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Gene Expression Studies:
Transcriptomics and Proteomics
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Levels of Biological Information
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Gene to cellular function
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Gene Expression Controls
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Gene to gene product
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RNA and Protein synthesis
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Post transcriptional modification
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Understanding cellular functions
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What is systems biology?
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Tools
Integral to understanding biological
systems is the ability to discover and
measure changes in the system
Mass spectrometry measures molecular
structure and abundance hence is utilized
in the measurement of DNA, RNA,
proteins and small molecule metabolites
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Proteome
Entire PROTEin complement expressed
by a genOME, or by a cell or tissue type;
including the modifications (e.g.
phosphorylation, ubiquitination, etc.)
dynamic and variable and describes the
functional state of a cell or tissue
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Dynamics and protein concentration rangeDynamics and protein concentration range
DNA mRNA ProteinsFunctional
Proteins
Genome Transcriptome Proteome
Transcription TranslationPost-translational
modifications
Human:
~ 30 000 genes ~300 000 transcripts ~ 3 000 000 proteins
Genomics on Obesity, Toulouse, 7Genomics on Obesity, Toulouse, 7--8 June 20078 June 2007
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complex
dynamic
PTMs
Genomics on Obesity, Toulouse, 7Genomics on Obesity, Toulouse, 7--8 June 20078 June 2007
Diverse properties of proteinsDiverse properties of proteins
Proteomics is a particularly rich source of biological information
The investigation of the properties and functions of proteins under
various conditions constitutes proteomics
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Same genomeSame genome
DifferentDifferent proteomesproteomes
ComplexityComplexity ofofproteomesproteomes
GenomicsGenomics onon ObesityObesity, Toulouse, 7, Toulouse, 7--88JuneJune 20072007
Every somatic cell of the butterfly and its caterpillar contains identical genetic
information. However, when the conversion of this genetic information takes place,
that is when the different genes are expressed into proteins, this leads to an
enormous individual phenotypic diversity. In other words, both animals have the
same genome, but they have different proteomes.
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Why study proteins?
Proteins are important components of
living organisms, since they are the
molecules responsible for the many
physiological processes and metabolicpathways of cells.
Protein quantity and some properties vary
with time and stresses introduced in itsenvironment.
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Limitations of genomics
Genomics and transcriptomics gives only a rough estimate ofthe genes level of expression into a protein since mRNAs areproduced in different conditions or may be degraded rapidly ortranslated inefficiently producing small quantities of proteins.
Proteins experience post-translational modifications that
profoundly affect their activities; for example some proteins arenot active until they become phosphorylated. Many transcripts give rise to more than one protein, through
alternative splicing or alternative post-translationalmodifications.
Many proteins form interacting partners with other proteins ormolecules and are fully functional in the presence of theseother molecules.
Protein degradation rate plays an important role in proteincontent.
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Proteomics
Proteomics is an attempt to describe or
explain biological state and qualitative
and quantitative changes of proteincontent of cells and extracellular biological
materials under different conditions to
further understand biological processes.
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Why proteomics
mRNA expression does not always reflectprotein expression level
Many biological samples (e.g. CSF, serum,
urine, etc. are not suitable for mRNA expressionanalysis.
Gene products are important determinants ofphysiological processes and phenomenon
Protein localization is important Protein modification cannot be detected at the
DNA or mRNA levels
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Goal of proteomics
Analysis of the varying proteomes of an
organism at different times, in order to
highlight differences between them.
Analysis of the structure and function of
biological systems
Veer away from the focused approach
which is on particular proteins but a broad-
based aproach
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Challenges
The analysis of a proteome is complex becausethe total number of proteins present in any givencell is very high. For the 30,000 genes of thehuman genome, the transcript number is ten-fold
higher, and the protein number is around 10-foldhigher than the transcriptome. There is also a large variation in the level of
expression of proteins in a cell. Proteins in lowabundance could be masked by those in highabundance and thus difficult to identify.
The amount of proteins in a cell cannot beamplified unlike DNA and RNA.
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From genes to proteins
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Examples of proteomic studies
Comparison of different states of protein
expression in the same cell subjected to different
conditions
Comparison of diseased or abnormal vs normalcontrols
Identification of a protein with specific activity
Understanding protein complexes Studies on post-translational modification
Protein biomarker discovery
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Example: diseased vs normal
controls (discovery proteomics)
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Sample experimental design
Usually serum or blood samples
Electrophoretic profiling
LC profiling
Mass spectrometry profiles as signature ofsample but medium to large proteins may not beobserved
Quantitative and qualitative analysis of small
peptides Qualitative and quantitative analysis of digests
for medium to large proteins
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Protein/ peptide characterization
Identify peptides in LC/MS profile
Match peptides between samples
Compare peptide variants Use simple statistics to find interesting
peptides
Use MS/MS to establish peptide sequenceand hence proteins
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Typical workflow for biomarker analysis of serum
proteins from diseased and normal controls
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Functional proteomics: studying
protein complexes
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Workplan
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Workplan (2)
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Sample preparation
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Protein sources
Animal sources
Soft
Hard tissues
Fluids/ secretions Plant sources
Succulent
Non succulent/ fibrous
Soft parts (e.g. meristem; flowers)
Microbial sources
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Nature of the protein
Where localized
Membrane bound
Cytoplasmic
Extracellular How localized
Bound
Unbound
With interacting
partners
General shape
Fibrous
Globular
PropertyAcidity/ basicity
Polar/ nonpolar
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Factors affecting stability of protein
during collection
Possible change Methods of prevention
Microbial degradation Addn of antimicrobials, e.g. Naazide
Storage at -20C
Enzyme denaturation Storage in 50% glycerol at lowtemp.
Storage in liquid nitrogen
Leakage of cellularcomponents
Separate cells asap; do not
freeze
Storage in isotonic soln
Oxidation Add antioxidant, e.g. DTT; 2-mercaptoethanol
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Factors affecting stability of protein
during collection (2)Possible change Methods of prevention
Enzymic conversion of
analyte
Add enzyme inhibitor
Store at -20C
Coagulation Add anticoagulant, e.g.heparin, EDTA
Gaseous loss Store under oil, e.g. liquidparaffin
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Extraction methods
Precipitation
Alcoholic
TCA and other acids
immunoprecipitation
Spin filtration
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Quantitative Methods
Total protein (Kjeldahl) Soluble protein
Biuret (least sensitive)
Lowry
Bradford
Bicinchoninic (affected by detergents)
Dye-binding methods
Methyl orange/ bromcresol green/ purple binds toalbumin
Coomasie blue
Immunologic methods (e.g. Western blotting, ELISA)
Precipitation
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Separation methods
There are several properties of proteins that can
be taken advantage of to separate proteins
usually by chromatographic methods. Proteins
can be separated by: size
shape
hydrophobicity
affinity to molecules
charge
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Classification of separation
techniquesMolecularcharacteristics
Property Separationtechnique
Polarity VolatilitySolubility
Absorptivity
GLC
Liquid-liquid chrom
Liquid-solid chrom
Ionic charge Ion exchange
Electrophoresis
Size diffusion Gel filtrationchrom, dialysis
Shape Sedimentation
Ligand-binding
Ultracentrifugn
Affinity chrom
f
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Examples of adsorbents and
applicationsAdsorbent Strength Applications
Silicic acid strong Steroids, amino acids,lipids
Charcoal Strong Peptides, CHOAluminum oxide Strong Steroids, ester,
alkaloids
Magnesium
carbonate
Medium porphyrins
Calcium phosphate Medium Proteins,polynucleotides
cellulose Weak proteins
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Electrophoresis
Gel electrophoresis is a technique used for theseparation of deoxyribonucleic acid (DNA),ribonucleic acid (RNA), or protein moleculesusing an electric current applied to a gel matrix.
Particles are separated according to chargeand/or size
Refers to the technique in which molecules are
forced across a span of gel, motivated by anelectrical current; activated electrodes at eitherend of the gel provide the driving force
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Polyacrylamide gels
F t Aff ti
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Factors Affecting
Electrophoresis SAMPLE MOBILITY
Charge to mass ratio
Ionic strength of solution
Temperature of the gel
P bl i t d ith hi h
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Problems associated with high
ionic strength Increased temperature
decreased viscosity and increased
current
buffer evaporation
sample denaturation
convective mixing
F t ff ti
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Factors affecting
electrophoresis (2) ELECTROPHORESIS SYSTEM
DYNAMICS
TYPES OF GEL SYSTEMS
Horizontal
Vertical
Slab
Disc
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Requirements
Must maintain a uniform electric field
across the gel
Provide cooling to prevent thermal
artifacts
Allow access to the gel for sample
loading and monitoring the run
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Slab gel electrophoresis
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2-D electrophoresis
2D2D electrophoresiselectrophoresis
GenomicsGenomics onon ObesityObesity, Toulouse, 7, Toulouse, 7--88JuneJune 20072007
1D:1D: separationseparation basedbased onon thethe pI ofpI ofproteinsproteins
2D:2D: separationseparation basedbased onon thethe molecularmolecularweightweight
ofofproteinsproteins
SeveralSeveral visualizationvisualization//detectiondetection possibilitiespossibilities
TheThe mostmostwidelywidely usedusedtechnicaltechnicalapproachapproach
=> Up to 10 000=> Up to 10 000proteinprotein spots/gelspots/gel
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2-D gel electrophoresis
S iti it f t i d t ti
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Sensitivity of protein detection
methods
Methods Sensitivity Linearity
Coomasie blue 100 ng low
Silver nitrate 200 pg low
Fluorescence 1 ng high
Fluorescentlabelling 250 pg high
Radiolabelling 1 pg high
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TAP tag purification system
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Purification
Column
Chromatography
HPLC
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2D-E 2D-LC
time consuming
reproductibility
staining
proteins of high MW
hydrophobicproteins
low quantityproteins
higher numberof proteins
quantityof sample (1-5 mg)
columns/buffers
differentialanalysis
mass spectrometry
severalproteinsin a fraction
GenomicsGenomics onon ObesityObesity, Toulouse, 7, Toulouse, 7--88JuneJune 20072007
LimitsLimitsofofeacheachproteomicproteomicapproachapproach
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Analysis: Mass spectrometry
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MS Technologies Used
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Advantages of MS
Shift in emphasis back to functional aspects of cellbiochemistry, gene
expression, and proteins in the cell. Mass Spec technology, because of its versatility will
enable identification, characterization and analysis ofproteins and biological molecules more easily andefficiently.
Attomole/Femtomole level sensitivity
Exact molecular weight measurement ofmolecules up to 150,000 500,000 Da canbe determined
New paradigm in peptide sequencing providesprotein ID in minutes instead of hours
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Mass spectrometry
Substances are ionized for analysis by MS Net charge can be either positive or negative
Mass-to-charge ratio of an ion (m/z) is thefundamental measurement
Sample prep is a key to success, requirementsare specific to the experiment and instrumenttype
Requires knowledge of sample history and
underlying biology Protein and MS Core Labs are plentiful withcurrent instrumentation
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Basic components of MS
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Ion source converts sample to ions by adding or taking
away one or more protons. Ions may be singly or multiply charged. Ions are easier to control in the mass spectrometer
than neutral molecules and are easier to detect.
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Ionization
Electrospray Ionization (ESI) Includes high (ml) and low (nl) flow rate liquid transfer
NanosprayTM Ion Source
ElectrosprayTM Source
Micro Ion SprayTM Source
Turbo Ion Spray TM Source
Matrix Assisted Laser Desorption Ionization (MALDI) Sample is dissolved with an energy transferring compound or
matrix
This is spotted on to a metal plate and allowed to crystallize When a laser is applied matrix crystals transfer energy facilitating
ionization
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Electrospray ionization (ESI)
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ESI spectrum of trypsinogen
Matrix assisted Laser Desorption
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Matrix-assisted Laser Desorption
Ionization (MALDI)
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MALDI/TOF Mass Spectrum of IgG
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ESI vs MALDI
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Mass Analyzers
Operate under high vacuum to keep ions
from bumping into gas molecules
Measures mass-to-charge ratio of ions
(m/z)
Key specifications are resolution, mass
accuracy, sensitivity and mass range
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Mass analyzers used in proteomics
Quadrupole (including multiple Quadrupoles): Unit resolution capabilities using frequencies to separate ions.Low mass accuracy and resolution. Limited mass range.
Time of Flight (including multiple TOF):
High resolution and accurate mass capabilities using time anddistance to separate ions. Unlimited mass range.
Ion Trap:
Unit and higher resolution capabilities using frequency to separateions. Moderate mass accuracy, limited mass range.
Fourier Transform (FT):
High resolution and mass accuracy using frequency to separate
ions. Hybrid:
Combination of different types of analyzers to achieve specificapplication
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Peptidomics