bc199lec-transcriptomics to proteomics2011

8/4/2019 BC199lec-Transcriptomics to Proteomics2011

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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


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


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

bc199lec-transcriptomics to proteomics2011

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