Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu

Recommended Reading : “The Expanding Role of mass Spectrometry in Biotechnology” Gary Siuzdak, MCC Press, San Diego, ISBN 0-9742451-0-0 “Ionization Methods in Organic Mass Spectrometry” Alison Ashcroft, RSC, Cambridge, UK, ISBN 0-85404-570-8 “Practical Organic Mass Spectrometry” 2nd Edn J R Chapman, Wiley, Chichester, UK, ISBN 0-471-95831-X “Spectroscopic Methods in Organic Chemistry” 4th Edn D H Williams, I Fleming, McGraw-Hill, ISBN 0-07-707212-X

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 nucleus Different atoms combine in different ways to form molecular sub-units called functional groups.

 Can work out molecular structure Chemistry 101 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 Mass Spectrometry Used For? Chemical Analysis and Identification Some Typical Applications Enviromental Monitoring and Analysis (soil, water and air pollutants, water quality, etc.) Geochemistry – age determination, Soil and rock Composition, Oil and Gas surveying Chemical and Petrochemical industry – Quality control Applications in Biotechnology Identify structures of biomolecules, such as carbohydrates, nucleic acids Sequence biopolymers such as proteins and oligosaccharides Determination of drug metabolic pathways

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 unit In many cases (such as small molecules), z = 1  measured m/z = mass of fragment But this is not always true For large bio-molecules analysed by electrospray (ESI), z >1 What happens in this case?

Multiple Charging Consider a peptide with MW of 10000 With ESI-MS, charges by H+ addition M + nH+  MnHn+ Resultant ions formed are :- When z = 1 m/z = (10000+1)/1 = 10001 When z = 2 m/z = (10000+2)/2 = 5002 When z = 3 m/z = (10000+3)/3 = 3334.3 When z = 4 m/z = (10000+4)/4 = 2501 When z = 5 m/z = (10000+5)/5 = 2001

Figure from The Expanding Role of MS in Bio-technology – G . Siuzdak

Multiple Charging Advantage 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 MS Computer Algorithms deconvolute m/z to original mass. Figure from The Expanding Role of MS in Biotechnology – G . Siuzdak

Mass Measurement Mass Spectrometers measure isotopic mass. They DO NOT measure average molecular mass!! (MW) e.g For a molecule with empirical formula C60H122N20O16S2 Average MW = 1443.8857 (weighted average for each isotope) Exact mass = 1442.8788 (exact mass of most abundant isotope) Nominal mass = 1442 (integer mass of most abundant isotope) Illustrated on next Slide

Resolution Influences achievable precision and accuracy of measurement Figure from The Expanding Role of MS in Bio-technology – G . Siuzdak

Resolution Influences achievable precision and accuracy of measurement R = ΔM/M Often expressed in ppm R = (ΔM/M) x106

Isotope Patterns Isotope patterns useful for identifying presence of certain elements Particularly useful for SMALL molecules Figure from The Expanding Role of MS in Bio-technology – G . Siuzdak

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: Sample Inlet Ion Source Mass Analyzer Detector Data System

How does it work? +4000 V 0 V e- e- e- e- e- Mass spectrometry accelerate separate ionise +4000 V 0 V e- Magnetic and/or electric field e- + + heavy vacuum light vapourise e- + A e- sample + B + C A+ B+ C+ Mass spectrometry e-

Analyser Types What is the analyser? Analyser is the section of instrument that separates ions of different m/z Many Different technologies Magnetic Sector, Quadrupole, Ion Trap, ToF All based on momentum separation

Analyser Types – Magnetic sector Easiest Conceptually to understand Separate electromagnetically “Electromagnetic Prism” Usually combined with ESA (energy focusing device) - enables high mass resolution (Double Focusing Instrument) – makes high accuracy mass measurements possible Large (Heavy!!), Expensive to operate Comparatively slow scan rates High Skill level required to operate and maintain Self-service use by users not possible

Analyser Types – Quadrupole Smaller, cheaper – computer controlled – Self service operation by trained users possible Electrostatic momentum separation by superimposed rf and dc voltages Rapid scan rates – enables measurement of transient samples introduced from chromatographic systems (GC, LC) Lower resolution – accurate mass NOT possible

Analyser Types – Quadrupole ion Trap Derivative of Quadrupole – cheap, small, rapid scanning Again, electrostatic momentum separation by rf and dc voltages Lower resolution – accurate mass not possible BUT – have ion trapping ability – can store and selectively eject ions Ions can be subjected to fragmented by CID and “daughter ions” analysed Allows MS-MS or MSn (Multiple levels of storage and trapping) Can perform both molecular ion analysis and structural determination

Analyser Types – Quadrupole ion Trap 3 Electrode system 2 x Endcap and 1x Ring Electrode Now have recent develpoment of Linear Ion Trap and orbitrap Developments on same theme.

Analyser Types – Quadrupole ion Trap Bruker HCT Ion Trap is very small – most of instrument is ion guides into the trap itself

Analyser Types – Time of Flight (ToF) Conceptual diagram!!!

Analyser Types – Time of Flight (ToF) Velocity separation - E= mv2 Ion packet given constant KE – ions of heavier mass take longer to pass down drift tube and reach detector Conceptually easy Allows very large masses to be measured (500,000Da) E= 1/2mv2 Time flight of ions through drift tube Ions of larger mass take longer to reach detector for constant E

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

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’s From 1980’s, start to have shift in focus towards analysis of samples that are larger and more bio-molecular in character Such 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

Electron Impact (EI) Mass Spectrometry Up until 1980’s, most mass spec is “chemical” analysis - performed using EI ionisation Bombard 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

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).

Electron Impact (EI) Mass Spectrometry

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 EI 2) 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 sample Often forms MH+ Still only applicable to volatile or Thermally stable samples.

CI-MS Comparison of EI and CI spectra

FAB-Mass Spectrometry Subsequent development of FAB (Fast Atom Bombardment) Still used for small delicate molecules Dissolve sample in liquid matrix and place on target Bombard with beam of fast atoms or ions (Xe or Cs+) Have secondary ion emission Low energy protonation of target molecules – very little excess energy – little fragmentation – readily observe parent ions. Now we’re getting somewhere.

FAB-Mass Spectrometry Problems with FAB Slow, Labor intensive, Very skilled. Matrix interference at low mass Generally observe MH+ (+ve ion mode) OR M-H (-ve ion mode)

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 work Will 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.

Electrospray Mass Spectrometry (ESI-MS) Solution phase technique - Can analyse both +ve and –ve ions (but not simultaneously) Samples usually dissolved in moderately polar solvent Typically MeOH or MeCN, often mixed H2O (up to 80%) DO NOT USE DMF, DMSO, THF, etc Do 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)

Electrospray Mass Spectrometry (ESI-MS) How does it work?

Electrospray Mass Spectrometry (ESI-MS) How does it work?

Electrospray Mass Spectrometry (ESI-MS) Thermo-Finnigan LCQ-Deca ESI-Ion Trap with LC System

Electrospray Mass Spectrometry (ESI-MS) Different versions of ESI (On-Axis / Orthoganal / Off Axis) Advantages Soft ionisation – limited fragmentation Multiple 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 mixtures Automated high throughput analysis of biological samples – 24/7 Can be coupled with many analysers – IT/Quadrupole /ICR / Orbitrap – vast range of different types of analysis possible

Electrospray Mass Spectrometry (ESI-MS) Can Deconvolute mass spectra as previously discussed

MALDI-ToF Mass Spectrometry Relatively simple technique Soft ionisation method that can be used to volatilise large macromolecules with minimum fragmentation Gives less multiple charging than ESI Samples 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

MALDI-ToF Mass Spectrometry Target irradiated by UV laser. Causes vaporisation of matrix and supersonic expansion of plume Dried sample is launched into the gas phase as matrix is vaporised UV 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

MALDI-ToF Mass Spectrometry

MALDI-ToF Mass Spectrometry Most MALDI-ToF are reflectron instruments Reflectron is energy focusing device (ion mirror) Increases resolution (and mass accuracy) – but limits mass range Linear ToF has low resolution but high mass range (up to m/z 300,000) Many Instruments are now ToF/ToF Can do MS/MS experiments

MALDI-ToF Mass Spectrometry Typical Current State of the Art Maldi-ToF Bruker Autoflex Now available as Tof/ToF Easy to use – walk up use after training. Highly automated Now can be used for imaging of Tissue samples

MALDI-ToF Mass Spectrometry - Conditions Suggested concentrations ~10 pmol @ <10 000 Da (pure) ~100 pmol @ >50 000 Da (pure) 10: 1 Ratio of Matrix : Sample (20nM-50uM of sample – typically 1-10uM) Several methods of target prep Multiple layer / co-mixed Spot 0.5uL of mixture on spot and allow to dry Analysis very dependant upon sample preparation

MALDI-ToF Mass Spectrometry - Matrices Matrix Application α-Cyano-4-hydroxycinnamic acid (CCA) peptides 3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid) proteins 2,5 Dihydroxybenzoic acid (DHB) peptides, proteins, polymers, sugars 3-Hydroxypicolinic acid (HPA) oligonucleotides Dithranol (anthralin) polymers

MALDI Contamination Limits Analysis is relatively insensitive to contaminants. Phosphate 20 mM EDTA 1 mM Detergents 0.1% Glycine 20 mM Glycerol 2% Sodium Citrate 20 mM Buffer (Tris)50 mM K phosphate 25 mM Guanidine 1 M Na phosphate 0.1M Na azide 1% Octyl glucoside 0.3% SDS 0.05% Ammon. Bicarb. 0.1M Suggested concentrations ~10 pmol @ <10 000 Da (pure) ~100 pmol @ >50 000 Da (pure)

MALDI –Characteristics Maldi-ToF Generally results in broader peak envelope than ESI This 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 500,000Da Maldi-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

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 spot Low mass spectra (<500MW) can be inhibited by interference from Matrix peaks – development of Naldi Spectra VERY dependant upon sample preparation and analysis conditions (especially laser power) – modern instruments have “fuzzy” logic to optimise analytical conditions on the fly

Biotechnology applications 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 oligo’s Carbohydrate analysis

Biotechnology applications 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)

Mouse Brain Digital Photo Before Matrix Addition

Mouse Brain H&E Stain After Molecular Imaging

Mouse Brain Full Molecular Spectrum 600-30,000 Da

Molecular Image of Lipid Mass m/z = 786

Molecular Image of Lipid Mass m/z = 1493

Molecular Image after Unsupervised PCA

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.

Any Questions?