Submitted to :Dr. Diwakar Singh, Ph.D. Assistant Professor (Biochemistry) Department of Biotechnology A.C.H.F, N.A.U.

, Navsari

Submitted by :Vanrajsinh H. Solanki Ph.D. Scholar Department of Biotechnology N. M. College of Agriculture N.A.U., Navsari

 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 Refers to the complete set of small-molecule metabolites Dynamic Intermediates and products of metabolism Examples include antibiotics, pigments, carbohydrates, fatty acids and amino acids Primary and secondary metabolites

Metabolome

Metabolites

HISTORY

2000-1500 BC 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

 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)

Combination

of Techniques

GC-MS HPLC-MS

Detection

Techniques

Nuclear Magnetic Resonance Spectroscopy (NMR) Mass Spectrometry (MS)

WHAT IS CHROMATOGRAPHY?
Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components.

Analyze
Separat e

Identify

Purify
Mixture Component s

Quantify

Types of Chromatography
Liquid Chromatography separates liquid samples

solvent (mobile phase) and a column (stationary phase) gas (mobile phase) and a column solid beads (stationary phase) solvent (mobile phase) and a

composed

with a liquid of solid beads

Gas Chromatography separates vaporized samples

with a carrier composed of a liquid or of

Paper Chromatography separates dried liquid

samples with a liquid paper strip (stationary phase)

Thin-Layer Chromatography separates dried liquid

samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

USES FOR CHROMATOGRAPHY

Chromatography is used by scientists to:

Analyze examine a mixture, its components, and their relations to one another Identify determine the identity of a mixture or components based on known components Purify separate components in order to isolate one of interest for further study Quantify determine the amount of the a mixture and/or the components present in the sample

1. HPLC
(High Performance Liquid Chromatography)

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

HPLC-UV

HPLC Pump Jacket for controlling column temperature 6-port valve

Mobile Phases A and B

Sample loop

HPLC column

syringe MP waste Detector

2. Gas Chromatography

INSTRUMENTATION
Instruments; Carrier Gas Flow regulators and meters Sample injection system Columns & ovens Detectors

18

SCHEMATIC DIAGRAM OF GAS CHROMATOGRAPH

19

GAS CHROMATOGRAPH COMPONENTS

top view Injection Port

Flame Ionization Detector

Column Oven

front view

CARRIER GAS
The mobile phase gas is called the carrier gas and must be chemically inert. Sample componet column detector mobile phase gas Helium ,argon ,nitrogen , carbon dioxide and hydrogen also used. Selection of the best crrier gas very important , because it effects both the column separation and detector performance . The ratio of viscosity of diffusion coefficient should be minimum for rapid analysis thats why H, He are prepared for a carrier gas .

21

Impurities in the carrier gas such as air water vapour and trace gaseous hydrocarbons can cause sample reaction, column character and affect the detector performance. The carrier gas system should contains a molecular sieve to remove water and other impurities. These gases are available in pressurized tanks. presure regulateres and flow meters are required to control the flow rate 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 often 22 used

PROCESS FLOW SCHEMATIC

Detector (flame ionization detector or FID)

Sample injection Carrier gas (nitrogen or helium)

Air Hydrogen

Long Column (30 m)

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 fraction of injection(1:50 to 1:500) Avaid over loading Slow injection & oversized samples cause band spreading & poor resolution

24

COLUMN CONFIGURATIONS

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.

25

COLUMN OVENS

Column temperature is very important in GC

The column is ordinarily housed in a thermostated oven.

they are usually formed as coils having diameters of 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).

26

COLUMN

Types of columns 1.packed columns 2. Open tubular or capillary.

Packed column-3m

Capillary column- 30m

27

PACKED COLUMNS
Packed columns are fabricated from glass, metal (stainless steel, copper, aluminum), or Teflon tubes that 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, that is coated with a thin layer (0.05 m) of the stationary liquid phase. In order to fit in a thermostating oven, the tubes are formed as coils having diameters of roughly 15 cm.

28

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

29

CAPILLARY (OR)OPEN TUBULAR COLUMNS

1.Wall-coated open tubular (WCOT) Capillary tubes coated with a thin layer of stationary phase Old: stainless steel, Al, Cu, plastic, glass. 2.Support-coated open tubular (SCOT) Inner surface of the capillary is lined with a thin film (~30m) of a support materials, like diatomaceous earth Lower efficiency than WCOT, higher than packed column 3.Fused-silica open tubular column (FSOT): Physical strength, low reactivity, flexibility. 0.32 to 0.25 mm

COLUMN STATIONARY PHASES:

Packed liquid coated silica particles (<100-300 mm diameter) in glass tube best for large scale but slow and inefficient Capillary/Open Tubular wall-coated (WCOT) <1 mm thick liquid coating on inside of silica tube support-coated (SCOT) 30 mm thick coating of liquidcoated support on inside of silica tube best for speed and efficiency but only small samples

31

32

THE STATIONARY PHASE

requirements are: Low vapor pressure Thermal stability Low viscosity (for fast mass transfer) High selectivity for compounds of interest

33

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 oC

34

TYPES OF DETECTORS
1. 2. 3. 4. 5.

6.
7. 8. 9. 10.

Thermal Conductivity Detector(TCD) Flame Ionization Detector(FID) Atomic Emission Detector(AED) Electron Capture Detector(ECD) Nitrogen Phosphoroes Detector(NPD) Photo Ionization Detector(PID) Flame Photometric detector(FPD) Electrolytic conductivity detector (Hall detector) Absolute Mass Detector(AMD) Thermionic Detector(TD)
35

APPLICATIONS
36

QUALITATIVE ANALYSIS: Retention time data should be useful for identification of mixtures Comparing the retention time of the sample as well as the standard Checking the purity of a compound: compar the standard and sample Additional peaks are obtained..impurities are present.compound is not pure

37

QUANTITATIVE ANALYSIS: Direct comparison method: -comparing the area of the peak, peak height, width of peak. Calibration curves: -standards of varying concentration are used determine 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 easily determined .

38

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 of components(amino acids ,plant extracts ,volatile oils)

39

3.CAPILLARY
ELECTROPHORESIS

ELECTROPHORESISAN 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 these factors independently or in combination.
-

WHAT IS CAPILLARY ELECTROPHORESIS

In

practical terms, a positive (anode) and negative (cathode) electrode are placed in a solution containing ions. Then, when a voltage is applied across the electrodes, solute ions of different charge, i.e., anions (negative) and cations (positive), will move through the solution towards the electrode of opposite charge. Capillary electrophoresis, then, is the technique of performing electrophoresis in buffer-filled, narrowbore capillaries, normally from 25 to 100 pm in internal diameter (ID).
42

CAPILLARY ELECTROPHORESIS THE BASICS OF INSTRUMENTATION

Electrophoresis

in a buffer filled, narrow-bore

capillaries Each capillary is about 25-100 m in internal diameter When a voltage is applied to the solution, the molecules move through the solution towards the electrode of opposite charge Depending on the charge, the molecules move through at different speeds

Separation is achieved

43

BASICS

CONT.

photocathode is then used to measure the absorbencies of the molecules as they pass through the solution The absorbencies are analyzed by a computer and they are represented graphically
44

THE ELECTROPHEROGRAM
The data output from CE is presented in the form of an electropherogram, which is analogous to a chromatogram. An electropherogram is a plot of migration time vs. detector response. The detector response is usually concentration dependent, such as UV-visible absorbance or fluorescence. The appearance of a typical electropherogram is shown in Figure for the separation of a threecomponent mixture of cationic, neutral and anionic solutes.

EQUIPMENT

Capillary tube Varied length but normally 25-50 cm Small bore and thickness of the silica play a role

Using a smaller internal diameter and thicker walls help prevent Joule Heating, heating due to voltage

EQUIPMENT
Detector
UV/Visible absorption Fluorescence Radiometric (for radioactive substances) Mass Spec.

CONT.

49

FACTORS AFFECTING THE EFFICIENCY OF CE

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
50

 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 can be measured in physiological solution such as urine and serum. These includes amino acids , nucleotides, nucleosides, bases, anions such as chlorides and sulphate (NO2- and NO3-) and cations such as Ca+2 and Fe+3 .

APPLICATIONS

Analysis of carbohydrates Analysis of inorganic anions/metal ions DNA profiling Protein identification

Advantages Fast Small Sample Relatively inexpensive Automated

Disadvantages Cannot identify neutral species Joule Heating Cannot discern shape

52

SUMMARY
1.

2.

3.

4. 5.

6.

7.

CE is based on the principles of electrophoresis. The speed of movement or migration of solutes in CE is determined by their size and charge. Small, highly charged solutes will migrate more quickly than large, less charged solutes. Bulk movement of solutes is caused by EOF. The speed of EOF can be adjusted by changing the buffer pH used. The flow profile of EOF is flat, yielding high separation efficiencies. The data output from CE is called an electropherogram.
53

CONCLUSION

It is the most efficient separation technique available for the analysis of both large and small molecules. DNA Profiling, protein identification, inorganic metals and ions can be detected easily by this method.

54

4. NMR
(Nuclear magnetic resonance (NMR) spectroscopy) (Molecular Spectroscopy)

Nuclear Magnetic Resonance (NMR) spectroscopy is the absorption of radio frequencies by atomic nuclei within a sample that is placed in a magnetic field. The types of sample that can be studied are liquids, solids and whole organisms. NMR spectroscopy finds applications in several areas of science and is routinely used to study chemical and biochemical structure and function using simple one-dimensional techniques and more complicated multidimensional techniques. Nuclear magnetic resonance was first described and measured in molecular beams by Isidor Rabi in 1938 (1944, Nobel Prize in physics). Felix Bloch and Edward Mills Purcell expanded the technique for use on liquids and solids, for which they shared the Nobel Prize in Physics in 1952.

MOLECULAR SPECTROSCOPY
Nuclear

magnetic resonance (NMR) spectroscopy: A spectroscopic technique that gives us information about the number and types 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.

NUCLEAR MAGNETIC RESONANCE

Resonance:

In NMR spectroscopy, resonance is the absorption of energy by a precessing nucleus and the resulting flip of its nuclear spin from a lower energy state to a higher energy state. The processing spins induce an oscillating magnetic field that is recorded as a signal by the instrument.

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

NMR SPECTROMETER

Schematic diagram of a nuclear magnetic resonance spectrometer.

NMR SPECTROMETER

Essentials of an NMR spectrometer are a powerful magnet, a radio-frequency generator, and a radio-frequency detector. The sample is dissolved in a solvent, most commonly CDCl3 or D2O, 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, a spectrum can be recorded in about 2 seconds.

NMR SPECTRUM
1H-NMR

spectrum of methyl acetate.

High frequency: The shift of an NMR signal to the left on the chart paper. Low frequency: The shift of an NMR signal to the right on the chart paper.

SIGNAL AREAS

Relative areas of signals are proportional to the number of H giving rise to each signal, Modern NMR spectrometers electronically integrate and record the relative area of each signal.

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 a frequency characteristic of the isotope. The resonant frequency, energy of the absorption and the intensity of the signal are proportional to the strength of the magnetic field. For example, in a 21 tesla magnetic field, protons resonate at 900 MHz. It is common to refer to a 21 T magnet as a 900 MHz magnet, although different nuclei resonate at a different frequency at this field strength in proportion to their nuclear magnetic moments.

4.1.1 Chemical shift

Depending on their local chemical environment, different nuclei in a molecule absorb at slightly different frequencies. Since this resonant frequency is directly proportional to the strength of the magnetic field, the shift is converted into a field-independent dimensionless value known as the chemical shift.

The chemical shift is reported as a relative measure from some reference resonance frequency.
For the nuclei 1H, 13C, and 29Si, TMS (tetramethylsilane) is commonly used as a reference.

4.1.2 J-coupling
Some of the most useful information for structure

determination in a one-dimensional NMR spectrum comes

from J-coupling or scalar coupling (a special case of spinspin 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.

4.1.3 Second-order (or strong) coupling Second-order effects decrease as the frequency difference between multiplets increases. NMR spectra display less distortion than lower frequency spectra.

Early spectra at 60 MHz were more prone to distortion than spectra from later machines typically operating at frequencies at 200 MHz or above.

4.1.4 Magnetic inequivalence

More subtle effects can occur if chemically equivalent spins (i.e., nuclei related by symmetry and so having the same NMR frequency) have different coupling relationships to external spins. Spins that are chemically equivalent but are not indistinguishable (based on their coupling relationships) are termed magnetically inequivalent. Magnetic inequivalence can lead to highly complex spectra which can only be analyzed by computational modeling.

4.2 Correlation spectroscopy

Correlation spectroscopy is one of several types of twodimensional nuclear magnetic resonance (NMR) spectroscopy or 2D-NMR. Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) is a set of nuclear magnetic resonance spectroscopy (NMR) methods which give data plotted in a space defined by two frequency axes rather than one. This type of NMR experiment is best known by its acronym, COSY. Other types of two-dimensional NMR include J-spectroscopy, exchange spectroscopy (EXSY), Nuclear Overhauser effect spectroscopy (NOESY), Total Correlation Spectroscopy (TOCSY) and heteronuclear correlation experiments, such as HSQC, HMQC, and HMBC.

4.3

Biomolecular NMR spectroscopy

4.3.1 NMR OF PROTEIN

Nuclear magnetic resonance spectroscopy of proteins (usually abbreviated protein NMR) is a field of structural biology in which NMR spectroscopy is used to obtain information about the structure and dynamics of proteins The field was pioneered by Richard R. Ernst and Kurt Wthrich Structure determination by NMR spectroscopy usually consists of several following phases each using a separate set of highly specialized techniques.

a.Sample Preparation b.Data Collection c.Resonance Assignment d.Restrain Generation e.Structure is calculated and validated

SAMPLE FOR NMR

CONT

Protein nuclear magnetic resonance is performed on aqueous samples of highly purified protein. Usually the sample consist of between 300 and 600 microlitres with a protein concentration in the range 0.1 3 millimolar. The source of the protein can be either natural or produced in an expression system using recombinant DNA techniques through geneticengineering. Recombinantly expressed proteins are usually easier to produce in sufficient quantity, and makes isotopic labelling possible.

APPLICATION To obtain high resolution 3-dimensional structures of the protein, similar to what can be achieved by X-ray crystallography (limited to proteins smaller than 35 kDa ). Only way to obtain high resolution information on partially or wholly intrinsically unstructured proteins. Common tool for the determination of Conformation Activity Relationships where the structure before and after interaction with, for example, a drug candidate is compared to its known biochemical activity.

4.3.2 NMR OF NUCLEIC ACID

Nuclear magnetic resonance spectroscopy of nucleic acids, often referred to as nucleic acid NMR, is the use of nuclear magnetic resonance spectroscopy to obtain information about the STRUCTURE AND DYNAMICS OF NUCLEIC ACID MOLECULES, such as DNA or RNA. NMR has advantages over the other resolution nucleic acid structure determination. method for high-

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

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

FTIR Fourier transform spectroscopy is a measurement technique whereby spectra are collected based on measurements of the coherence of a radiative source, using time-domain or space-domain measurements of the electromagnetic radiation or other type of radiation.

5. Mass Spectroscopy (MS)

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 in 1922). 1948-52 - Time of Flight (TOF) mass analyzers introduced. 1955 - Quadrupole ion filters introduced by W. Paul, also invents 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. 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 a simpler 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 subunits 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 what groups are present in the original molecule and how they are combined together

Can work out molecular structure

What is Mass Spectrometry?

Mass spectrometry is a powerful technique for chemical analysis that is used to identify unknown compounds, to quantify known compounds, and to elucidate molecular structure
Principle of operation

A Mass spectrometer is a MOLECULE SMASHER Measures molecular and atomic masses of whole molecules, molecular fragments and atoms by generation and detection of the corresponding gas phase ions, separated according to their mass-to-charge ratio (m/z). Measured masses correspond to molecular structure and atomic composition of parent molecule allows determination and elucidation of molecular structure.

What is Mass Spectrometry?

May also be used for quantitation of MOLECULAR SPECIES. VERY SENSITIVE TECHNIQUE - Works with minute quantities of samples (as low as 10-12g, 10-15 moles) and is easily interfaced with chromatographic separation methods for identification of components in a mixture Mass spectrometry provides valuable information to a wide range of professionals: chemists, biologists, physicians, astronomers, environmental health specialists, to name a few.
Limitation is a Destructive technique cannot reclaim sample

What is a Mass Spectrometer? Many different types each has different advantages, draw-backs and applications

All 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 and data processing under computer control Data system and Computer Control is often overlooked most significant advance in MS allows 24/7 automation and development of modern powerful analytical techniques.

What is a Mass Spectrometer?

All Instruments Have:

1. Sample Inlet
2. Ion Source 3. Mass Analyzer Detector Data System

80

How does it work?

accelerate
ionise

separate

ee+

+4000 V

0V
Magnetic and/or electric field

+
v a c u u m

heavy

81

vapourise

light

eesample

A+

B
+

A+

B+

C+

MASS SPECTROMETRY

e-

Analyser Types
Analyser is the section of instrument that separates ions of different m/z Many Different technologies
A. Magnetic Sector B. Quadrupole

C. Ion Trap
D. ToF

All based on momentum separation

A. Magnetic Sector

83

B. Quadrupole

C. Quadrupole ion Trap

3 Electrode system 2 x Endcap and 1x Ring Electrode

Now have recent develpoment of Linear Ion Trap and orbitrap 85 Developments on same theme.

Analyser Types Quadrupole ion Trap

Ion Trap is very small most of instrument is ion guides into the trap itself

86

MASS SPECTROMETER INSTRUMENT DESIGN

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, advantages and disadvantages Selection of appropriate ionization method and analyzer are critical and defines MS applications. Wide range of MS applications
87

APPLICATIONS of ms
Advances in Proteomics and other areas in biotechnology made possible by development of soft ionisation Maldi and ESI MS techniques Protein and peptide analysis for MW determination Protein Identification and profiling using digests and data base searching major development in Proteomics Protein post-translational modification Protein structure characterisation Maldi-Imaging Oligo-nucleotide analysis Confirmation of purity of synthetic oligos Carbohydrate analysis

CONT

Automated high throughput analysis

Screening of biological samples

Pharmicokinetics LC-MS seperation and identification of components of complex mixtures Normally LC-ESI, now increasingly LC-Maldi-ToF Intact virus analysis Cell imaging (Maldi) Tissue Imaging (Maldi)

MS PRINCIPLES
Find

a way to charge an atom or molecule (ionization) charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer) ions using microchannel plate or photomultiplier tube

Place

Detect

MASS SPECTROMETRY (MS)

Introduce

sample to the instrument Generate ions in the gas phase Separate ions on the basis of differences in m/z with a mass analyzer Detect ions

Generalized Protein Identification by MS

Spot removed from gel Fragmented using trypsin Spectrum of fragments generated

Library

MATCH

Artificial spectra built

Artificially trypsinated

Database of sequences (i.e. SwissProt)

Methods for protein identification

MS PRINCIPLES

Different elements can be uniquely identified by their mass

MS PRINCIPLES

Different compounds can be uniquely identified by their mass

Butorphanol N -CH2OH HO HO L-dopa Ethanol

COOH -CH2CH-NH2

CH3CH2OH

HO MW = 327.1 MW = 197.2 MW = 46.1

MASS SPECTROMETRY
Analytical

method to measure the molecular or atomic weight of samples

MASS SPECTROMETRY

For small organic molecules the MW can be determined to within 5 ppm or 0.0005% which is sufficiently accurate to confirm 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

Sample

+ _

Ionizer

Mass Analyzer

Detector

MASS SPECTROMETERS
Linear Time Of Flight tube
ion source

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) DetectionsOffrom Reflector Time Flight tube multiple laser bursts are averaged. Multiple laser
ion source

detector

time of flight

Tandem MS- MS/MS -separation and identification of compounds in complex mixtures - 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
detector

reflector

time of flight

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

mass to charge ratio (m/z)
M/Z RATIO:

Molecular weight divided by the charge on this protein

THANK YOU