techniques for metabolic study

8/2/2019 Techniques for Metabolic Study

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Submitted to :-

Dr. Diwakar Singh, Ph.D.

Assistant Professor (Biochemistry)

Department of BiotechnologyA.C.H.F, N.A.U., Navsari

Submitted by :-Vanrajsinh H. Solanki

Ph.D. ScholarDepartment of BiotechnologyN. M. College of Agriculture

N.A.U., Navsari


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Metabolomics Newly emerging field of 'omics' research

Comprehensive and simultaneous systematic determination ofmetabolite levels in the metabolome and their changes overtime as a consequence of stimuli

Metabolome Refers to the complete set of small-molecule metabolites Dynamic

Metabolites Intermediates and products of metabolism

Examples include antibiotics, pigments, carbohydrates, fattyacids and amino acids

Primary and secondary metabolites


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2000-1500 BC

The first paper was titled, Quantitative Analysis of Urine Vapor

and Breath by Gas-Liquid Partition Chromatography, byRobinson and Pauling in 1971.

The name metabolomics was coined in the late 1990s (the firstpaper using the word metabolome is Oliver, S. G., Winson, M. K.,Kell, D. B. & Baganz, F. (1998). Systematic functional analysis ofthe yeast genome.

Many of the bioanalytical methods used for metabolomics have

been adapted (or in some cases simply adopted) from existingbiochemical techniques.

Human Metabolome project first draft of human metabolome in2007

HISTORY


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Four main points in Analysis of metabolomics data :

Efficient and unbiased

Separation of analytes

Detection

Identification and quantification


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Separation Techniques

Gas Chromatography (GC)

Capillary Electrophoresis (CE) High Performance Liquid Chromatography (HPLC)

Combination of Techniques

GC-MS

HPLC-MS

Detection Techniques

Nuclear Magnetic Resonance Spectroscopy (NMR)

Mass Spectrometry (MS)


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WHATIS CHROMATOGRAPHY?

Chromatography is a technique for

separating mixtures into their componentsin order to analyze, identify, purify, and/orquantify the mixture or components.

Separate

Analyze

Identify

Purify

QuantifyComponents

Mixture


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Types of Chromatography

Liquid Chromatographyseparates liquid samples with a liquidsolvent (mobile phase) and a column composed of solid beads(stationary phase)

Gas Chromatographyseparates vaporized samples with a carrier

gas (mobile phase) and a column composed of a liquid or ofsolid beads (stationary phase)

Paper Chromatographyseparates dried liquid samples with a liquidsolvent (mobile phase) and a paper strip (stationary phase)

Thin-Layer Chromatographyseparates dried liquid samples witha liquid solvent (mobile phase) and a glass plate covered with athin layer of alumina or silica gel (stationary phase)


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USESFOR CHROMATOGRAPHY

Chromatography is used by scientists to:

Analyze examine a mixture, its components, andtheir relations to one another

Identify determine the identity of a mixture orcomponents based on known components

Purify separate components in order to isolate one ofinterest for further study

Quantifydetermine the amount of the a mixtureand/or the components present in the sample


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1.HPLC(High Performance Liquid Chromatography)


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1. High-Performance Liquid Chromatography

Mobile phase reservoirs

HPLC Pump(s)

Mixing valves

Sample injector (manual or auto)

Column

Detector

Plumming

Mobile phase waste container


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HPLC-UV

MobilePhases

A and B

HPLCPump

syringe

6-portvalve

Sampleloop

HPLCcolumn

Detector

MP waste

Jacket forcontrolling columntemperature


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2.

Gas Chromatography


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Instruments;

Carrier Gas

Flow regulators and meters

Sample injection system

Columns & ovens

Detectors

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INSTRUMENTATION


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SCHEMATIC DIAGRAM OF GASCHROMATOGRAPH


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CARRIER GAS

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The mobile phase gas is called the carrier gas andmust be chemically inert.

Sample componet column detector

mobile phase gasHelium ,argon ,nitrogen , carbon dioxide and

hydrogen also used.

Selection of the best crrier gas very important ,

because it effects both the column separation anddetector performance .

The ratio of viscosity of diffusion coefficient shouldbe minimum for rapid analysis thats why H, He

are prepared for a carrier gas .


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Impurities in the carrier gas such as air water vapour and tracegaseous hydrocarbons can cause sample reaction, column

character and affect the detector performance.

The carrier gas system should contains a molecular sieve toremove water and other impurities.

These gases are available in pressurized tanks. presureregulateres and flow meters are required to control the flowrate of the gas.

The gases are supplied from the high pressure gas cylinder ,being stored at pressure up to 300psi

carrier gas should be better then 99.99%and 99.999% is oftenused

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PROCESS FLOW SCHEMATIC

Carrier gas(nitrogen orhelium)

Sample injection

Long Column (30 m)

Detector (flameionizationdetector or FID)

HydrogenAir


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SAMPLE INJECTION PORT

Calibated Microsyringes are used to

inject liquid sample Purge :volatile components are

removed from sample by gentle heating

Rubber or silicone diaphragm(septum)

Sample port T: 50C Packed C: sample sizes-1 to 20 L

Capillary C: 10 to 30 mL

splitter is used to deliver a fractionof injection(1:50 to 1:500)

Avaid over loading

Slow injection & oversized samplescause band spreading & poorresolution

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COLUMN CONFIGURATIONS

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Two types of columns are used in gas

chromatography, packed and open tubular or

capillary.

Packed column length from less than 2 m to 5 m

Capillary columns from few m to 100 m

They are constructed of stainless steel, glass, fused

silica, or Teflon.


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COLUMN OVENS

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Column temperature is very important in GC

The column is ordinarily housed in a thermostated

oven.

they are usually formed as coils having diametersof 10 to 30 cm.

The optimum column temperature depends upon the

boiling point of the sample and the degree of

separation required. Roughly, a temperature equal to or slightly above the

average boiling point of a sample results in a

reasonable elution time (2 to 30 min).


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COLUMN

Types of columns

1.packed columns

2. Open tubular or capillary.

27Capillary column- 30mPacked column-3m


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PACKEDCOLUMNS

Packed columns are fabricated from glass, metal(stainless steel, copper, aluminum), or Teflon tubesthat typically have

Lengths------ 2 to 3 m

Inside diameters ------- 2 to 4 mm.

These tubes are densely packed with a uniform,finely divided packing material, or solid support, thatis coated with a thin layer (0.05 m) of thestationary liquid phase.

In order to fit in a thermostating oven, the tubes areformed as coils having diameters of roughly 15 cm.

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OLDER PACKED COLUMNS

Older packed columns uniform silica particles(150-250 m)

required to ensure uniform path lengths

usually 1/8 (3.2 mm OD, 2.2 mm I.D.) diameter, 1

2 m length

max flow rate about 1 mL/min or 8 cm/min

The columns themselves were either glass or

stainless steel

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CAPILLARY (OR)OPEN TUBULAR COLUMNS

1.Wall-coated open tubular (WCOT)

Capillary tubes coated with a thin layer ofstationary phase

Old: stainless steel, Al, Cu, plastic, glass.

2.Support-coated open tubular (SCOT)

Inner surface of the capillary is lined with a thinfilm (~30m) of a support materials, likediatomaceous earth

Lower efficiency than WCOT, higher than packedcolumn

3.Fused-silica open tubular column (FSOT):

Physical strength, low reactivity, flexibility. 0.32 to0.25 mm


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COLUMN STATIONARY PHASES:

Packed

liquid coated silica particles (


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THE STATIONARY PHASE

requirements are:

Low vapor pressure

Thermal stability

Low viscosity (for fast mass transfer) High selectivity for compounds of interest

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DETECTORS Use: Detect the difference between a pure carrier gas

&eluted compound Ideal detector:

High sensitivity to even small concentrtion

linerity, ie, less response to low concentration&proportional response to high concentration

Large linear dynamic range

Useful at a range of temperatures

Good stability and reproducibility

Rapid response time

Easy to use Stable, Predictable response

Inexpensive

operation from RT to 400 oC34


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TYPES OFDETECTORS

1. Thermal Conductivity Detector(TCD)2. Flame Ionization Detector(FID)

3. Atomic Emission Detector(AED)

4. Electron Capture Detector(ECD)

5. Nitrogen Phosphoroes Detector(NPD)6. Photo Ionization Detector(PID)

7. Flame Photometric detector(FPD)

8. Electrolytic conductivity detector (Hall detector)

9. Absolute Mass Detector(AMD)

10. Thermionic Detector(TD)

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APPLICATIONS


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QUALITATIVE ANALYSIS:

Retention time data should be useful foridentification of mixtures

Comparing the retention time of the sample as wellas the standard

Checking the purity of a compound: compar thestandard and sample

Additional peaks are obtained..impurities are

present.compound is not pure

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QUANTITATIVE ANALYSIS:

Direct comparison method:

-comparing the area of the peak, peak height,width of peak.

Calibration curves:

-standards of varying concentration are useddetermine peak areas .

o Internal standard method:

-A known concentration of the internal standard

is added separately to the standard solution-The peak area ratio of sample and internal

standard.unknown concentration is easilydetermined .

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ELEMENTAL ANALYSIS

Determination of C,H ,O ,S and N .

Determination of mixture of drugs

Isolation and identification of drugs

Isolation and identification of mixture ofcomponents(amino acids ,plant extracts ,volatileoils)

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3.CAPILLARYELECTROPHORESIS

ELECTROPHORESISAN OVERVIEW


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ELECTROPHORESIS AN OVERVIEW

Definition: The differential movement for migration of ions

by attraction or repulsion in an electric field.

- Separation of components of a mixture using an electric field

v=Eq/f

v = velocity of molecule

E = electric field

q = net charge of molecule

f = friction coefficient

o Can determine the size, shape, and charge of a molecule

o Different forms of electrophoresis are used for each of thesefactors independently or in combination.


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WHAT IS CAPILLARY ELECTROPHORESIS

In practical terms, a positive (anode) and negative

(cathode) electrode are placed in a solutioncontaining ions.

Then, when a voltage is applied across theelectrodes, solute ions of different charge, i.e.,

anions (negative) and cations (positive), willmove through the solution towards the electrodeof opposite charge.

Capillary electrophoresis, then, is the technique of

performing electrophoresis in buffer-filled, narrow-bore capillaries, normally from 25 to 100 pm ininternal diameter (ID).


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CAPILLARY ELECTROPHORESIS

THE BASICS OF INSTRUMENTATION

Electrophoresis in a buffer filled, narrow-borecapillaries

Each capillary is about 25-100 m in internaldiameter

When a voltage is applied to the solution, themolecules move through the solution towards theelectrode of opposite charge

Depending on the charge, the molecules move

through at different speeds Separation is achieved


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BASICS CONT.

A photocathode is thenused to measure theabsorbencies of the

molecules as they passthrough the solution

The absorbencies areanalyzed by a computer

and they are representedgraphically


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THE ELECTROPHEROGRAM


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THE ELECTROPHEROGRAM

The data output from CE ispresented in the form of anelectropherogram, which isanalogous to a chromatogram.

An electropherogram is a plotof migration time vs. detectorresponse.

The detector response is usuallyconcentration dependent, suchas UV-visible absorbance orfluorescence.

The appearance of a typical

electropherogram is shown inFigure for the separation of athreecomponent mixture ofcationic, neutral and anionicsolutes.


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EQUIPMENT

Capillary tube

Varied length butnormally 25-50 cm

Small bore andthickness of the silicaplay a role

Using a smaller internaldiameter and thicker walls

help prevent JouleHeating, heating due tovoltage


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EQUIPMENT CONT.

Detector

UV/Visible absorption

Fluorescence

Radiometric (for radioactive

substances) Mass Spec.


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FACTORS AFFECTING THE EFFICIENCY OFCE

Buffers

Additives for CZE Additives for HPCE

Hints

Ionic Strength in HPCE

PKa Values of Common Buffers

Proteins, Choosing a Proper Buffer

Capillaries

Conditioning

Dimensions, Changing

How to Properly Cut A Capillary

Storage

Data Analysis

Migrating Peak Correction
http://www.microsolvtech.com/cehint15.asphttp://www.microsolvtech.com/cehint2.asphttp://www.microsolvtech.com/cehint3.asphttp://www.microsolvtech.com/cehint14.asphttp://www.microsolvtech.com/cehint6.asphttp://www.microsolvtech.com/cehint16.asphttp://www.microsolvtech.com/cehint5.asphttp://www.microsolvtech.com/cehint13.asphttp://www.microsolvtech.com/cutcap.asphttp://www.microsolvtech.com/cehint4.asphttp://www.microsolvtech.com/cehint1.asphttp://www.microsolvtech.com/cehint1.asphttp://www.microsolvtech.com/cehint4.asphttp://www.microsolvtech.com/cutcap.asphttp://www.microsolvtech.com/cehint13.asphttp://www.microsolvtech.com/cehint5.asphttp://www.microsolvtech.com/cehint16.asphttp://www.microsolvtech.com/cehint6.asphttp://www.microsolvtech.com/cehint14.asphttp://www.microsolvtech.com/cehint3.asphttp://www.microsolvtech.com/cehint2.asphttp://www.microsolvtech.com/cehint15.asp


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Peptide analysis routinaly carried out

Diagnostic and gene cloning experiments For DNA sequencing and the polymerase chain reaction

(PCR) Point mutation in DNA such as occur in a human

disease Chiral compounds can be resolved

A range of small molecules, drug and metabolites canbe measured in physiological solution such as urine andserum. These includes amino acids , nucleotides,nucleosides, bases, anions such as chlorides andsulphate (NO2

- and NO3-) and cations such as Ca+2 and

Fe+3 .

APPLICATIONS


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APPLICATIONS

Analysis of carbohydrates

Analysis of inorganicanions/metal ions

DNA profiling

Protein identification

Advantages

Fast

Small Sample

Relatively inexpensive

Automated

Disadvantages

Cannot identify neutralspecies

Joule HeatingCannot discern shape

S


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SUMMARY

1. CE is based on the principles of electrophoresis.2. The speed of movement or migration of solutes in

CE is determined by their

3. size and charge. Small, highly charged solutes will

migrate more quickly than large, less chargedsolutes.

4. Bulk movement of solutes is caused by EOF.

5. The speed of EOF can be adjusted by changing thebuffer pH used.

6. The flow profile of EOF is flat, yielding highseparation efficiencies.

7. The data output from CE is called anelectropherogram.

C


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CONCLUSION

It is the most efficient separation techniqueavailable for the analysis of both large and smallmolecules.

DNA Profiling, protein identification,

inorganic metals and ions can be detectedeasily by this method.


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4.NMR(Nuclear magnetic resonance (NMR)

spectroscopy)(Molecular Spectroscopy)

Nuclear Magnetic Resonance (NMR) spectroscopy is theb i f di f i b i l i i hi l


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absorption of radio frequencies by atomic nuclei within a samplethat is placed in a magnetic field.

The types of sample that can be studied are liquids, solids andwhole organisms.

NMR spectroscopy finds applications in several areas of scienceand is routinely used to study chemical and biochemical structure

and function using simple one-dimensional techniques and morecomplicated multidimensional techniques.

Nuclear magnetic resonance was first described and measuredin molecular beams by Isidor Rabi in 1938 (1944, Nobel Prize in

physics).

Felix Bloch and Edward Mills Purcell expanded the technique foruse on liquids and solids, for which they shared the Nobel Prize inPhysics in 1952.

M S


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MOLECULAR SPECTROSCOPY

Nuclear magnetic resonance (NMR)spectroscopy: A spectroscopic technique thatgives us information about the number andtypes of atoms in a molecule, for example, about

the number and types of

hydrogen atoms using 1H-NMR spectroscopy.

carbon atoms using 13C-NMR spectroscopy.

phosphorus atoms using 31P-NMR spectroscopy.


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NUCLEAR MAGNETIC RESONANCE

Resonance: In NMR spectroscopy, resonance isthe absorption of energy by a precessing nucleusand the resulting flip of its nuclear spin from alower energy state to a higher energy state.

The processing spins induce an oscillatingmagnetic field that is recorded as a signal by theinstrument.

Signal: A recording in an NMR spectrum of a nuclearmagnetic resonance.


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NMR SPECTROMETER Schematic diagram of a nuclear magnetic resonance

spectrometer.

NMR S C O


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NMR SPECTROMETER

Essentials of an NMR spectrometer are a powerful magnet,

a radio-frequency generator, and a radio-frequencydetector.

The sample is dissolved in a solvent, most commonlyCDCl

3or D

2O, and placed in a sample tube which is then

suspended in the magnetic field and set spinning.

Using a Fourier transform NMR (FT-NMR) spectrometer, aspectrum can be recorded in about 2 seconds.

NMR S


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NMR SPECTRUM

1H-NMR spectrum of methyl acetate.

High frequency: The shift of an NMR signal to theleft on the chart paper.

Low frequency: The shift of an NMR signal to theright on the chart paper.


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SIGNAL AREAS

Relative areas of signals are proportional to thenumber of H giving rise to each signal, ModernNMR spectrometers electronically integrate andrecord the relative area of each signal.


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4.1. Basic NMR techniques of nmr

When placed in a magnetic field, NMR active nuclei (such

as 1H or 13C) absorb electromagnetic radiation at afrequency characteristic of the isotope.

The resonant frequency, energy of the absorption and the

intensity of the signal are proportional to the strength of themagnetic field.

For example, in a 21 tesla magnetic field, protons resonateat 900 MHz. It is common to refer to a 21 T magnet as a900 MHz magnet, although different nuclei resonate at adifferent frequency at this field strength in proportion totheir nuclear magnetic moments.

4 1 1 Chemical shift
http://en.wikipedia.org/wiki/Electromagnetic_radiationhttp://en.wikipedia.org/wiki/Isotopehttp://en.wikipedia.org/wiki/Tesla_(unit)http://en.wikipedia.org/wiki/Protonhttp://en.wikipedia.org/wiki/Megahertzhttp://en.wikipedia.org/wiki/Nuclear_magnetic_momenthttp://en.wikipedia.org/wiki/Nuclear_magnetic_momenthttp://en.wikipedia.org/wiki/Nuclear_magnetic_momenthttp://en.wikipedia.org/wiki/Nuclear_magnetic_momenthttp://en.wikipedia.org/wiki/Nuclear_magnetic_momenthttp://en.wikipedia.org/wiki/Nuclear_magnetic_momenthttp://en.wikipedia.org/wiki/Megahertzhttp://en.wikipedia.org/wiki/Protonhttp://en.wikipedia.org/wiki/Tesla_(unit)http://en.wikipedia.org/wiki/Isotopehttp://en.wikipedia.org/wiki/Electromagnetic_radiationhttp://en.wikipedia.org/wiki/Electromagnetic_radiationhttp://en.wikipedia.org/wiki/Electromagnetic_radiation


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4.1.1 Chemical shift

Depending on their local chemical environment, differentnuclei in a molecule absorb at slightly differentfrequencies. Since this resonant frequency is directlyproportional to the strength of the magnetic field, the shiftis converted into a field-independentdimensionless valueknown as the chemical shift.

The chemical shift is reported as a relative measure fromsome reference resonance frequency.

For the nuclei1

H,13

C, and29

Si, TMS (tetramethylsilane)is commonly used as a reference.
http://en.wikipedia.org/wiki/Chemical_shifthttp://en.wikipedia.org/wiki/Tetramethylsilanehttp://en.wikipedia.org/wiki/Tetramethylsilanehttp://en.wikipedia.org/wiki/Chemical_shifthttp://en.wikipedia.org/wiki/Chemical_shifthttp://en.wikipedia.org/wiki/Chemical_shift


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Some of the most useful information for structuredetermination in a one-dimensional NMR spectrum comes

from J-coupling or scalar coupling (a special case of spin-

spin coupling) between NMR active nuclei. This coupling

arises from the interaction of different spin states through the

chemical bonds of a molecule and results in the splitting of

NMR signals.

4.1.2 J-coupling

4 1 3 Second-order (or strong) coupling
http://en.wikipedia.org/wiki/Spin-spin_couplinghttp://en.wikipedia.org/wiki/Spin-spin_couplinghttp://en.wikipedia.org/wiki/Spin-spin_couplinghttp://en.wikipedia.org/wiki/Spin-spin_couplinghttp://en.wikipedia.org/wiki/Spin-spin_couplinghttp://en.wikipedia.org/wiki/Spin-spin_couplinghttp://en.wikipedia.org/wiki/Spin-spin_coupling


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4.1.3 Second-order (or strong) coupling

Second-order effects decrease as the frequency

difference between multiplets increases.

NMR spectra display less distortion than lowerfrequency spectra.

Early spectra at 60 MHz were more prone to distortionthan spectra from later machines typically operating atfrequencies at 200 MHz or above.

v


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4.1.4 Magnetic inequivalence

More subtle effects can occur if chemically equivalentspins (i.e., nuclei related by symmetry and so having thesame NMR frequency) have different couplingrelationships to external spins.

Spins that are chemically equivalent but are notindistinguishable (based on their coupling relationships)are termed magnetically inequivalent.

Magnetic inequivalence can lead to highly complexspectra which can only be analyzed by computationalmodeling.

4 2 Correlation spectroscopy


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4.2 Correlation spectroscopy

Correlation spectroscopy is one of several types of two-dimensional nuclear magnetic resonance (NMR) spectroscopyor 2D-NMR.

Two-dimensional nuclear magnetic resonancespectroscopy (2D NMR) is a set of nuclear magnetic resonance

spectroscopy (NMR) methods which give data plotted in a spacedefined by two frequency axes rather than one.

This type of NMR experiment is best known byits acronym, COSY. Other types of two-dimensional NMR include

J-spectroscopy, exchange spectroscopy (EXSY), NuclearOverhauser effect spectroscopy (NOESY), Total CorrelationSpectroscopy (TOCSY) and heteronuclear correlationexperiments, such as HSQC, HMQC, and HMBC.

4.3 Biomolecular NMR spectroscopy
http://en.wikipedia.org/wiki/Correlation_spectroscopyhttp://en.wikipedia.org/wiki/2D-NMRhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Acronymhttp://en.wikipedia.org/wiki/Correlation_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_Overhauser_effecthttp://en.wikipedia.org/wiki/Nuclear_Overhauser_effecthttp://en.wikipedia.org/wiki/HSQChttp://en.wikipedia.org/wiki/HMQChttp://en.wikipedia.org/wiki/HMBChttp://en.wikipedia.org/wiki/HMBChttp://en.wikipedia.org/wiki/HMQChttp://en.wikipedia.org/wiki/HSQChttp://en.wikipedia.org/wiki/Nuclear_Overhauser_effecthttp://en.wikipedia.org/wiki/Nuclear_Overhauser_effecthttp://en.wikipedia.org/wiki/Nuclear_Overhauser_effecthttp://en.wikipedia.org/wiki/Nuclear_Overhauser_effecthttp://en.wikipedia.org/wiki/Nuclear_Overhauser_effecthttp://en.wikipedia.org/wiki/Correlation_spectroscopyhttp://en.wikipedia.org/wiki/Acronymhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/2D-NMRhttp://en.wikipedia.org/wiki/2D-NMRhttp://en.wikipedia.org/wiki/2D-NMRhttp://en.wikipedia.org/wiki/2D-NMRhttp://en.wikipedia.org/wiki/Correlation_spectroscopyhttp://en.wikipedia.org/wiki/Correlation_spectroscopyhttp://en.wikipedia.org/wiki/Correlation_spectroscopy


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Nuclear magnetic resonance spectroscopy of

proteins (usually abbreviated protein NMR) is a fieldof structural biology in which NMR spectroscopy is used toobtain information about the structure and dynamics ofproteinsThe field was pioneered by Richard R. Ernst and Kurt

WthrichStructure determination by NMR spectroscopy usuallyconsists of several following phases each using a separateset of highly specialized techniques.

a.Sample Preparationb.Data Collectionc.Resonance Assignmentd.Restrain Generatione.Structure is calculated and validated

4.3.1 NMR OF PROTEIN

CONT
http://en.wikipedia.org/wiki/Structural_biologyhttp://en.wikipedia.org/wiki/NMR_spectroscopyhttp://en.wikipedia.org/wiki/Richard_R._Ernsthttp://en.wikipedia.org/wiki/Kurt_W%C3%BCthrichhttp://en.wikipedia.org/wiki/Kurt_W%C3%BCthrichhttp://en.wikipedia.org/wiki/Kurt_W%C3%BCthrichhttp://en.wikipedia.org/wiki/Kurt_W%C3%BCthrichhttp://en.wikipedia.org/wiki/Kurt_W%C3%BCthrichhttp://en.wikipedia.org/wiki/Richard_R._Ernsthttp://en.wikipedia.org/wiki/Richard_R._Ernsthttp://en.wikipedia.org/wiki/Richard_R._Ernsthttp://en.wikipedia.org/wiki/Richard_R._Ernsthttp://en.wikipedia.org/wiki/Richard_R._Ernsthttp://en.wikipedia.org/wiki/Richard_R._Ernsthttp://en.wikipedia.org/wiki/NMR_spectroscopyhttp://en.wikipedia.org/wiki/NMR_spectroscopyhttp://en.wikipedia.org/wiki/NMR_spectroscopyhttp://en.wikipedia.org/wiki/Structural_biologyhttp://en.wikipedia.org/wiki/Structural_biologyhttp://en.wikipedia.org/wiki/Structural_biology


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Protein nuclear magnetic resonance is performed on

aqueous samples of highly purified protein.

Usually the sample consist of between 300 and 600microlitres with a protein concentration in the range 0.1 3 millimolar.

The source of the protein can be either natural orproduced in an expression system using recombinantDNA techniques through geneticengineering.

Recombinantly expressed proteins are usually easier toproduce in sufficient quantity, andmakes isotopiclabelling possible.

SAMPLE FOR NMR

APPLICATION
http://en.wikipedia.org/wiki/Protein_purificationhttp://en.wikipedia.org/wiki/Mole_(unit)http://en.wikipedia.org/wiki/Protein_expressionhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Genetic_engineeringhttp://en.wikipedia.org/wiki/Protein_expressionhttp://en.wikipedia.org/wiki/Isotopehttp://en.wikipedia.org/wiki/Isotopic_labelinghttp://en.wikipedia.org/wiki/Isotopic_labelinghttp://en.wikipedia.org/wiki/Isotopehttp://en.wikipedia.org/wiki/Protein_expressionhttp://en.wikipedia.org/wiki/Genetic_engineeringhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Protein_expressionhttp://en.wikipedia.org/wiki/Protein_expressionhttp://en.wikipedia.org/wiki/Protein_expressionhttp://en.wikipedia.org/wiki/Mole_(unit)http://en.wikipedia.org/wiki/Protein_purification


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To obtain high resolution 3-dimensional structures of the

protein, similar to what can be achieved by X-raycrystallography (limited to proteins smaller than 35 kDa ).

Only way to obtain high resolution information on partiallyor wholly intrinsically unstructured proteins.

Common tool for the determination of Conformation ActivityRelationships where the structure before and after interactionwith, for example, a drug candidate is compared to its known

biochemical activity.

APPLICATION

4.3.2 NMR OF NUCLEIC ACID
http://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/KDahttp://en.wikipedia.org/wiki/Intrinsically_unstructured_proteinshttp://en.wikipedia.org/wiki/Conformation_Activity_Relationshiphttp://en.wikipedia.org/wiki/Conformation_Activity_Relationshiphttp://en.wikipedia.org/wiki/Conformation_Activity_Relationshiphttp://en.wikipedia.org/wiki/Conformation_Activity_Relationshiphttp://en.wikipedia.org/wiki/Conformation_Activity_Relationshiphttp://en.wikipedia.org/wiki/Conformation_Activity_Relationshiphttp://en.wikipedia.org/wiki/Conformation_Activity_Relationshiphttp://en.wikipedia.org/wiki/Intrinsically_unstructured_proteinshttp://en.wikipedia.org/wiki/Intrinsically_unstructured_proteinshttp://en.wikipedia.org/wiki/Intrinsically_unstructured_proteinshttp://en.wikipedia.org/wiki/Intrinsically_unstructured_proteinshttp://en.wikipedia.org/wiki/Intrinsically_unstructured_proteinshttp://en.wikipedia.org/wiki/KDahttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallography


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Nuclear magnetic resonance spectroscopy of nucleic acids, oftenreferred to as nucleic acid NMR, is the use of nuclear magnetic

resonance spectroscopy to obtain information about the STRUCTUREAND DYNAMICS OF NUCLEIC ACID MOLECULES, such as DNA orRNA.

NMR has advantages over the other method for high-

resolution nucleic acid structure determination.

X-ray crystallography, in that the molecules are being OBSERVED INTHEIR NATURAL STATE OF BEING IN SOLUTION, rather than ina crystal lattice which may affect the molecule's structural properties.

Nucleic acid NMR is USEFUL FOR MOLECULES OF UP TO 100NUCLEOTIDES. As of 2003, nearly half of all known RNA structureshad been determined by NMR spectroscopy.

4.3.2 NMR OF NUCLEIC ACID

FTIR
http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/Nucleic_acid_structure_determinationhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/Crystal_latticehttp://en.wikipedia.org/wiki/Crystal_latticehttp://en.wikipedia.org/wiki/Crystal_latticehttp://en.wikipedia.org/wiki/Crystal_latticehttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/Nucleic_acid_structure_determinationhttp://en.wikipedia.org/wiki/Nucleic_acid_structure_determinationhttp://en.wikipedia.org/wiki/Nucleic_acid_structure_determinationhttp://en.wikipedia.org/wiki/Nucleic_acid_structure_determinationhttp://en.wikipedia.org/wiki/Nucleic_acid_structure_determinationhttp://en.wikipedia.org/wiki/Nucleic_acid_structure_determinationhttp://en.wikipedia.org/wiki/Nucleic_acid_structure_determinationhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopyhttp://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_spectroscopy


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FTIRFourier transform spectroscopy is a measurementtechnique whereby spectra are collected based on

measurements of the coherence of a radiative source,using time-domain or space-domain measurements ofthe electromagnetic radiation or other type of radiation.
http://en.wikipedia.org/wiki/Coherence_(physics)http://en.wikipedia.org/wiki/Radiatehttp://en.wikipedia.org/wiki/Time-domainhttp://en.wikipedia.org/wiki/Electromagnetic_radiationhttp://en.wikipedia.org/wiki/Electromagnetic_radiationhttp://en.wikipedia.org/wiki/Electromagnetic_radiationhttp://en.wikipedia.org/wiki/Electromagnetic_radiationhttp://en.wikipedia.org/wiki/Time-domainhttp://en.wikipedia.org/wiki/Time-domainhttp://en.wikipedia.org/wiki/Time-domainhttp://en.wikipedia.org/wiki/Radiatehttp://en.wikipedia.org/wiki/Coherence_(physics)


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5. Mass Spectroscopy

(MS)

MS HISTORY


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MS HISTORY

JJ Thomson built MS prototype to measure m/z of

electron, awarded Nobel Prize in 1906 MS concept first put into practice by Francis Aston, a

physicist working in Cambridge England in 1919.Designed to measure mass of elements (Nobel Prize in1922).

1948-52 - Time of Flight (TOF) mass analyzers introduced.

1955 - Quadrupole ion filters introduced by W. Paul, alsoinvents the ion trap in 1983 (wins 1989 Nobel Prize)

1968 - Tandem mass spectrometer appears Mass spectrometers are now one of the MOST

POWERFUL ANALYTIC TOOLS IN CHEMISTRY

All chemical substances are combinations of atoms


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All chemical substances are combinations of atoms.

Atoms of different elements have different masses (H = 1, C = 12,

O = 16, S = 32, etc.)

An element is a substance that cannot be broken down into asimpler species by chemical means - has a unique atomic number

corresponding to the number of protons in the nucleus

Different atoms combine in different ways to form molecular sub-

units called functional groups. Mass of each group is the combined mass of the atoms forming the

group (often unique).

e.g. phenyl (C6H5) mass = 77, methyl (CH3) mass = 15, etc.

So:- If you break molecule up into constituent groups and measure

the mass of the individual fragments (using MS) - Can determine whatgroups are present in the original molecule and how they are combinedtogether

Can work out molecular structure

What is Mass Spectrometry?


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Mass spectrometry is a powerful technique for

chemical analysis that is used to identify unknown compounds,to quantify known compounds, and to elucidate molecularstructure

Principle of operation

A Mass spectrometer is a MOLECULE SMASHER

Measures molecular and atomic masses of wholemolecules, molecular fragments and atoms by generation anddetection of the corresponding gas phase ions, separated

according to their mass-to-charge ratio (m/z).

Measured masses correspond to molecular structureand atomic composition of parent molecule allowsdetermination and elucidation of molecular structure.



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May also be used for quantitation of MOLECULAR

SPECIES.

VERY SENSITIVE TECHNIQUE - Works with minutequantities of samples (as low as 10-12g, 10-15 moles) and is

easily interfaced with chromatographic separation methodsfor identification of components in a mixture

Mass spectrometry provides valuable information to awide range of professionals: chemists, biologists, physicians,

astronomers, environmental health specialists, to name afew.

Limitation is a Destructive technique cannot reclaimsample

What is a Mass Spectrometer?


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Many different types each has different advantages,

draw-backs and applicationsAll consist of 4 major sections linked together

Inlet Ionization source Analyser Detector

All sections usually maintained under high vacuum

All functions of instrument control, sample acquisition anddata processing under computer control

Data system and Computer Control is often overlooked

most significant advance in MS allows 24/7 automationand development of modern powerful analyticaltechniques.

What is a Mass Spectrometer?


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80

All Instruments Have:

1. Sample Inlet

2. Ion Source

3. Mass Analyzer

Detector

Data System

accelerate separate

How does it work?


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81

e-

+

e-

e-

+4000 V 0 V

+

e-

e-

heavy

light

Magnetic and/orelectric field

sample

vapourise

ionisep

+A

+

B

+

CA+ B+ C+

vac

uum

MASSSPECTROMETRY

Analyser Types


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Analyser Types

Analyser is the section of instrument that separates ionsof different m/z

Many Different technologies

A. Magnetic SectorB. Quadrupole

C. Ion Trap

D. ToF

All based on momentum separation

A. Magnetic Sector


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83


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C. Quadrupole ion Trap


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85

3 Electrode system

2 x Endcap and 1x Ring Electrode

Now have recent develpoment ofLinear Ion Trap and orbitrap

Developments on same theme.

Analyser Types Quadrupole ion Trap


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86Ion Trap is very small most of instrument is ion guides intothe trap itself

MASS SPECTROMETER INSTRUMENT DESIGN


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DIFFERENT TYPES OF IONIZATION SOURCE

EI, CI, FAB, ESI, Maldi, (APCI, DESI, DART)(Also sources for inorganic analysis ICP, GD, etc.)

DIFFERENT TYPES OF ANALYSER

Magnetic Sector, Quadrupole, Ion Trap, ToF

Different sources and analysers have different properties, advantagesand disadvantages

Selection of appropriate ionization method and analyzer are critical anddefines MS applications.

Wide range of MS applications


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APPLICATIONS of ms


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Advances in Proteomics and other areas in biotechnology

made possible by development of soft ionisation Maldi andESI MS techniques

Protein and peptide analysis for MW determination

Protein Identification and profiling using digests and data

base searching major development in ProteomicsProtein post-translational modification

Protein structure characterisation

Maldi-Imaging

Oligo-nucleotide analysis Confirmation of purity ofsynthetic oligos

Carbohydrate analysis

APPLICATIONS of ms

CONT


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Automated high throughput analysisScreening of biological samples

Pharmicokinetics

LC-MS seperation and identification of componentsof complex mixtures Normally LC-ESI, nowincreasingly LC-Maldi-ToF

Intact virus analysis

Cell imaging (Maldi)

Tissue Imaging (Maldi)

CONT

MS PRINCIPLES


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MS PRINCIPLES

Find a way to charge an atom or molecule(ionization)

Place charged atom or molecule in a magnetic

field or subject it to an electric field andmeasure its speed or radius of curvaturerelative to its mass-to-charge ratio (massanalyzer)

Detect ions using microchannel plate orphotomultiplier tube

MASS SPECTROMETRY (MS)


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Introduce sample to the instrument

Generate ions in the gas phaseSeparate ions on the basis of differences in m/z

with a mass analyzer

Detect ions

Generalized Protein Identification by MS


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Artificialspectra built

Artificiallytrypsinated

Database ofsequences

(i.e. SwissProt)

Spot removed

from gel

Fragmented

using trypsin

Spectrum offragmentsgenerated

MATCH

Library


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Methods for

proteinidentification

MS PRINCIPLES


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S C S

Different elements can be uniquely identified by theirmass

MS PRINCIPLES


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Different compounds can be uniquely identified bytheir mass

CH3CH2OH

N

OH

HO

-CH2-

-CH2CH-NH2

COOH

HO

HO

Butorphanol L-dopa Ethanol

MW = 327.1 MW = 197.2 MW = 46.1

MASS SPECTROMETRY


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MASS SPECTROMETRY

Analytical method to measure themolecular or atomic weight of samples

M S


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MASS SPECTROMETRY

For small organic molecules the MW can be determined towithin 5 ppm or 0.0005% which is sufficiently accurate toconfirm the molecular formula from mass alone

For large biomolecules the MW can be determined within

an accuracy of 0.01% (i.e. within 5 Da for a 50 kD protein)

Recall 1 dalton = 1 atomic mass unit (1 amu)

MASS SPEC PRINCIPLES


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MASS SPEC PRINCIPLES

Ionizer

Sample

+_

Mass Analyzer Detector

MASSSPECTROMETERS


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Linear Time Of Flight tube

Reflector Time Of Flight tube

detector

reflector

ion source

ion source

detector

time of flight

time of flight

Time of flight (TOF) (MALDI) Measures the time required for ions to fly down the length

of a chamber.

Often combined with MALDI (MALDI-TOF) Detections frommultiple laser bursts are averaged. Multiple laser

Tandem MS- MS/MS-separation and identification of compounds in complexmixtures- induce fragmentation and mass analyze the fragment ions.- Uses two or more mass analyzers/filters separated by a

collision cell filled with Argon or Xenon

Different MS-MS configurations

Quadrupole-quadrupole (low energy)

Magnetic sector-quadrupole (high)

Quadrupole-time-of-flight (low energy)

Time-of-flight-time-of-flight (low energy)

All proteins are sorted based on a


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M/ZRATIO:

Molecular weight divided by the chargeon this protein

p

mass to charge ratio (m/z)


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THANK YOU

techniques for metabolic study

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