such databases by following these links.

We would discuss a few “Interesting ones”.

•World 2-D PAGE

•NCIFCRF

•DEAMBULUM-Protein Databases

•Ludwig Institute of Cancer Research

•Phoretix

World 2-D Page:Index of 2-D page Databases-ExPaSy

•Basically a link to various 2-D Page databases.

•Has a useful tool called 2-D Hunt where one could search for 2-DE related sites on the web.

•Indexed as databases for multi species, mammalia, yeast, plant,bacteria,viruses and parasites, cell lines.

Swiss 2-D Page

•Basically a protein databank for 2-D page and SDS page reference maps.

•May give the exact location of the protein in the map or the region in the map assuming the fact that it has a Swissprot entry.

•Options: Search by keywords, Accession number, spot clicking,full text,author,Swiss-2D Page spot serial number,SRS.Most of them being self-explanatory.

•Protein list for a particular reference map(table)(can be downloaded).It gives details on the gene name,protein description,S-2DP reference number,S-2DP accession number,identification method,Exp. Molecular weights and Pis for each entity found.

•We can also locate the location of a protein sequence in all/one/selected reference maps available.If it is not found a temporary virtual entry is created on the ExPASy server.

SWISS 2D-PAGE (contd)

•It gives cross reference to Medline and a few other databases.•In addition to this textual data, SWISS-2DPAGE provides several 2-D PAGE images showing the experimentally determined location of the protein, as well as a theoretical region computed from the sequence protein, indicating where the protein might be found in the gel.

•Genbio (Geneva Bioinformatics) gives subscription(PAID) for the Swiss 2D PAGE to Commercial Institutions.

•Vital Statistics

¯ Current release(15.0) has 861 entries in 33 reference maps.

¯Vital stats continued...

¯Sources of reference maps:

¯Human( Liver, plasma, HepG2, RBC, Lymphoma, HepG2 Secreted Proteins, CBF, Macrophage like Cell Line, Erythroleukemia cell, platelet, kidney, promycelocytic leukemia cells, colorectal epithelia cells, colorectal adenocarcinoma cell line(DL-1), Soluble nuclear proteins and matrix from liver tissue)

¯Mouse( Liver, gastrocnemius muscle, pancreatic islet cells,brown adipose tissue, white adipose tissue,soluble nuclear proteins, matrix from liver tissue).

¯Arabidopsis thaliana

¯Dictyostelium discoideum

¯Escherichia coli(for 7 pI ranges: 3.5-10,4-5,4.5-5.5,5-6,5.5-6.7,6-9,6-11)

¯Saccharomyces cerevisiae

Swiss 2D Page(cont..)

There have been some recent additions to the database.

SDS and 2-D Page of nuclear proteins from Human HeLa cells have been added to the growing list of reference maps.It is still an ongoing project.Information about known proteins found within that gel stretch has been mapped(see beloe: right-SDS, left-PAGE)

Swiss 2D Page(cont)

Some Useful abbreviations:

-ID line: comprises of ID, Entry name,Entry class and the method(2Dgel) in the order as mentioned.They follow a specific nomenclature.

-AC line basically contains Accession numbers seperated by a semi colon.It’s a stable way of identifying entries with each release.

-DT Line specifies date( self explanatory!).

-DE Line gives a descriptive information about the protein.If the complete sequence was not determined then last line would spell as “Fragment”.

Some useful abbreviations(cont…)

-The IM line

The IM (Images) lines list the 2-D PAGE and SDS-PAGE images which are associated to the entry. These may be, for example, TUMORAL LIVER, NORMAL LIVER or just LIVER.

-RP(Reference Position) line: Describes extent of work carried out by the author.Eg: Protein sequence, amino acid composition, mapping on gel, characterisation and review.

-The “O” series contains organism species(OS),taxonomy(OX) and classification(OC).

-MT(Master) line has information about types of maps used(Eg: Plasma, liver etc).

Methods used for zeroing on the identified spots.

•Total of 3398 identified spots(as of the latest version).

•Amino acid composition has identified 5.3% of these spots.

•Co-migration: 2.6%

•Gel-matching: 46.7%

•Immunoblotting: 20%

•Microsequencing: 15.5%

•Peptide mass fingerprinting: 26.3%

•Tandem mass spectroscopy: 2.3%

Well..does it carry a message?

Browsing the Swiss 2-D Page using spot clicking

-We could get information about a known protein by clicking on one of the “checks” in the extensive list of image maps available.

-On clicking it throws a tailor-ready image map showing the accurate/approximate position of that protein with respect to all the image maps available.But for obvious reasons the best view can be obtained from the reference image map we initially clicked.

-A hypertext link can then be used to obtain the full SWISS-PROT entry for that protein, displaying protein sequence, domain structure, information on known post-translational processing and modifications, and references.

Image clicking(continued…)

-From SWISS-PROT, the user can select a link to SWISS-3DIMAGE to see the three-dimensional structure of the protein, if it is known, or to submit the sequence to the SWISS-MODEL three-dimensional modelling tool or view the domain structure .

- Also, from SWISS-PROT, the user can select links to pertinent information from DNA sequence databases (EMBL/Genbank), chromosomal and genomic maps (GDB Genome Database), bibliographic references and abstracts (Medline), and databases on the association of human proteins with diseases (OMIM Online Mendelian Inheritance in Man).

Diagramatic representation of Image Clicking... Here we click on

this spot in reference map of the Colorectal epithelia cell

Throws a screenshowing

the pictures of different image

maps with respect to that protein

Diagramatic representation(cont…)Protein

identification on chosen reference map The red

rectangle is the expected region of the protein on the gel.

Spots are the proteins identified

Dotted lines are extensions of the possible regions if the protein is acetylated, phosphorylated or glycosylated.

Enough of Swiss 2D PAGE!!!

-On image clicking we could also calculate the theoretical pI and Molecular weight of different sequence fragments with desired end points.One could specify the N-Terminal and C-Terminal values in the options available in the screen.By default it would compute it for the entire sequence available.

-Swiss 2D page also has a cross reference to another popular 2D Gel database in Siena 2D Gel database.

Now bye bye! Swiss 2D PAGE.

Biobase/Julio Celis Database

(Very well structured & lucid!!!)

Preparation and labelling of Human keratinocytes-please visit:

http://biosun.biobase.dk/~pdi/procedures/procedure_label.html

- Hosted at Danish Center of Human Genome Research.

-Have the distinction of constructing the first 2D Gel database(HeLa cells) in 1981(Bravo, R., Bellatin, J. and Celis, J.E).

-Human and Mouse 2D PAGE Databases.

-annotated 2-D gel pattern of fluids from different species can be found in the fluid gallery.

-One can find 2D-Gel immunoblots of selected proteins against various antibodies.

-2D Gel gallary of various human cell types and fluids.(Includes tumors, keratinocytes and post-translational modifications.).

Biobase/Julio Celis Database(cont…)

Human 2-D PAGE Database:

The keratinocyte 2D PAGE database constructed using carrier ampholytes, is the largest of its kind and currently list 3625 cellular (2313 isoelectric focusing, IEF; 954 non equilibrium pH gradient electrophoresis, NEPHGE), and externalised polypeptides (358, IEF) of which 1285 have been identified using a combination of techniques including immunoblotting [32], Edman degradation of internal peptides [33, 34], and mass spectrometry [35]. (Might be outdated!!!!)

Representation through flow charts to follow...

(Biobase Cont..)

By clicking on each of the available reference gels,we can get information(links to medline,swissprot,PDB,cellular location,Knockout,method used) on the available proteins(checked spots) on the gel.

Databases for study of skin biology

HK-IEF d’base HK-NEPHGE d’base KP present in medium IEF Database

865 IP 372 IP

59 IP

Biobase(cont…)

Database for study of Bladder Cancer

TCC-IEF database

TCC-NEPHGE d’base

BSCC-IEF d’base

Urine-IEF d’base

449 IP

144 IP

309 IP

197 IP

Biobase(cont…)

Other 2D Page Databases

Human MRC-5-Fibroblasts-IEF

D’base

Human MRC-5-Fibroblasts-NEPHGE

D’base

262 IP 84 IP

Biobase(cont…)

Search Options:

Seacrh by protein name, keyword, sample spot number,

Relative Molecular mass, pI, organelle /component.

Other options relating listing of proteins,views of the gels are quite self explanatory.

Other utilities of the Database:

Has links to

-NCBI’S Human-Mouse Homology maps through its Mouse 2D-PAGE Databases.

-Interesting studies like Mouse-Genome Informatics(Jackson’s lab) and Mouse Atlas Projects.

NCIFCRF(National Cancer Institute…..could not sphere out what FCRF was!!!..sorry)

*Seems a very exhaustive and useful source.Lots of things still to study.*

2D Protein Gel Databases

Maintained by Image Processing

Section

Maintain thegel analysis

software-GELLAB II

WebGel Flicker dbEngine

WebGel:WebGel is an Internet-based, interactive, qualitative and

quantitative gel database analysis system.

A WebGel database contains previously quantified gel data generated from a

stand-alone quantitataive gel analysis system.

wbdemoDB

demonstration

database

of serum

proteins in a

fetal alcohol

syndrome study.

melanie2DB

demonstration

database

of E.coli gelsfrom the

Melanie 2.3

demonstration

database.

fasDB

database

of serum proteins

in a fetal alcohol

syndrome study

Flicker comparing two Plasma 2D-PAGE Gels.

FLICKER

“Flicker is a method for comparing images from different Internet sources on your Web browser. In the case of 2D protein electrophoretic gel images, maps identifying proteins in these gels are becoming increasingly available. Visually comparing 2D sample gels against these 2D gel database maps may suggest putative protein spot identification in many cases. Flicker was originally developed for comparing 2D protein gels across the Internet.”

-Part of the description in their web site.

dbEngine

It is a simple database search engine which may be used to quickly create a searchable database on a World Wide Web (WWW) server. Data may be prepared from spreadsheet programs (such as Excel, etc.) or from tables exported from relational database systems. This Common Gateway Interface (CGI-BIN) program is used with a WWW server such as available commercially, or from NCSA or CERN. Its capabilities include: 1) searching records by combinations of terms connected with ANDs or ORs; 2) returning search results as hypertext links to other WWW database servers; 3) mapping lists of literature reference identifiers to the full references; 4) creating bidirectional hypertext links between pictures and the database.

Manual

SIENA 2-D PAGE

-Similar to Swiss 2D PAGE when it comes to browsing the Database.

-Point to be noted: Last update was June 2000.

-Their Gel entries include many human protein maps.

-For further information please toy around with their site!

PROTEOME Inc.

-Available databases are:

-Caenorhabditis elegans Proteome Database(YPD)

-Saccharomyces cerevisiae Proteome Database (WormPD)

-S.pombe (PombePD)

-Human PSD(Quite interesting!!!):

Its sort of a survey database.It has greater than 17,000 human, mouse and rat proteins.Within this their PDtm(Protein Coupled Receptor database) has around 600 GPCRs.

As with the case with most companies(Trick up their sleeve!)…their Human PS database is available only for subscription.

HSC-2D PAGE (Harefield)

-Hosted at Heart Science Center at Harefiled.

-Individual gel databases available:

Human Heart

Human Endothelial cell

Rat Heart

Dog Heart

-Well..visit their site,again quite similar to the Swiss 2D-PAGE.

Ventricles

PPMDB at Sphinx

•Warning!!! “Its no more updated”….but could still be a useful source.

•Was aimed to create directory of Arabidopsis plasma membrane proteins and to obtain expression and sequence data.

•Includes->

•a 2-D map agreeing to the rules of federated 2-D databases

•a repertoire of plasma membrane proteins not-present on the map

•protein sequence data linked to EST or cDNA data

•and protein expression data according to ecotypes and plant organs.

Sphinx(cont…)

-Available reference gels include Callus 2D gels,Cationic and Anionic detergent 2D gels,analytical plant organs,analytical solubization methods,analytical ecotypes and preparative.

-One could query a sequence against PPMdb sequences to find matches.

-On clicking on sequence matching we can find detailed information about procedures like 2DE,Running,staining and scanning.

-We could also perform a more elaborate search on available gels by entering user options like protein name,sub-cellular location,accession number,hydrophobicity properties,Gel family,MW,pI etc.

Challenges/shortcomings in 2D-Gel Databases

(Food for thought!)

-Detection of very low abundancy polypeptides.

-The resolution of very basic and high molecular weight proteins

-The lack of satisfactory quantitation procedures for the analysis of all the proteins resolved in the gels.

-Storage of image analysis results

-Data integrity- Merging data from different sources?

-Explanatory notes on the reference maps?Subscribe to swiss-flash newsletter to keep yourself updated on 2D gel and other resources available to ExPASy!!!

Try these links

•http://www.2dgels.com/

•http://www.genomicglossaries.com/content/lifesciences_databasesdirectory.asp

•http://www.expasy.ch/ch2d/2d-index.html

•http://www.infobiogen.fr/services/deambulum/english/db4.html#GELS2D

•http://www.bio-mol.unisi.it/2d/2d.html

•http://biobase.dk/cgi-bin/celis

•http://www.phoretix.com/customers/wl_2d_specific_sites.htm

•http://sphinx.rug.ac.be:8080/ppmdb/index.html

and then Time to chill!!!