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Mass spectrometryand related technologies. James Dalton Ian Donaldson Rhidhwan Wahab Qian Zhen MRes Bioinformatics. 2002. University of Leeds, UK.
Mass SpectrometryBy James Dalton. What is Mass Spectrometry? What information can it provide? Where are Mass Spectrometers used? The biochemical applications How does a Mass Spectrometer work? Some nice internet sites
What is Mass Spec? Analytical tool measuring molecular weight (MW) of sample Only picomolar concentrations required Within an accuracy of 0.01% of total weight of sample and within 5 ppm for small organic molecules For a Mr of 40 kDa, there is a 4 Da error This means it can detect amino acid substitutions / post-translational modifications
What sort of info is returned? Structural information can be generated Particularly using tandem mass spectrometers Fragment sample & analyse products Useful for peptide & oligonucleotide sequencing Plus identification of individual compounds in complex mixtures
Where are they used? Biotechnology: analysis of proteins, peptides, oligonucleotides Pharmaceutical: drugs discovery, combinatorial chemistry, pharmokinetics, drug metabolism Clinical: neonatal screening, haemoglobin analysis, drug testing Environmental: water, food, air quality (PCBs etc) Geological: oil composition
In biochemistry Accurate molecular weight measurements: purity of sample, detection of amino acid substitutions, post-translational modifications, and disulphide bridges Reaction monitoring: enzyme activity, chemical modification, protein digestion Amino acid sequencing Oligonucleotide sequencing Protein structure: protein folding (H/D exchange), protein-ligand complex formation, macromolecular structure determination
How does a Mass Spectrometer work? 3 fundamental parts: the ionisation source, the analyser, the detector Samples easier to manipulate if ionised Separation in analyser according to mass-to-charge ratios (m/z) Detection of separated ions and their relative abundance Signals sent to data system and formatted in a m/z spectrum
Simplified Schematic The analyser, detector and ionisation source are under high vacuum to allow unhindered movement of ions Operation is under complete data system control
m/z spectrum example Leucine Enkephalin, Pentapeptide, YGGFL Expected Mr = 555.3 Da, Calculated Mr = 555.1 Da (dominant ions at 556.1)
Sample Introduction& Ionisation Direct into ionisation source or via chromatography for component separation (HPLC, GC, capillary electrophoresis) Ionisation can be positively charged (for proteins) or negatively charged (for saccharides and oligonucleotides)
Ionisation methods Atmospheric Pressure Chemical Ionisation (APCI) Chemical Ionisation (CI) Electron Impact (EI) Electrospray Ionisation (ESI) Fast Atom Bombardment (FAB) Field Desorption / Field Ionisation (FD/FI) Matrix Assisted Laser Desorption Ionisation (MALDI) Thermospray Ionisation
Detection & Recording of Ions Detector monitors ion current, amplifies it and then transmits signal to data system Common detectors: photomultiplier, electron multiplier, micro-channel plate
Peptide Sequencing Peptides of 2.5 kDa or less give best data Protein sample often taken from 2-D gels and digested A protein digest can be analysed as entire mix Initial MS spectrum showing Mr of all components in digest (peptide map) may be enough for a database search and identification Peptides fragmented along the amino acid backbone in tandem mass spectrometry Some peptides generate enough info for full sequence, others only generate partial sequences of 4-5 amino acids Often this “tag” sequence is sufficient for database identification
Internet sites www.astbury.leeds.ac.uk/Facil/MStut/mstutorial.htm(Dr Alison E. Ashcroft at Leeds) www.asms.org (The American Society for Mass Spectroscopy) www.spectroscopynow.com (Base Peak) Mass Spec tools www.expasy.ch/tools/#proteome http://prowl.rockefeller.edu www.mann.embl-heidelberg.de
Introduction to Tandem Mass Spectrometry. By Ridhwan Wahab. Mass spectrometry is a very powerful method to analyse the structure of organic compounds, but suffers from 3 major limitations: 1 Compounds cannot be characterised without clean samples 2 This technique has not the ability to rovide sensitive and selective analysis of complex mixture 3 For big molecules like peptides spectra are very complex and very difficult to interpret
History of the technique MS/MS or tandem mass spectrometry or 3D MS was first introduced in the 1970’s and was quickly accepted in analytical community. The technique has been used for structure elucidation of unknown and for analysis of complex mixture with minimum sample clean up. Development in the mid-1980’s and advancing the popularity of MS/MS included the availability of powerful data system able to control the MS/MS experiment.
Principle The introduction of the complex mixture in the inlet of a mass spectrometer always gives a complex spectrum which is difficult to interpret. We often observe a lot of peaks and the most important correspond to the major constituent. That’s why it is very difficult to identify one precise compound in the spectrum of a mixture. The principle of MS/MS method is the use of Filiation Reactions. This method introduce a further specificity to recognise a substance in a mixture.
In Tandem MS – normally has 2 mass spectrometers in series. In first mass spectrometer (MS1) is used to SELECT, from the primary ions, those of a particular m/z value which then pass into the Fragmentation Region. The ion selected by the MS1 is the parent ion and can be a molecular ion resulting from the primary fragmentation. DISSOCIATION occurs in the fragmentation region. The daughter ions are analysed in the Second Spectrometer (MS2). In fact, the MS1 can be viewed as an ion source for MS2. MS2 MS1
In MS1, ionisation is made by electron impact. Then one ion is selected by a particular system (quadrupole, magnetic sector, ion cyclotron resonance). This only one type of ion go through the outlet to the fragmentation region. In the fragmentation region, there is a neutral gas (i.e He, Ar, N2….) on a high pressure. The ions selected interact with these molecules of gas. During the collision, the kinetic energy of ions is transformed into internal energy which permits the fragmentation into daughter ions. In MS2, the daughter ions are detected and the final spectrum shows the peaks of selected ion and all its daughters. appear in the front and daughter peaks behind them. The MOST important peaks
Instrumentation for MS/MS A tandem mass spectrometer can be thought of as two mass spectrometers in series connected by a chamber that can break a molecule into pieces perhaps like a puzzle. This chamber is known a collision cell. A sample is “sorted” and “weighted” in the first mass spectrometer (MS1), then broken into pieces in the collision cell, and a piece or pieces sorted and weighted in the second mass spectrometer (MS2).
Types of sources ESI (Electrospray) FAB (Fast Atom Bombardment) LSI (Liquid Secondary Ion) These terms indicate the way the compound is placed into the tandem mass spectrometer. Dissolvation-nebulization sources : ESI (“electrospray”) (flow rate : 1 to 10 mL/min). Different parameters of “spray” also exist : “ Thermospray ” source “ APCI ” (Atmospheric Pressure Chemical Ionisation) “ Particle beam ” source “ Nanospray ” (2 to 20 nL/min)
Ionisation-desorption sources Based on the secondary emission : the bombardment of a solid or liquid sample with a primary beam (ion, atom or photon) induce the secondary emission of particles : electrons, neutral particles and ions. Only these particles are analysed by mass spectroscopy. The name of the sources depends on the nature of the incident beam : SIMS (“Secondary Ion Mass Spectrometry”), ION FAB (“Fast Atom Bombardment”), ATOM LD (“Laser Desorption”), PHOTON MALDI (“Matrix Assisted Laser Desorption Ionisation”) In FAB, the sample can be mixed with the matrix. (This type of sources are used to analyse compounds which have a high molecular weigh, especially the polymers and the biological compounds) Types of sources
Analysers A tandem mass spectrometer is a mass spectrometer that has more than one analyser, in practice usually two. The two analysers are separated by a collision cell into which an inert gas (for example, argon or xenon) is admitted to collide with the selected sample ions and brings about their fragmentation. Most Common combinations being: quadrupole-quadrupole magnetic sector – qudrupole magnetic sector – magnetic sector quadrupole- time-of-flight (TOF)
Tandem Quadrupoles TQMS are the most commonly used : it’s a sequential arrangement of two mass analysing quadrupoles with a third quadrupole interposed as a gas cell. A quadrupole is made up of four electrodes where a continued potential U and a radio-frequency potential Vcos (wt) are applied. (Each ion adopts an oscillating path whose amplitude depends on the ratio U/V and on the ratio m/z. We can stabilize or destabilize the trajectory of ions with scanning of U and V but with the ratio U/V which is always constant. Only ions which have a stable trajectory can be detected.) The first quadrupole (MS1) allows the selection of precursor ions according to m/z value. The third sector MS2 is a mass filter too for the daughter ions. In the intermediate quadrupole, there exists a high pressure. This role is to induce ionic oscillations for ions to have an important linear speed. This quadrupole is not selective according to the mass, it is a RF-only quadrupole (alternative alimentation of radio-frequency).
Sector Mass Spectrometers Ion kinetics energies are fundamental importance to the mass analysing properties of sector mass spectrometers. They are double focusing instruments and they use a magnet (B) with electric sector (E). When the electric sector takes place between the source and the magnet, the geometry is normal (EB) and when it is between the magnet and the detector, the geometry is inverse (BE), for example MIKES (Mass analysed Ion Kinetic Energy Spectrometry). In the EB system : The ions issued from source are placed into electric sector where they are accelerated by a potential V. Then, they are placed into a magnetic sector where the magnetic field is perpendicular to the direction of their movement. So, the ions describe an arc of the circle. In practical, scanning the magnetic sectors allows to focus the ion fragments on the slit of the analyser. The analysers with electric and magnetic sectors have a good resolution but are too big and expensive.
Ion Trap The principle of Ion Trap is next to this of tandem quadrupoles. The difference is that ions can be formed directly in the Trap : so, the source and the analyser are the same instrument. The excellent sensibility of the Ion Trap involves its use to detect and quantify traces. This technique is relatively new and we can find it in environmental laboratories (analysis of pesticides, micro pollutants). Ion Cyclotron Resonance (ICR) It is the same principle as Ion Trap. With this type of analyser, we can study ions which have a ratio m/z bigger than 5000. The pressure inside the cell is low (10-9 torr). The performances of ICR are promising but they are very expensive.
Collision cell The collisional activation can be divided into two categories, involving high or low energy, to which different types of collision cell are appropriate. In sector instruments, where high energy collisions are most common, the cell is usually a tight chamber of 1-3 cm length with entrance and exit slits which transmit the ion beam. Good pumping is essential to maintain a low pressure outside the cell. In some instruments, the collision cell has been electrically insulated from the mass spectrometer and can be held at a high potential to retard the ion beam and reaccelerated it on exit. This allows control of the collision energy and also reduces the kinetics energy spread of daughter ions formed.
Types of detector The detector’s purpose is to translate the ion arrival into an electric signal measured by the electronic system of the mass spectrometer. It exists two different classical types of detector. 1. Electrons multiplying The principal sorts of electron multiplying are the “channeltron” and the “micro channel plate”. The principle is based on the impact of an ion on a surface composed by half conductors. This impact creates electrons which are accelerated to another surface where they also create other electrons, etc…Those electrons are recovered and the number of them is proportional to the signal’s intensity. 2. Photodetectors Electrons are created in the same way and interact with a phosphorecent surface which generate photons. Those photons are also recovered and the number of them is proportional to the signal’intensity.
Applications of Tandem Mass Spectrometry Analysis of Food constituents Labows and Shuhan (1983) - volatiles substances (i.e limonene/ethyl butyrate). Warburton (1981) - identification of cholesterol in egg york & citric acid in lemon. Walther (1983) - presence of caffeine in leaf tea. Protein identification Structural studies Sherman et al. (1985)- phosphoinositides, representing an important class of glycerolphospholipids. Analysis of drug residue International Olypic Committee - detection of anabolic steroids such as stanozol [10418-03-8].
Applications of Tandem Mass Spectrometry Analysis for contaminants Plattner and Bennett (1983) - rapid detection of mycotoxins in food and animal feeds. Steiner et al (1980) - detection of parathion (pesticides residues in food) Lau et al (1985) - identification of arsenobetaine and arsenocholine (organometallic compounds) in fish.
Tandem GC/MS/MS SRM: (Single Reaction Monitoring) MRM:(Multiple Reaction Monitoring)
Bibliography Internet sites : http://www.google.com http://www.bmss.org.uk/what_is/whatis.html http://www.duke.edu/~mdfeezor/NSHome/inform/msms1.html http://www.astbury.leeds.ac.uk/Facil/MStut/mstutorial.htm http://ms.mc.vanderbilt.edu/tutorials/ms/3.htm http://www.garvan.unsw.edu.au/public/corthals/book/IPMS.html http://www.micromass.co.uk/basics/Glossary.html
Ionization Methods —MALDI and ESI QIAN ZHEN
Ionization Methods Sort of ionization methods APCI—Atmospheric Pressure Chemical Ionization CI—Chemical Ionization EI——Electron Ionization ESI—Electrospray Ionization FAB—Fast Atom Bombardment FD—Field Desorption FI—Field Ionization MALDI—Matrix Assisted Laser Desorption Ionization TSP—Thermospray Ionization
Ionization Methods Two Samples —MALDI and ESI Main Advantage: Soft Ionization
Ionization Methods Soft Ionization: They do not (in the most part) degrade the molecule during the ionization process.
Ionization Methods—MALDI http://www.fbsmres.leeds.ac.uk/users/bmbqz/5070/Movie4.swf
Ionization Methods—MALDI The Mechanism of MALDI —Ion Desorption The Formation of a ‘Solid Solution’ Matrix Excitation Analyte Ionization
Ionization Methods—MALDI Advantage: Rapid and convenient molecular weight determination. Limitations: 1) MS/MS difficult. 2) Requires a mass analyzer that is compatible with pulsed ionization techniques. 3) Not easily compatible with LC/MS. Mass range: typically less than 500,000 Da
Ionization Methods—ESI http://www.fbsmres.leeds.ac.uk/users/bmbqz/5070/Movie5.swf
Ionization Methods—ESI The Mechanism of ESI —Ion Evaporation
Ionization Methods—ESI Advantages: 1) Good for charged, polar or basic compounds. 2) Permits the detection of high-mass compounds at mass-to-charge ratios that are easily determined by most mass spectrometers (m/z typically less than 2000 to 3000). 3) Best methods for analysing multiply charged compounds. 4) Very low chemical background leads to excellent detection limits. 5) Can control presence or absence of fragmentation by controlling the interface lens potentials. 6) Compatible with MS/MS methods.
Ionization Methods—ESI Limits: 1) Multiply charged species require interpretation and mathematical transformation. 2) Very sensitive to contaminants such as alkali metals or basic compounds. 3) Relatively low ion currents. 4) Relatively complex hardware compared to other ion sources. Typically less than 200,000 Da.
Ionization Methods—Applications 1) Proteins and peptides: - Monitoring biomolecular reactions. - Analysis of protein conjugates. - Analysis of high molecular weight biopolymers. - Nested-PSD for interpretation of peptide sequences. 2) Polymers. - Analysis of synthetic polymers. - Analysis of epoxy resins. - Structural determination in MALDI by examination of the Neutral Spectrum.
Ionization Methods Further Reading 1. For MALDI beginner: http://www.srsmaldi.com/Maldi/Guide.html 2. For MALDI lab user: http://www.srsmaldi.com/Maldi/Lab.html 3. For MALDI tutorial: http://ms.mc.vanderbilt.edu/tutorials/maldi/maldi-ie_files/frame.htm 4. Ionization Methods 1: http://www.jeol.com/ms/docs/ionize.html 5. Ionization Methods 2: http://www.waters.com/Waters_Website/Applications/lcms/lcms_itq.htm
SELDI-TOF-MS Surface-enhanced laser desorption/ionization- time of flight-mass spectrometry. By Ian Donaldson.
What is SELDI-TOF-MS? Surface-Enhanced Laser Desorption/Ionization-time of flight-mass spectrometry. Advantages over the MALDI technology:- Use of complex biological material- No pre-processing or labelling- Chromatography on-chip- Uses small amounts (< 1?l/ 500-1000 cells) of sample (biopsies, microdissected tissue)
ProteinChipTM by Ciphergen Biosystems Inc. Sample fractionation accomplished by ‘Retentate’ chromatography. Sample deposited on spots, each chip has different surface interactions. Chromatographic: Biological: - Cation exchange - Antibody/Antigen - Anion exchange - Receptor/Ligand - Metal affinity capture - DNA/Protein - Normal phase - Enzyme (Hydrophobic) (bound to preactivated surface) - Reverse phase (Hydrophilic)
Slide adapted from http://www.ciphergen.com/tech_doc11.2.html ProteinChipTM. Protein purification by ‘Retentate mapping chromatography’.
4. Detection by TOF-MS 2. Washing 3. Add EAM 1. Add crude extract SELDI process. gifs from http://www.bmskorea.co.kr/new01_21-1.htm EAM = Energy absorbing molecule
Applications of SELDI? Fingerprinting/Biomarker discoveryDetect proteins from two different states e.g. disease versus normal tissue samples. ToxicologyDetection of toxicity biomarkers Protein characterisationIncluding the identification of novel ligands and protein characteristics. Assay developmentSimplification or advancement of existing methodologies. On-chip peptide mapping with trypsin digestion (ProFound).
Definition of Antigenic Determinants: Epitope Mapping Step 4: Bound epitopes identified by SELDI Step 3: Fragments (outside of Ab binding site) removed Step 1: Bind protein antigen(s) to affinity capture device (Ab) on ProteinChip™ array In Situ Digest Step 2 SELDI ProteinChipTM Arrays: -----for Epitope Mapping Wash away unbound fragments Slide adapted from http://www.ciphergen.com/images/slide.PPT
ProteinChipTM Software Analysis. Data Acquisition.- Protocol design- Automated mass data collection and creation of a ‘retentate map’. Data views.Several presentation formats (‘data views’).- Spectrum view or ‘retentate map’- Peak map- Difference map view- Gel view- 3-D overlays- Internal protein calibrants (based on pI/MW)
Difference map view. Gel view.
SELDI: Summary. Improvements over existing technologies: Small amount of starting material Sample does not need to be processed Versatile applications and analysis
Selected reviews on SELDI: Merchant M and Weinberger S (2000) Recent advancements in surface-enhanced laser desorption/ionization-time of flight-mass spectrometry. Electrophoresis 21: 1164-1167. Fung E, Thulasiraman V, Weinberger SR, Dalmasso EA (2001) Protein biochips for differential profiling. Current Opinion in Biotechnology 12: 65-69. Weinberger SR, Dalmasso EA, Fung ET (2002) Current achievements using ProteinChipTM array technology. Current Opinion in Chemical Biology 6: 86-91.
SELDI Web sites: Molecular Analytical Systems (MAS). http://www.seldi.org/ Manufacturers of ProteinChip(R) http://www.ciphergen.com/
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