Peter Oledzki

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-Presented by Peter
Oledzki John Pinney
Ashwin Sivakumar
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Proteomics

• 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.

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Proteomics and genomics are inter-dependent
Genome Sequence
Proteomics
Genomics
mRNA
Protein Fractionation
Primary Protein products
2-D Electrophoresis
Proteomics
Functional protein products
Protein Identification
Post-Translational Modification
Determination of gene

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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.

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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.

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1-D Gel electrophoresis

• Separation in only 1 dimension size.
• Smaller molecules travel further through the gel
  then large molecules, thus separation.

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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.

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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).

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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.

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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!

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2-D Proteomics Example

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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.

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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.

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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 todays 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.

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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.

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Spot detection

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

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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.

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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.

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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.

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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.

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Links

• Z3 system (Compugen) - http//www.2dgels.com/
• Melanie3 (SIB) - http//us.expasy.org/melanie/
• ProteomWeaver (Definiens) - http//www.proteomweav
  er.com/
• PDQuest (Bio-Rad) - http//www.biorad.com/
• Delta2d (Decodon) - http//www.decodon.com/

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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
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• 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-M
  ajor 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).

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• 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

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• World 2-D PageIndex 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.

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• 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.

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• 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.

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• 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

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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)
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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.Its
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.
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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).
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• 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?
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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.
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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).
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Diagramatic representation of Image Clicking...
Here we click on this spot in reference map of
the Colorectal epithelia cell
Throws a screen showing the pictures of
different image maps with respect to that
protein
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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.
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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.
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Biobase/Julio Celis Database
(Very well structured lucid!!!)
- 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. -annotat
ed 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.).
Preparation and labelling of Human
keratinocytes-please visit http//biosun.biobase.
dk/pdi/procedures/procedure_label.html
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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...
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(Biobase Cont..) By clicking on each of the
available reference gels,we can get
information(links to medline,swissprot,PDB,cellula
r location,Knockout,method used) on the available
proteins(checked spots) on the gel.
Databases for study of skin biology
HK-IEF dbase
HK-NEPHGE dbase
KP present in medium IEF Database
372 IP
865 IP
59 IP
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Biobase(cont)
Database for study of Bladder Cancer
TCC-NEPHGE dbase
Urine-IEF dbase
TCC-IEF database
BSCC-IEF dbase
144 IP
197 IP
449 IP
309 IP
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Biobase(cont)
Other 2D Page Databases
Human MRC-5- Fibroblasts-IEF Dbase
Human MRC-5- Fibroblasts-NEPHGE Dbase
84 IP
262 IP
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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 -NCBIS Human-Mouse
Homology maps through its Mouse 2D-PAGE
Databases. -Interesting studies like Mouse-Genome
Informatics(Jacksons lab) and Mouse Atlas
Projects.
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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
WebGel
Flicker
dbEngine
Maintain the gel analysis software- GELLAB II
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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
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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.
Flicker comparing two Plasma 2D-PAGE Gels.
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dbEngine
Manual
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.
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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!
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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.
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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
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• 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-gt
• 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.

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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.
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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!!!
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• Try these links
• http//www.2dgels.com/
• http//www.genomicglossaries.com/content/lifescien
  ces_databasesdirectory.asp
• http//www.expasy.ch/ch2d/2d-index.html
• http//www.infobiogen.fr/services/deambulum/englis
  h/db4.htmlGELS2D
• http//www.bio-mol.unisi.it/2d/2d.html
• http//biobase.dk/cgi-bin/celis
• http//www.phoretix.com/customers/wl_2d_specific_s
  ites.htm
• http//sphinx.rug.ac.be8080/ppmdb/index.html

and then Time to chill!!!