Gene Expression Studies: Transcriptomics and Proteomics

Levels of Biological Information

Gene to cellular function

Gene Expression Controls

Gene to gene product

RNA and Protein synthesis

Post transcriptional modification

Understanding cellular functions

What is systems biology?

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

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

Dynamics and protein concentration range

DNA

mRNA

Proteins

Functional Proteins

Genome

Transcriptome

Proteome

Transcription

Translation

Post-translational modifications

Human: ~ 30 000 genes ~300 000 transcripts ~ 3 000 000 proteins

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Diverse properties of proteins

Proteomics is a particularly rich source of biological information

complex dynamic PTMs

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The investigation of the properties and functions of proteins under various conditions constitutes proteomics

Complexity of proteomes

Same genome

Different proteomes
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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.

Why study proteins?

Proteins are important components of living organisms, since they are the molecules responsible for the many physiological processes and metabolic pathways of cells. Protein quantity and some properties vary with time and stresses introduced in its environment.

Limitations of genomics
Genomics and transcriptomics gives only a rough estimate of the genes level of expression into a protein since mRNAs are produced in different conditions or may be degraded rapidly or translated inefficiently producing small quantities of proteins. Proteins experience post-translational modifications that profoundly affect their activities; for example some proteins are not active until they become phosphorylated. Many transcripts give rise to more than one protein, through alternative splicing or alternative post-translational modifications. Many proteins form interacting partners with other proteins or molecules and are fully functional in the presence of these other molecules. Protein degradation rate plays an important role in protein content.

Proteomics
Proteomics is an attempt to describe or explain biological state and qualitative and quantitative changes of protein content of cells and extracellular biological materials under different conditions to further understand biological processes.

Why proteomics
mRNA expression does not always reflect protein expression level Many biological samples (e.g. CSF, serum, urine, etc. are not suitable for mRNA expression analysis. Gene products are important determinants of physiological processes and phenomenon Protein localization is important Protein modification cannot be detected at the DNA or mRNA levels

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 broadbased aproach

Challenges
The analysis of a proteome is complex because the total number of proteins present in any given cell is very high. For the 30,000 genes of the human genome, the transcript number is ten-fold higher, and the protein number is around 10-fold higher than the transcriptome. There is also a large variation in the level of expression of proteins in a cell. Proteins in low abundance could be masked by those in high abundance and thus difficult to identify. The amount of proteins in a cell cannot be amplified unlike DNA and RNA.

From genes to proteins

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 normal controls Identification of a protein with specific activity Understanding protein complexes Studies on post-translational modification Protein biomarker discovery

Example: diseased vs normal controls (discovery proteomics)

Sample experimental design

Usually serum or blood samples Electrophoretic profiling LC profiling Mass spectrometry profiles as signature of sample but medium to large proteins may not be observed Quantitative and qualitative analysis of small peptides Qualitative and quantitative analysis of digests for medium to large proteins

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 sequence and hence proteins

Typical workflow for biomarker analysis of serum proteins from diseased and normal controls

Functional proteomics: studying protein complexes

Workplan

Workplan (2)

Sample preparation

Protein sources
Animal sources
Soft Hard tissues Fluids/ secretions

Plant sources
Succulent Non succulent/ fibrous Soft parts (e.g. meristem; flowers)

Microbial sources

Nature of the protein

Where localized
Membrane bound Cytoplasmic Extracellular

General shape
Fibrous Globular

Property
Acidity/ basicity Polar/ nonpolar

How localized
Bound Unbound With interacting partners

Factors affecting stability of protein during collection

Possible change Microbial degradation Enzyme denaturation Leakage of cellular components Oxidation Methods of prevention
Addn of antimicrobials, e.g. Na azide Storage at -20C Storage in 50% glycerol at low temp. Storage in liquid nitrogen Separate cells asap; do not freeze Storage in isotonic soln Add antioxidant, e.g. DTT; 2mercaptoethanol

Factors affecting stability of protein during collection (2)

Possible change Enzymic conversion of analyte Coagulation Gaseous loss Methods of prevention Add enzyme inhibitor Store at -20C Add anticoagulant, e.g. heparin, EDTA Store under oil, e.g. liquid paraffin

Extraction methods
Precipitation
Alcoholic TCA and other acids immunoprecipitation

Spin filtration

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 to albumin Coomasie blue Immunologic methods (e.g. Western blotting, ELISA) Precipitation

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

Classification of separation techniques

Molecular characteristics Polarity Ionic Size Shape Property
Volatility Solubility Absorptivity

Separation technique
GLC Liquid-liquid chrom Liquid-solid chrom

charge diffusion Sedimentation Ligand-binding

Ion exchange Electrophoresis

Gel filtration chrom, dialysis Ultracentrifugn Affinity chrom

Examples of adsorbents and applications

Adsorbent Silicic acid Charcoal Strength strong Strong Applications
Steroids, amino acids, lipids

Peptides, CHO
Steroids, ester, alkaloids

Aluminum oxide Strong

Magnesium carbonate

Medium

porphyrins
Proteins, polynucleotides

Calcium phosphate Medium

cellulose

Weak

proteins

Electrophoresis
Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix. Particles are separated according to charge and/or size Refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current; activated electrodes at either end of the gel provide the driving force

Polyacrylamide gels

Factors Affecting Electrophoresis

SAMPLE MOBILITY
Charge to mass ratio Ionic strength of solution Temperature of the gel

Problems associated with high ionic strength

Increased temperature decreased viscosity and increased current buffer evaporation sample denaturation convective mixing

Factors affecting electrophoresis (2)

ELECTROPHORESIS SYSTEM DYNAMICS
TYPES OF GEL SYSTEMS Horizontal Vertical
Slab Disc

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

Slab gel electrophoresis

2-D electrophoresis
2D electrophoresis
The most widely used technical approach

1D: separation based on the pI of proteins 2D: separation based on the molecular weight of proteins Several visualization/detection possibilities visualization/

=> Up to 10 000 protein spots/gel

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2-D gel electrophoresis

Sensitivity of protein detection methods

Methods Coomasie blue Silver nitrate Fluorescence Fluorescent labelling Radiolabelling Sensitivity 100 ng 200 pg 1 ng 250 pg 1 pg Linearity low low high high high

TAP tag purification system

Purification
Column Chromatography

HPLC

Limits of each proteomic approach

2D-E
time consuming reproductibility staining proteins of high MW hydrophobicproteins low quantity proteins

2D-LC

higher number of proteins quantity of sample (1-5 mg) columns/buffers differentialanalysis mass spectrometry several proteins in a fraction
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Analysis: Mass spectrometry

MS Technologies Used

Advantages of MS
Shift in emphasis back to functional aspects of cell biochemistry, gene expression, and proteins in the cell. Mass Spec technology, because of its versatility will enable identification, characterization and analysis of proteins and biological molecules more easily and efficiently. Attomole/Femtomole level sensitivity Exact molecular weight measurement of molecules up to 150,000 500,000 Da can be determined New paradigm in peptide sequencing provides protein ID in minutes instead of hours

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 the fundamental measurement Sample prep is a key to success, requirements are specific to the experiment and instrument type Requires knowledge of sample history and underlying biology Protein and MS Core Labs are plentiful with current instrumentation

Basic components of MS

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.

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

Electrospray ionization (ESI)

ESI spectrum of trypsinogen

Matrix-assisted Laser Desorption Ionization (MALDI)

MALDI/TOF Mass Spectrum of IgG

ESI vs MALDI

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

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 and distance to separate ions. Unlimited mass range. Ion Trap: Unit and higher resolution capabilities using frequency to separate ions. 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 specific application

Peptidomics