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Genomica Funzionale
10-‐14 Febbraio 2014
Topics covered
-‐ Metabolic Engineering;
-‐ Concept of Metabolomics;
-‐ Metabolomic plaAorms (LC-‐MS, GC-‐MS, NMR, ICP-‐MS etc); -‐ Set up of a metabolomic protocol and database;
-‐ ApplicaLons in plant-‐/food science field;
-‐ BioinformaLcs applied to Metabolomic data.
Preliminary DefiniLons
• Metabolomics – “Newly” emerging field of 'omics' research – Comprehensive and simultaneous systematic determination of metabolite levels in
the metabolome and their changes over time as a consequence of stimuli (J.K. Nicholson, 1999)
• Metabolome – Refers to the complete set of small-molecule metabolites – Dynamic
• Metabolites – Intermediates and products of metabolism – Examples include antibiotics, pigments, carbohydrates, fatty acids and amino acids – Primary and secondary metabolites
• Metabonomics – Technique allowing determination of cell metabolomic changes, induced by
phenology/physiology or stress-related responses (S. Oliver, 2002)
Metabolomics
Metabolome Reflects the State of the Cell, Organ or Organism
n Change in the metabolome is a direct consequence of protein acLvity changes • Not necessarily true for genomic, proteomic or transcriptomic changes
n Disease, environmental factors, Drugs, etc., perturbs the state of the metabolome • Provides a system-‐wide view of the organism or cell’s response
• The first paper was titled, “Quantitative Analysis of Urine Vapor
and Breath by Gas-Liquid Partition Chromatography”, by Robinson and Pauling in 1971.
• The name metabolomics was coined in the late 1990s (the first paper using the word metabolome is Oliver, S. G., Winson, M. K., Kell, D. B. & Baganz, F. (1998). Systematic functional analysis of the yeast genome.
• Many of the bioanalytical methods used for metabolomics have been adapted (or in some cases simply adopted) from existing biochemical techniques.
• Human Metabolome project – first draft of human metabolome in 2007
• Aracyc – first plant metabolic database in 2010
History
Modeling Approaches in metabolomics
• Top down (data-‐driven): – Omic-‐approach., with determina<on of large sets of data in a specific condi<on (for
instance, responses to bio<c or abio<c stresses). – The system is dissected in sub-‐systems.
• BoWom up (experimental hypothesis-‐driven): – Starts with few elementary components according an experimental hypothesis
interconnec<ng all of them. – Data integra<ons proceeds step-‐by-‐step in order to generate model in which
experimental elements are combined with literature etc.
Metabolomic plaAorm sensiLvity
Data gathering (I)
• Four main points in Analysis of metabolomics data : – Efficient and unbiased – Separation of analytes – Detection – Identification and quantification
• Separation Techniques – Gas Chromatography (GC) – Capillary Electrophoresis (CE) – High Performance Liquid Chromatography (HPLC) – Ultra Performance Liquid Chromatography (UPLC)
• Detection Techniques – Nuclear Magnetic Resonance Spectroscopy (NMR) – Mass Spectrometry (MS)
Data gathering (II)
Uses of mass spectrometer
Ø Used in industry and academia for both rouLne and research purposes. Brief summary of the major mass spectrometric applicaLons:
• Biotechnology: the analysis of metabolites, proteins, pepLdes, oligonucleoLdes
• PharmaceuLcal: drug discovery, combinatorial chemistry, pharmacokineLcs, drug metabolism • Clinical: neonatal screening, hemoglobin analysis, drug tesLng • Environmental: PAHs, PCBs, water quality, food contaminaLon • Geological: oil composiLon
Mass spectrometry for biochemists
Ø Accurate molecular weight measurements:
§ confirmaLon of sample, determinaLon of purity of a molecule, metabolic profiling, metabolite purificaLon, verifying amino acid subsLtuLons, detecLon of post-‐translaLonal modificaLons, calculaLng number of disulphide bridges .
Ø ReacLon monitoring:
§ to monitor enzyme reacLons, chemical modificaLon, protein digesLon
Ø Amino acid sequencing:
§ sequence confirmaLon, de novo characterisaLon of pepLdes, idenLficaLon of proteins by database searching with a sequence “tag” from a proteolyLc fragment
Ø OligonucleoLde sequencing:
§ the characterizaLon or quality control of oligonucleoLdes
Ø Protein structure:
§ protein folding monitored by H/D exchange, protein-‐ligand complex formaLon under physiological condiLons, macromolecular structure determinaLon
Separation Technique – GC (I)
• Mostly in Organic Chemistry • Require chemical
derivatization • Mobile and stationary phase
Separation Technique – GC (I)
Kind of autosamplers: -‐ Liquid -‐ Sta<c head-‐space by syringe
technology -‐ Dynamic head-‐space by transfer-‐line
technology
-‐ Solid-‐phase microextrac<on (SPME)
• Biochemistry and analytical chemistry • Wide range analytes • Mobile and stationary phase • Retention time
Separation Technique – HPLC (I)
HPLC compared to UPLC
Detection Technique - MS
• To identify and to quantify metabolites
• Serves to both separate and to detect
• Mass to charge ratios • Using electron beam • Ion source, mass
analyzer and detector
SPECIFIC MS DEFINITIONS (I)
-‐ M/Z: between the mass of a given ion and the number of elementary charges that it carries; -‐ MONOISOTOPIC MASS: the monoisotopic mass is the mass of the isotopic peak whose elemental composi<on is composed of the most abundant isotopes of those elements; -‐ AVERAGE MASS: The average mass is the weighted average of the isotopic masses weighted by the isotopic abundances; -‐ MASS SPECTRUM: an intensity vs m/z (mass-‐to-‐charge ra<o) plot.
-‐ Accurate mass: the experimentally measured mass value; -‐ Exact mass: the calculated mass based on adding up the masses of each atom in the molecule; -‐ Atomic mass: mass of a par<cle/molecule. Each element is determined rela<ve to Carbon having a mass of exactly 12.0000; -‐ Mass defect: the difference between the mass of the individual components of the nucleus alone, and the mass of the bound nucleus -‐ Mass error = (exact mass) – (accurate mass) • Mass error in parts per million (ppm) = (mass error) (exact mass) X 106
SPECIFIC MS DEFINITIONS (II)
-‐MASS RESOLUTION: resolu<on measures of the ability to dis<nguish two peaks of slightly different mass-‐to-‐charge ra<os ΔM, in a mass spectrum (R = M/dM); -‐ SENSITIVITY: func<on of analyte concentra<on or amount.
SPECIFIC MS DEFINITIONS (III)
Sample introducLon
IonizaLon Minimize collisions, interferences
Separate masses
Count ions Collect results
Mass spectrometry IntroducLon
Overview of Mass Spectrometry
Mass Spectrum
Mass Analyzer
Ioniza<on M+/Fragmenta<on Sample Molecule (M)
ProtonaLon : M + H+ MH CaLonizaLon : M + Cat+ MCat+ DeprotonaLon: MH M-‐ + H+ Electron EjecLon: M M+. + e-‐ Electron Capture: M + e-‐ M-‐.
Mechanism of Ioniza<on
MS divided into 3 fundamental parts
Mass spectrometer
Data system
IonizaLon source e.g. electrospray(ESI), Atmospheric pressure chemical (APCI), Matrix assisted laser Desorp<on(MALDI)
Analyser Mass to charge,m/z e.g. quadrupole, Time of flight, magnet, FT-‐ICR
Detector e.g. photomul<plier, Microchannel plate, Electron mul<plier Orbitrap
Working of Mass Spectrometry
Ø Divided into three fundamental parts: § IonizaLon source § Analyzer § Detector
Ø Sample is introduced in the ionizaLon source where they are ionized. ( It is easier to manipulate ions than neutral molecules).
Ø Ions separated according to their mass to charge raLo in the analyzer. The separated ions are detected and this signal sent to a data system where the m/z raLos are stored together with their relaLve abundance for presentaLon in the format of a m/z spectrum.
Ø In posiLve ionizaLon mode, a trace of formic acid is oben added to aid protonaLon of the sample molecules.
Ø In negaLve ionizaLon mode a trace of ammonia soluLon or a volaLle amine is added to aid deprotonaLon of the sample molecules.
Ø Proteins and pepLdes are usually analyzed under posiLve ionisaLon condiLons and saccharides and oligonucleoLdes under negaLve ionisaLon condiLons.
Ø In all cases, the m/z scale must be calibrated by analyzing a standard sample.
PosiLve or NegaLve IonizsaLon? § If the sample has funcLonal groups that readily accept a proton (H+) then
posiLve ion detecLon is used e.g. amines R-‐NH2 + H+ ® R-‐NH3+ as in proteins, pepLdes
§ If the sample has funcLonal groups that readily lose a proton then negaLve ion detecLon is used e.g. carboxylic acids R-‐CO2H ® R-‐CO2– and alcohols R-‐OH ® R-‐O– as in saccharides, oligonucleoLdes
PosiLve or negaLve ionisaLon?
Sample ionizaLon methods Ø The ionisaLon methods used for the majority of biochemical analyses are
§ Electrospray IonisaLon (ESI)
§ Matrix Assisted Laser DesorpLon IonisaLon (MALDI).
Other IonisaLon methods include:
§ Atmospheric Pressure Chemical IonisaLon (APCI)
§ Chemical IonisaLon (CI)
§ Electron Impact (EI)
§ Fast Atom Bombardment (FAB)
§ Field DesorpLon / Field IonisaLon (FD/FI)
§ Thermo spray IonisaLon (TSP)
• Matrix Assisted Laser DesorpLon IonisaLon (MALDI) (F. Hillenkamp, M. Karas, R. C. Beavis, B. T. Chait, Anal. Chem., 1991, 63, 1193) deals well with thermolabile, non-‐vola<le organic compounds especially those of high molecular mass and is used successfully in biochemical areas for the analysis of proteins, pepLdes, glycoproteins, oligosaccharides, and oligonucleoLdes.
• It is rela<vely straighcorward to use. The mass accuracy depends on the type and
performance of the analyser of the mass spectrometer, but most modern instruments should be capable of measuring masses to within 0.01% of the molecular mass of the sample, at least up to ca. 40,000 Da.
• MALDI is based on the bombardment of sample molecules with a laser light to bring about sample ionisaLon. The sample is pre-‐mixed with a highly absorbing matrix compound for the most consistent and reliable results, and a low concentra<on of sample to matrix works best.
• The matrix transforms the laser energy into excitaLon energy for the sample, which leads to spufering of analyte and matrix ions from the surface of the mixture. In this way energy transfer is efficient and also the analyte molecules are spared excessive direct energy that may otherwise cause decomposi<on. Most commercially available MALDI mass spectrometers now have a pulsed nitrogen laser of wavelength 337 nm.
Matrix Assisted Laser DesorpLon IonisaLon
"Somehow, a peak seems to have appeared." Tanaka reported at the weekly Monday team meeLng on February 2nd, 1985, half a year aber the project had started.
Six O'clock in the Evening on October 9th 2002 News arrived saying that Koichi Tanaka had won the Nobel Prize in Chemistry 2002 On October 9th, the Royal Swedish Academy of Sciences announced their decision to award the Nobel Prize in Chemistry 2002 to three people for their development of methods for idenLficaLon and structure analyses of biological macromolecules. -‐ Koichi Tanaka (at the Lme : Life Science Laboratory Assistant Manager of Shimadzu CorporaLon), Prof. John B. Fenn (Virginia Commonwealth University, USA) and Prof. Kurt Wuthrich (Swiss Federal InsLtute of Technology)
IonizaLon in MALDI
Ø IonizaLon is separated into two divisions § primary ion formaLon
iniLal ions formed during laser pulse
frequently matrix molecules
§ secondary ion formaLon
ions formed during subsequent reacLons
may be matrix-‐matrix reacLons or matrix-‐analyte reacLons
Ø ResulLng analyte ions are usually § protonated § CaLonized § radical caLons
PosiIve ionisaIon MALDI m/z spectrum of a pepIde mixture using alpha-‐cyano-‐4-‐hydroxycinnamic acid as matrix
MALDI in small molecule analyses
67 Isoprenoids (PDA)
lycopene
prolycopene
Ø Generate analyte ions in solu<on before the analyte reaches the mass spectrometer. Ø The LC eluent is sprayed (nebulized) into a chamber at atmospheric pressure in the presence of a strong electrosta<c field and heated drying gas. Ø The electrosta<c field causes further dissocia<on of the analyte molecules. Ø The heated drying gas causes the solvent in the droplets to evaporate. As the droplets shrink, the charge concentra<on in the droplets increases. Ø Eventually, the repulsive force between ions with like charges exceeds the cohesive forces and ions are ejected (desorbed) into the gas phase. Ø These ions are afracted to and pass through a capillary sampling orifice into the mass analyzer.
Electron Spray IonizaLon(ESI) (J. Fenn, J. Phys. Chem., 1984, 88, 4451)
Ø The micro droplet shrinks due to solvent evaporaLon The resulLng increase in charge density of the droplet, forces the charged analyte ion out of the soluLon before the droplet breaks up.
Ø Electrospray ionizaLon can produce mulLply charged ions with the number of charges tending to increase as the molecular weight increases.
“The Taylor Cone”
Electron spray ionizaLon (ESI)
ESI applicaLon for polar molecules
Ø In APCI, the LC eluent is sprayed through a heated (typically 250°C – 400°C) vaporizer at atmospheric pressure. Ø The heat vaporizes the liquid. The resul<ng gas-‐phase solvent molecules are ionized by electrons discharged from a corona needle. Ø The solvent ions then transfer charge to the analyte molecules through chemical reac<ons (chemical ioniza<on). Ø The analyte ions pass through a capillary sampling orifice into the mass analyzer.
Atmospheric pressure chemical ionizaLon (APCI)
APCI applicaLon to profile non polar compounds
Atmospheric pressure photoionization(APPI)
Ø Atmospheric pressure photoioniza<on (APPI)for LC/MS is a rela<vely new technique. Ø As in APCI, a vaporizer converts the LC eluent to the gas phase. A discharge lamp generates photons in a narrow range of ioniza<on energies. Ø The range of energies is carefully chosen to ionize as many analyte molecules as possible while minimizing the ioniza<on of solvent molecules. Ø The resul<ng ions passthrough a capillary sampling orifice into the mass analyzer.
APPI applicaLon in lipidomics
ESI/APCI Benefits
Ø Good for charged, polar or basic compounds
Ø Permits the detecLon of high-‐mass compounds at mass-‐to-‐charge raLos that are easily determined by most mass spectrometers (m/z typically less than 2000 to 3000).
Ø Best method for analyzing mulLply charged compounds.
Ø Very low chemical background leads to excellent detecLon limits (ESI).
Ø Can control presence or absence of fragmentaLon by controlling the interface lens potenLals.
Ø CompaLble with MS/MS methods.
ESI/APCI LimitaLons
Ø MulLply charged species require interpretaLon and mathemaLcal transformaLon (can be difficult someLmes).
Ø Complementary. Not good for uncharged, non-‐basic, low-‐polarity compounds (ESI; e.g. steroids).
Ø Very sensiLve to contaminants such as alkali metals or basic compounds.
Ø RelaLvely low ion currents Ø RelaLvely complex hardware compared to other ion sources
Mass range
Ø Low-‐high Typically less than 200,000 Da.
Flavonoids
“Common” func<ons -‐ An<oxidant
-‐ Protec<on from UV/aging
-‐ Preven<on of lipid oxida<on
-‐ Scavenging of free radicals
Natural Sources -‐ Fruits (berries) -‐ Wine and juices
-‐ Stroke preven<on -‐ An<cancer ac<vity
-‐ Synergic and benefic effects with vitamins A, C and E
-‐ Fer<lity preserva<on
“In animal” func<ons
“In plant” func<ons -‐ Photo-‐quenching
-‐ Insect/animal afrac<on -‐ Symbio<c interac<ons
Flavonoid biosynthesis
Winkel-Shirley, 2001 Jaakola et al., 2001
Cocciolone et al., 2002 Farzad et al., 2003
1. Structural genes
-‐ Maize (P1) (Cocciolone et al., 05)
5-‐fold
2. Regulatory genes
Metabolic engineering of flavonoids
-‐ Tomato (STS) (Schijlen et al., 06)
-‐ Tomato (CHI) (Muir et al., 00)
HPLC of flavonoids in processed tomatoes
60-‐fold
-‐ Tomato (LC/C1) (Bovy et al., 02)
20-‐fold
-‐ Canola (STS) (Husken et al., 05) 360ug/gm
The Purple Tomato
Butelli et al., 08
Construct and fruit phenotype
HPLC of anthocyanins
In vivo experiments
Ripening-‐phenotype in purple tomatoes
