Presentation on theme: "Chemical Analysis by Mass Spectrometry"— Presentation transcript:
1 Chemical Analysis by Mass Spectrometry Dr Phil MortimerChemistry Department Mass Spectrometry Facility
2 Recommended Reading :“The Expanding Role of mass Spectrometry in Biotechnology”Gary Siuzdak, MCC Press, San Diego, ISBN“Ionization Methods in Organic Mass Spectrometry”Alison Ashcroft, RSC, Cambridge, UK, ISBN“Practical Organic Mass Spectrometry” 2nd EdnJ R Chapman, Wiley, Chichester, UK, ISBN X“Spectroscopic Methods in Organic Chemistry” 4th EdnD H Williams, I Fleming, McGraw-Hill, ISBN X
3 Chemistry 101 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 nucleusDifferent atoms combine in different ways to form molecular sub-units called functional groups.
4 Can work out molecular structure Chemistry 101Mass 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
5 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 structurePrinciple of operationA 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.
6 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, moles) and is easily interfaced with chromatographic separation methods for identification of components in a mixtureMass 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
7 What is Mass Spectrometry Used For? Chemical Analysis and IdentificationSome Typical ApplicationsEnviromental Monitoring and Analysis (soil, water and air pollutants, water quality, etc.)Geochemistry – age determination, Soil and rock Composition, Oil and Gas surveyingChemical and Petrochemical industry – Quality controlApplications in BiotechnologyIdentify structures of biomolecules, such as carbohydrates, nucleic acidsSequence biopolymers such as proteins and oligosaccharidesDetermination of drug metabolic pathways
8 How Does it Work?Generate spectrum by separating gas phase ions of different mass to charge ratio (m/z)m=molecular or atomic mass, z = electrostatic charge unitIn many cases (such as small molecules), z = 1 measured m/z = mass of fragmentBut this is not always trueFor large bio-molecules analysed by electrospray (ESI), z >1What happens in this case?
9 Multiple Charging Consider a peptide with MW of 10000 With ESI-MS, charges by H+ additionM + nH+ MnHn+Resultant ions formed are :-When z = 1 m/z = ( )/1 = 10001When z = 2 m/z = ( )/2 =When z = 3 m/z = ( )/3 =When z = 4 m/z = ( )/4 =When z = 5 m/z = ( )/5 =
10 Figure from The Expanding Role of MS in Bio-technology – G . Siuzdak
11 Multiple ChargingAdvantage in that allows measurement of high mass ions with instruments of limited m/z range.Particularly true for ESI-MS – Advantage for analysis of high mass samples that take multiple charges – brings sample m/z down into measurable range of MSComputer Algorithms deconvolute m/z to original mass.Figure from The Expanding Role of MS in Biotechnology – G . Siuzdak
12 Mass Measurement Mass Spectrometers measure isotopic mass. They DO NOT measure average molecular mass!! (MW)e.g For a molecule with empirical formula C60H122N20O16S2Average MW =(weighted average for each isotope)Exact mass =(exact mass of most abundant isotope)Nominal mass = 1442(integer mass of most abundant isotope)Illustrated on next Slide
13 Resolution Influences achievable precision and accuracy of measurement Figure from The Expanding Role of MS in Bio-technology – G . Siuzdak
14 Resolution Influences achievable precision and accuracy of measurement R = ΔM/MOften expressed in ppmR = (ΔM/M) x106
15 Isotope PatternsIsotope patterns useful for identifying presence of certain elementsParticularly useful for SMALL moleculesFigure from The Expanding Role of MS in Bio-technology – G . Siuzdak
16 What is a Mass Spectrometer? Many different types – each has different advantages, draw-backs and applicationsAll consist of 4 major sections linked togetherInlet – Ionization source – Analyser – DetectorAll sections usually maintained under high vacuumAll functions of instrument control, sample acquisition and data processing under computer controlData system and Computer Control is often overlooked – most significant advance in MS – allows 24/7 automation and development of modern powerful analytical techniques.
17 What is a Mass Spectrometer? All Instruments Have:Sample InletIon SourceMass AnalyzerDetectorData System
18 How does it work? +4000 V 0 V e- e- e- e- e- Mass spectrometry accelerateseparateionise+4000 V0 Ve-Magnetic and/or electric fielde-++heavyvacuumlightvapourisee-+Ae-sample+B+CA+B+C+Mass spectrometrye-
19 Analyser Types What is the analyser? Analyser is the section of instrument that separates ions of different m/zMany Different technologiesMagnetic Sector, Quadrupole, Ion Trap, ToFAll based on momentum separation
20 Analyser Types – Magnetic sector Easiest Conceptually to understandSeparate electromagnetically“Electromagnetic Prism”Usually combined with ESA (energy focusing device) - enables high mass resolution (Double Focusing Instrument) – makes high accuracy mass measurements possibleLarge (Heavy!!), Expensive to operateComparatively slow scan ratesHigh Skill level required to operate and maintainSelf-service use by users not possible
22 Analyser Types – Quadrupole Smaller, cheaper – computer controlled – Self service operation by trained users possibleElectrostatic momentum separation by superimposed rf and dc voltagesRapid scan rates – enables measurement of transient samples introduced from chromatographic systems (GC, LC)Lower resolution – accurate mass NOT possible
23 Analyser Types – Quadrupole ion Trap Derivative of Quadrupole – cheap, small, rapid scanningAgain, electrostatic momentum separation by rf and dc voltagesLower resolution – accurate mass not possibleBUT – have ion trapping ability – can store and selectively eject ionsIons can be subjected to fragmented by CID and “daughter ions” analysedAllows MS-MS or MSn (Multiple levels of storage and trapping)Can perform both molecular ion analysis and structural determination
24 Analyser Types – Quadrupole ion Trap 3 Electrode system2 x Endcap and 1x Ring ElectrodeNow have recent develpoment of Linear Ion Trap and orbitrapDevelopments on same theme.
25 Analyser Types – Quadrupole ion Trap Bruker HCTIon Trap is very small – most of instrument is ion guides into the trap itself
26 Analyser Types – Time of Flight (ToF) Conceptual diagram!!!
27 Analyser Types – Time of Flight (ToF) Velocity separation - E= mv2Ion packet given constant KE – ions of heavier mass take longer to pass down drift tube and reach detectorConceptually easyAllows very large masses to be measured (500,000Da)E= 1/2mv2Time flight of ions through drift tubeIons of larger mass take longer to reach detector for constant E
28 Mass Spectrometer Instrument Design Different types of Ionization sourceEI, CI, FAB, ESI, Maldi, (APCI, DESI, DART)(Also sources for inorganic analysis – ICP, GD, etc.)Different types of analyserMagnetic Sector, Quadrupole, Ion Trap, ToFDifferent sources and analysers have different properties, advantages and disadvantagesSelection of appropriate ionization method and analyzer are critical and defines MS applications.Wide range of MS applications
29 Development of Mass Spectrometry Until 1980’s, most mass spec geared primarily towards “traditional” chemical analysis (small molecules)- MS primarily conducted using EI ionisation – unchanged since 30’s and 40’sFrom 1980’s, start to have shift in focus towards analysis of samples that are larger and more bio-molecular in characterSuch samples are often more delicate and easily fragmented.This results in the development of “softer” ionisation techniques and analysers capable of extended mass ranges.Allows MS determination of high mass parent ions (such as intact proteins, etc.).Strongly influences development of Proteomics field
30 Electron Impact (EI) Mass Spectrometry Up until 1980’s, most mass spec is “chemical” analysis - performed using EI ionisationBombard gaseous sample with high energy (70eV) e-Results in ejection of e- from target molecule to form gas phase ion species – which is then passed to analyser for analysis.e- + M -> 2e- +M+Sample normally introduced via heated probe, GC, or leak (frit) inlet
31 Electron Impact (EI) Mass Spectrometry Problems with EI ionisation– requires sample be in the gas phase before ionisation- limits samples to those already existing in the gas phase or thermally stable samples that are easily volatised (for probe introduction)2) – High Energy (Hard) Ionisation – lots of excess energy given to target – causes fragmentation to lose energy and become stable – resulting in lots of characteristic fragments ions, but little parent ion (useful for structural characterisation).
33 Overcoming problems with EI-MS – Use of CI How to overcome limitations?Derivatize sample to make more volatile and thermally stable derivative that can be analysed by EI2) Develop other ionisation techniques using lower ionisation energies and other means of introducing sample.Intermediate method was Chemical Ionisation (CI)Uses bath gas (CH3/NH4/CH3(CH2)2CH3) to protonate sampleOften forms MH+Still only applicable to volatile or Thermally stable samples.
35 FAB-Mass Spectrometry Subsequent development of FAB (Fast Atom Bombardment)Still used for small delicate moleculesDissolve sample in liquid matrix and place on targetBombard with beam of fast atoms or ions (Xe or Cs+)Have secondary ion emissionLow energy protonation of target molecules – very little excess energy – little fragmentation – readily observe parent ions.Now we’re getting somewhere.
36 FAB-Mass Spectrometry Problems with FABSlow, Labor intensive, Very skilled.Matrix interference at low massGenerally observe MH+ (+ve ion mode)ORM-H (-ve ion mode)
37 Current Mass Spectrometry – Biochemical MS Today, majority of MS is of bio-chemiccal / biological samples performed using either Electrospray MS or Maldi-toF MS.Other methods exist, but these perform bulk of the workWill concentrate on these for the rest of the lecture.Both are “soft” (low energy) ionisation methods that usually yield little fragmentation and so are useful for determination of parent mass of delicate molecules.Both are condensed phase techniques and require that samples are soluble.
38 Electrospray Mass Spectrometry (ESI-MS) Solution phase technique - Can analyse both +ve and –ve ions (but not simultaneously)Samples usually dissolved in moderately polar solventTypically MeOH or MeCN, often mixed H2O (up to 80%)DO NOT USE DMF, DMSO, THF, etcDo NOT use involatile buffers.Typical concentration 1-10uM (can be 20nM-50uM depending on sample)Usually requires addition of volatile buffer (0.1-1%)Typically AcOH or TFA (+ve ion) / NH4OH (-ve ion)
39 Electrospray Mass Spectrometry (ESI-MS) How does it work?
40 Electrospray Mass Spectrometry (ESI-MS) How does it work?
41 Electrospray Mass Spectrometry (ESI-MS) Thermo-Finnigan LCQ-DecaESI-Ion Trap with LC System
42 Electrospray Mass Spectrometry (ESI-MS) Different versions of ESI (On-Axis / Orthoganal / Off Axis)AdvantagesSoft ionisation – limited fragmentationMultiple charging with peptides / proteins / oligionucleotides(Analysis of molecules with MW > mass range of instrument)Can be linked with LC – acts as inlet – allows MS identification of components of mixturesAutomated high throughput analysis of biological samples – 24/7Can be coupled with many analysers – IT/Quadrupole /ICR / Orbitrap – vast range of different types of analysis possible
43 Electrospray Mass Spectrometry (ESI-MS) Can Deconvolute mass spectra as previously discussed
44 MALDI-ToF Mass Spectrometry Relatively simple techniqueSoft ionisation method that can be used to volatilise large macromolecules with minimum fragmentationGives less multiple charging than ESISamples co-deposited on target plate with matrix (and often an additive) and allowed to dry.Many samples can be on plate.Plate inserted into instrument vacuum
45 MALDI-ToF Mass Spectrometry Target irradiated by UV laser.Causes vaporisation of matrix and supersonic expansion of plumeDried sample is launched into the gas phase as matrix is vaporisedUV energy absorbed by matrix causes it to dissociate and typically transfers a proton to sample molecule within the plume to form MH+Now have protonated target, which is accelerated into analyser for seperation and detection
47 MALDI-ToF Mass Spectrometry Most MALDI-ToF are reflectron instrumentsReflectron is energy focusing device (ion mirror)Increases resolution (and mass accuracy) – but limits mass rangeLinear ToF has low resolution but high mass range (up to m/z 300,000)Many Instruments are now ToF/ToFCan do MS/MS experiments
48 MALDI-ToF Mass Spectrometry Typical Current State of the Art Maldi-ToFBruker AutoflexNow available as Tof/ToFEasy to use – walk up use after training.Highly automatedNow can be used for imaging of Tissue samples
49 MALDI-ToF Mass Spectrometry - Conditions Suggested concentrations~10 < Da (pure)~100 > Da (pure)10: 1 Ratio of Matrix : Sample(20nM-50uM of sample – typically 1-10uM)Several methods of target prepMultiple layer / co-mixedSpot 0.5uL of mixture on spot and allow to dryAnalysis very dependant upon sample preparation
51 MALDI Contamination Limits Analysis is relatively insensitive to contaminants.Phosphate 20 mM EDTA 1 mMDetergents 0.1% Glycine 20 mMGlycerol 2% Sodium Citrate 20 mMBuffer (Tris)50 mM K phosphate 25 mMGuanidine 1 M Na phosphate 0.1MNa azide 1% Octyl glucoside 0.3%SDS 0.05% Ammon. Bicarb. 0.1MSuggested concentrations~10 < Da (pure)~100 > Da (pure)
52 MALDI –Characteristics Maldi-ToF Generally results in broader peak envelope than ESIThis is particularly true at high mass.Low mass Maldi-ToF (<20,000Da) – can use reflectron – get high resolution (R>10,000)High MW Maldi – requires use of linear mode – lower resolution – Higher Mass range (up to 500,000DaMaldi-ToF generally results in generation of singly charged species (z = 1)However, often requires desalting, otherwise have broad mass envelop addition due to multiple slated peaks forming – particularly prevalent for proteins
53 MALDI –Characteristics Analysis is rapid – therefore, is often used for high throughput analysis and screening applications – many samples on one plate.Sensitivity enhanced by using “AnchorChip” Plates – concentrates sample solution in small spotLow mass spectra (<500MW) can be inhibited by interference from Matrix peaks – development of NaldiSpectra VERY dependant upon sample preparation and analysis conditions (especially laser power) – modern instruments have “fuzzy” logic to optimise analytical conditions on the fly
54 Biotechnology applications Advances in Proteomics and other areas in biotechnology made possible by development of soft ionisation Maldi and ESI MS techniquesProtein and peptide analysis for MW determinationProtein Identification and profiling using digests and data base searching – major development in ProteomicsProtein post-translational modificationProtein structure characterisationMaldi-ImagingOligo-nucleotide analysis – Confirmation of purity of synthetic oligo’sCarbohydrate analysis
55 Biotechnology applications Automated high throughput analysisScreening of biological samplesPharmicokineticsLC-MS – seperation and identification of components of complex mixtures – Normally LC-ESI, now increasingly LC-Maldi-ToFIntact virus analysisCell imaging (Maldi)Tissue Imaging (Maldi)
56 Mouse Brain Digital Photo Before Matrix Addition
62 Practical Analytical MS Considerations Know what you are trying to achieve – Structural analysis? Accurate Mass Determination? Prepare sample according to given preparation protocols Pay attention to sample amount / concentration Best results with purified samples – Mixtures of components give reduced spectra intensity and difficult to identify sample components Remember : - you know most about your sample – not the analyst – give any and all available required information.