Introduction to Mass Spectrometry Table of Contents 1. What is Mass spectrometry 2. Mass Spectrometry History 3. Basic Components in a Mass Spectrometer 4. Sample Inlets 5. Ionization Technologies 6. Mass Analyzers 7. Mass Detectors 8. Mass Resolution, and Accuracy 9. Isotope Effect on Mass Spectra 10. Tandem Mass Spectrometry 11. Typical Mass Spectra Ying Ge, Ph.D. Human Proteomics Program School of Medicine and Public Health University of Wisconsin-Madison * This presentation is solely used for public education purpose.
What is Mass Spectrometry? Mass spectrometry is the art of measuring atoms and molecules to determine their molecular weight. Such mass or weight information is sometimes sufficient, frequently necessary, and always useful in determining the identity of a species. To practice this art one puts charge on the molecules of interest, i.e., the analyte, then measures how the trajectories of the resulting ions respond in vacuum to various combinations of electric and magnetic fields. Clearly, the sine qua non of such a method is the conversion of neutral analyte molecules into ions. For small and simple species the ionization is readily carried by gas-phase encounters between the neutral molecules and electrons, photons, or other ions. In recent years, the efforts of many investigators have led to new techniques for producing ions of species too large and complex to be vaporized without substantial, even catastrophic, decomposition. John B. Fenn, 2002 Nobel Laureate in Chemistry
Mass Spectrometry History In 1899, J.J. Thompson built the first mass spectrometer, a cathode ray tube In 1918, Dempster developed electron impact (EI) ionization and magnetic focusing MS In 1919, Aston weighed isotopes of inactive element using mass spectrometry In 1946, Stephens described the first time-of-flight (TOF) mass spectrometer In 1952, Johnson and Nier designed the first double focusing mass spectrometer In 1953, Wolfgang Paul introduced quadrupole mass spectrometers and ion trap detectors In 1956, McLafferty proposed a mechanism for γ-h transfer (McLafferty Rearrangement) In 1966, Biemann, et. al. began peptide sequencing by mass spectrometry In 1966, Munson and Field developed chemical ionization (CI) In 1968, Dole introduced electrospray ionization (ESI) on macroions In 1969, Beckey developed field desorption (FD) MS for organic molecules In 1974, McFarlane and Torgerson developed plasma desorption (PD) MS In 1974, Comisarow and Marshall developed FT-ICR MS In 1978, Yost and Enke developed triple quadrupole MS In 1981, Barber developed fast atom bombardment (FAB) In 1983, Tanaka, Karas, and Hillenkamp developed matrix-assistant laser desorption/ ionization (MALDI) In 1984, Fenn et. al. developed electrospray ionization (ESI) on biomolecules
Nobel Prize History in Mass Spectrometry In 1899, J.J. Thompson built the first mass spectrometer awarded Physics Nobel Prize in 1906 In 1919, Francis Anston discovered isotopes using mass spectrometry, awarded Chemistry Nobel Prize in 1922 In 1953, Wolfgang Paul invented ion quadrupole and ion trap mass spectrometers, awarded Physics Nobel Prize in 1989 In 1988-9, John Fenn and Koich Tanaka developed ESI for ionizing large molecules, awarded Chemistry Nobel Prize in 2002
Basic Components in a Mass Spectrometer High Vacuum (10-6 10-9 Torr) Sample Inlet Ion transfer Ion Source Mass Analyzer Detector Recorder A mass spectrometer is composed of five essential parts: 1. Inlet: introducing samples from ambient room pressure into ion source 2. Ion source: converting sample molecules to ions 3. Mass analyzer: separating ions according to their mass 4. Detector: detecting ions and amplifying the signal 5. Recorder: receiving signal from detector, further amplifying, recording, creating mass spectrum
Sample Inlet Systems An inlet system is needed to transfer the sample from the atmospheric pressure (760 Torr) into the source as mass spectrometers are operated in vacuum (~10-6 -10-9 Torr). Common Inlet Systems: 1. Chromatography: gas chromatography (GC) liquid chromatography (LC) capillary electrophoresis (CE) 2. Syringe for direct infusion in ESI 3. MALDI probe or MALDI plate HPLC NanoLC CapLC www.agilent.com
Ionization Sources Basic Type Name and Acronym Ionizing Agent Gas Phase (for Volatile compounds) Condensed Phase Desorption (for nonvolatile compounds) Electron Ionization (EI) Chemical Ionization (CI) Field ionization (FI) Field desorption (FD) Plasma desorption (PD) Secondary ion mass spectrometry (SIMS) Fast atom bombardment (FAB) Electrospray ionization (ESI) Atmospheric pressure chemical ionization (APCI) Matrix-assisted desorption/ionization (MALDI) Energetic electrons Reagent gaseous ions High-potential electrode High-potential electrode Fission fragment from 252 Cf Energetic beam of ions Energetic atomic beam High electrical field High electric field Laser Laser Beam
Electron Ionization (EI) - Hard Ionization M + e - (60-70 ev) M + +2e - 1. The sample is thermally vaporized -> gas phase sample. 2. Electrons ejected from a heated filament are accelerated through an electric field at 70 V to form a continuous electron beam. 3. High energy electron beam passes through the gas phase sample molecules. 4. The electrons collide with the neutral sample molecules -> knock off electron in the sample molecule -> ionization, positively charged molecule ion M + 5. Excess internal energy in the molecule leads to some degree of fragmentation.
Electron Ionization (EI) - Hardest Ionization EI, also known as electron impact ionization, is routinely used for analysis of small, hydrophobic, thermally stable molecules. The sample must be delivered as a gas from a probe via thermal desorption, or by introduction of a gas through a capillary- the output of a capillary column from gas chromatography instrumentation (GC/MS). The utility of EI decreases significantly for compounds >400 Da as thermal desorption of the sample often leads to thermal decomposition before vaporization occurs. Advantages: Well-understood ( oldest method) Suitable for all volatile compounds Reproducible mass spectra Fragmentation provides structural information Libraries of mass spectra available for EI mass spectral "fingerprint" Disadvantages Sample must be thermally volatile and stable Molecular ion may be weak or absent for many compounds Low mass range (<1,000 Da)
Chemical Ionization (CI) CI - applied to samples similar to those analyzed by EI, primarily used to enhance the abundance of the molecular ion. CI uses gas phase ion-molecule reactions within the vacuum of MS to produce ions from the sample molecule. CI is initiated with a reagent gas such as methane, isobutane, or ammonia, ionized by electron impact (EI). High gas pressure in the ionization source results in ion-molecule reactions between the reagent gas ions and reagent gas neutrals. Some of the products of the ion-molecule reactions can react with the analyte molecules to produce ions. A possible mechanism for ionization in CI occurs as follows: Reagent (R) + e - R + + 2 e - R + + RH RH + + R RH + + Analyte (A) AH + + R
Fast Atom/Ion Bombardment (FAB) Soft ionization Fast Atom/Ion Bombardment Gary Siuzdak,,Scripps Center for Mass Spectrometry 1. The analyte is dissolved in a liquid matrix 2. Place a small amount (about 1 microliter) on a target/probe. 3. The target/probe is bombarded with a fast atom beam (e.g. 6 kev xenon atoms) that desorb molecular-like ions and fragments from the analyte. 4. Cluster ions from the liquid matrix are also desorbed and produce a chemical background that varies with the matrix used. Disadvantages: 1. High chemical background Advantages: 2. Difficult to distinguish low MW 1. Suitable for polar thermally labile compound compounds from chemical background 2. Rapid and simple sample preparation 3. Analyte must be soluble in liquid matrix 3. Relatively tolerant of sample variation 4. Not applicable for higher MW (>7 kda) 4. Mass range from ~300 7000 Da molecules 5. Strong ion currents good for high resolution mass measuremnt
Matrix Assisted Laser Desorption Ionization (MALDI) UV or IR laser Matrix (M) Analyte (A) Desorption + + + + + Desolvation + + H+ + Proton transfer + MH + + A M+ AH + MH - + A M+ AH - 1. Analyte is mixed with excess matrix acid 2. Laser pulses -> matrix molecules absorb photon energy -> excite stage, 4. Localized heating caused microexplosion of the matrix material, producing matrix neutral and matrix ions e.g. M +, MH +, (M-H) - 5. Collision with the neutral analytes facilitate charge transfer from matrix molecules Analyte molecules are ionized by gas phase proton transfer, AH +, (A+Na) +, (A-H) -
Common MALDI Matricies MALDI matrix -- A nonvolatile solid material facilitates the desorption and ionization process by absorbing the laser radiation. As a result, both the matrix and any sample embedded in the matrix are vaporized. The matrix also serves to minimize sample damage from laser radiation by absorbing most of the incident energy. MALDI plate for matrix/sample deposition Gary Siuzdak,,Scripps Center for Mass Spectrometry
Matrix Assisted Laser Desorption Ionization (MALDI) MALDI, good for higher MW compounds such as peptides, proteins, and oligonueotides. A MALDI mass spectrum mainly consists of singly charged ions, e.g. [M+H]+, [M+Na] +, [M+K]+ for each sample component. [M+H ]+ Cytochrome C (12.3 kda)
Matrix Assisted Laser Desorption Ionization (MALDI) Advantages: Practical mass range of up to 300 kda Soft ionization with little or no fragmentation Good sensitivity (picomole-femtomole) Tolerance of salts in millimolar concentration Suitable for analysis of complex mixture Disadvantages: Relatively low resolution Matrix interference, especially for low MW Requires a mass analyzer compatible with pulsed ionization techniques MS/MS difficult Not easily compatible with LC/MS
Surface Enhanced Laser Desorption/Ionization (SELDI) SELDI, similar to MALDI, except the surface of SELDI protein chip contains arrays of chromatographic surfaces with different properties, e.g. hydrophobic, cation exchange, anion exchange & metal affinity. Samples goes directly onto the ProteinChip TM Array (Ciphergen) Protein captured and retained on the chip by affinity capture Energy absorbing molecules added to the chip Laser desorption/ionization of the molecules on the chip Advantages: 1. on-chip analysis of proteins, 2 high throughput Disadvantages: 1. low coverage (mainly most abundant proteins) 2. poor reproducibility http://www.evms.edu/vpc/seldi/seldiprocess/index.html
Electrospray Ionization (ESI) Softest Ionization ESI Process 1. Droplet formation -> 2. Droplet shrinkage -> 3. Gaseous ion formation A solution of the analyte passing through a capillary (needle) held at high potential. -> generate a mist of highly charged droplets -> traveling down a potential and pressure gradient towards MS analyzer -> the droplets reduced in size by evaporation of the solvent or by Coulomb explosion -> complete evaporation of the solvent results in fully desolvated gas phase ions.
3000 2500 Electrospray Ionization (Cont d) 2000 ESI, routinely used for analysis of proteins, peptides, carbohydrates, lipids. oligonucleotides, and synthetic polymers, produces singly charged small 1500 molecules but well known for the formation of multiply-charged large molecules 1000 500 18 + 17 + 16 + 15 + 14 + 13 + ESI/MS of Cytochrome C (12.3 kda) 0 600 700 800 900 m/z Advantages: Good for charged, polar or basic compounds Practical mass range of up to 100 kda Soft ionization, capable of observing MI and non-covalent interactions Good sensitivity (femtomole-attomole) Excellent detection limits (low chemical background) Fragmentation controllable Compatible with LC/MS & MS/MS 1000 1100 1200 Disadvantages: Multiply charged species require interpretation No good for uncharged, non-basic, lowpolarity compounds (e.g.steroids) complementary to APCI. Very sensitive to contaminants Low salt tolerance Relatively low ion currents
Nano Electrospray Ionization (NanoESI) http://www.newobjective.com/electrospray/index.html In nanoesi, the spray needle has been made very small and is positioned close to the entrance to the mass analyzer, resulting in the increased sensitivity and detection limit (up to attomole!). Flow rate: ESI (1-20 ul/min); NanoESI (1-50 nl/min) Commercially available nanospray sources: 1. The PicoView nanospray source from New Objective 2. Nanomate from Advion Biosciences 3. Nanospray sources by Thermo and others.
Atmospheric Pressure Chemical Ionization (APCI) Similar interface to that used for ESI. In APCI, a corona discharge is used to ionize the analyte in the atmospheric pressure region. Advantages: Good for less-polar compounds Better for compounds w/o N Gentle vaporization of the analyte intense MH +, minimal fragmentation Enables coupling MS and LC with flow rate up to 1 ml/min, better for normal phase LC Compatible with MS/MS methods Disadvantages: Low mass range (<2000 Da), not good for proteins Sensitivity depends strongly upon analyte Increased fragmentation compared to ESI Complementary to ESI
Comparison of Sensitivity and Mass Ranges by Different Ionization Techniques NanoESI highest sensitivity EI, APCI for lower mass range ESI, MALDI for higher mass range the most common ionization sources for biomolecular mass spectrometry/proteomics Gary Siuzdak, Scripps Center for Mass Spectrometry
General Comparison of Common Ionization Sources Ionization Source Typical Mass Range Matrix Interference Degradation LC/MS Amenable Sensitivity EI 500 none Thermal degradation CI 500 None Thermal degradation FAB 7,000 Yes, severe Matrix reaction & thermal degradation MALDI 300,000 yes Photo degradation APCI 1,200 none thermal degradation Very limited GC/MS Very limited Very limited possible excellent picomole picomole nanomole Low to high femtomole high femtomole ESI (nanoesi) ~70, 000 none none excellent femtomole -attomole
Mass Analyzer - Basic Types (a). Magnetic Sector Magnetic field affect radius of curvature of ions -> m/z (b). Time of Flight (TOF) Flight time - correlated directly with ion s m/z (c). Quadupole (d). Ion Trap (e). Ion Cyclotron Resonance (ICR) Scan radio frequency field -> m/z Scan radio frequency field -> m/z Image current ion cyclotron frequency -> m/z
Mass Analyzer - Hybrid Instruments 1. Triple Quadrupole API 4000 from Sciex/Applied Biosystems TSQ Quantum from Thermo Micromass Quattro Premier from Waters Triple Quadrupole from Agilent 2. Quadrupole Time-of-Flight (Q-TOF) Q-Tof Premier from Waters QSTAR from Sciex/Applied Biosystems Q-q-TOF from Bruker Q-TOF from Agilent 3. Time-of-Flight/Time-of-Flight (TOF/TOF) 4700 Proteomics Analyzer, 4800 TOF/TOF Analyzer from Sciex/ABI TOF/TOF from Bruker 4. Quadrupole Fourier Transform Mass Spectrometer (Q-FTMS) Qq-FTMS from Bruker 5. Ion Trap Fourier Transform Mass Spectrometer (IT-FTMS) LTQ FT and LTQ-orbitrap from Thermo?
Magnetic Sector - The Oldest Mass Analyzer In magnetic sector, ions were separated with a magnetic field. E k Lorentz force law Accelerating potential 1. Ions are accelerated into a magnetic field 2. The radius (r) of an ion depends on the velocity of the ion (v), the magnetic field strength (B), and the ion s m/e. 3. MS Spectrum were obtained by scanning the magnetic field and monitoring ions striking a fixed point detector.
Double Focusing Magnetic Sector Mass Analyzer Magnetic sector 2005 Paul Gates, University of Bristol Electrostatic sector They consist of a large magnetic sector, and an electrostatic sector. The electric sector serves as a kinetic energy focusing point allowing only ions of a certain kinetic energy to pass through its field irrespective of m/z Advantages: 1. Classic mass spectrometer 2. Very high reproducibility 3. High resolution and sensitivity 4. High dynamic range 5. Reproducible high energy MS/MS Disadvantages: 1. Large size and high cost 2. Not well-suited for pulsed ionization method, i.e. MALDI 3. Poor resolution in MS/MS spectra
Time of Flight (TOF) Mass Spectrometer Sample Ion beam Laser pulse Field-free region Flight Tube d m/z = 2Vt 2 /d 2 Advantages: Simplest/fastest MS analyzer Well suited for pulsed ionization methods (e.g. MALDI) High ion transmission MS/MS information from post-source decay Highest practical mass range of all analyzers Detector Time-of-flight m/z = 2Vt 2 /d 2 m/z determined from ion s time of arrival Smaller ions - higher V reach the detector earlier than larger ions Disadvantages: Requires pulsed ionization method or ion beam switching Limited dynamic range of fast digitizers Limited MS/MS experiments
Time of Flight Reflectron Mass Analyzer Gary Siuzdak,Scripps Center for Mass Spectrometry The reflectron combines time-of-flight technology with an electrostatic mirror. The reflectron serves to increase the amount of time (t) ions need to reach the detector while reducing their kinetic energy distribution, thereby reducing the temporal distribution t. Since resolution is defined by the mass of a peak divided by the width of a peak or m/ m (or t/ t since m is related to t), increasing t and decreasing t results in higher resolution. Advantage: 1. Higher resolution (>5000); 2. MS/MS option - PSD (Post Source Decay) Disadvantage: Decreased sensitivity at higher masses (typically above 5000 m/z).
Quadrupole Time of Flight (Q-TOF) Mass Analyzer Gary Siuzdak,Scripps Center for Mass Spectrometry Quadrupole-TOF combines the quadrupole s ability to select a particular ion and the ability of TOF-MS to achieve simultaneous and accurate measurements of ions across the full mass range. The quadrupole can act as any simple quadrupole analyzer to scan across a specified m/z range and also be used to selectively isolate a precursor ion and direct that ion into the collision cell. The resultant fragment ions are then analyzed by the TOF reflectron mass analyzer. Advantages: 1. high resolving power (~10,000); 2. high accuracy (10 ppm) 3. upper m/z limit in excess of 10,000 4. high sensitivity; 5 MS/MS options
Quadrupole Mass Spectrometer Resonant ions (detected) Nonresonant ions to detector Agilent MSD 2000, Paul Gates Source slits Quadrupole rods Analyte ions of different M/Z The quadrupole mass analyzer is a "mass filter". DC and RF potentials combination on the quadrupole rods set to only pass ions with selected mass-to-charge ratio -> focused on the detector. All other ions do not have a stable trajectory through the quadrupole mass analyzer, eventually collide with the quadrupole rods -> never reaching the detector. Varying strength and frequencies of the electric field (rf scan), different ions will be detected.
Triple-Quadrupole Mass Spectrometer Ion Beam Collision Gas N 2 Ar 2 Detector Q1 Q2 Q3 (mass filter) Ion Selection (collision cell) Ion Fragmentation (mass filter) Ion detection Advantages: Classical mass spectra Good reproducibility Relatively small and low-cost systems Low-energy CID MS/MS available in triple-quadrupole Disadvantages: Limited resolution Peak heights variable as a function of mass (mass discrimination). Not well suited for pulsed ionization methods Low-energy MS/MS depend strongly on energy, collision gas, pressure, etc.
Quadrupole Ion Trap (QIT) Mass Analyzer Three hyperbolic electrodes: Ring electrode entrance end-cap electrode exit end-cap electrode QIT: Roughly the size of a tennis ball! The ions are trapped in a 3-D electrical field through entrance endcap electrode The RF (applied on ring electrodes to create 3D quadrupolar potential field) is scanned to resonantly excite and eject ions through small holes in the end-cap to a detector Advantages: 1. MS n capability 3. High sensitivity 2. Compatible with LC/MS/MS Disdvantages: 1. Low resolution 2. Limited dynamic range
Linear Ion Trap (LIT) Mass Analyzer The major difference between LIQ and 3D QIT: LIQ confines ions along the axis of a quadrupole mass analyzer using a two-dimensional radio frequency field with potentials applied to end electrodes. Gary Siuzdak, Scripps Center for Mass Spectrometry The primary advantage to the linear trap over the 3D trap: Larger analyzer volume => improves dynamic ranges =>good for quantitative analysis.
Fourier Transform Ion Cyclotron Resonance (FTICR) Mass Spectrometer Advantages of FT-ICR MS: 1) High resolution (> 1,000,000) 2) High mass accuracy (< 1 ppm) 3) High sensitivity (attomole) 4) Simultaneous detection of all ions 5) Many MS/MS options Disadvantages: Limited dynamic range Subject to space charge effects Many parameters need to be tuned (excitation/trapping/detection) Labor intensive not yet automated! A mass analyzer and a detector Superconducting Magnet ESI source FTICR cell Ion guide Turbo/Cryo pumps skimmer Capillary
How Does FTICR Work? Detection Magnetic field Trapping B Excitation In FT MS, all the ions were trapped, excited and detected in the same ICR cell, therefore it is not only a mass analyzer but also a detector. The detected time-domain signal will be Fourier transform to frequency domain. ω = In the trapping mode, RF applied to excite all the ions to the coherent orbital. In the detection mode, RF stopped, ions of the same m/z form an ion packet and induce image current which can be detected by the diode plates and amplified, digitized and Fourier transformed to frequency domain. The frequency is in inverse proportional to m/z. The amplitude depends on the total number of charges. qb m
Magnetic Field Effect On FTMS Performance FTMS attribute Effect of Magnetic Field Strength B Which means: Resolution (m/ m) Directly proportional to B Improves mass accuracy and the ability to get isotopic resolution on large macromolecules. Kinetic energy Directly proportional to B 2 Increases the fragmentation and also ability to fragment larger macromolecules. Ion capacity Directly proportional to B 2 Can store more ions before spacecharge adversely affects performance
Bruker 7T FTMS FTMS Console 7T Superconducting Electromagnet NanoESI Source FT ICR Infinity Cell
Thermo LTQ/FT Ion Trap based Fourier Transform ICR Mass Spectrometer FTICR (FTMS) on LC timescale Accurate Mass High Sensitivity Ultra-high Resolution Above All -Easy to Operate
New Mass Analyzer - Orbitrap Orbitrap: a new type of mass analyzer employed trapping in an electric field 1. The field potential distribution is a combination of quadrupole and logarithmic potentials. 2. Ion stability is achieved only due to ions orbiting around an axial electrode in absence of magnetic or rf fields. 3. Orbiting ions also perform harmonic oscillations along the electrode with frequency in proportional to (m/z) -1/2 4. Oscillations are detected using image current detection similar to FT. LTQ/Orbitrap Alexander Makarov, Anal. Chem. 2000, 72, 1156-1162
Mass Detectors Mass detector detects a current signal generated from the incident ions. Different mass detectors are used to detect ions depending on the type of mass spectrometer. Most Commonly Used Detectors: Electron Multiplier Faraday Cup Photomultiplier Conversion Dynode High-Energy Dynode Detector (HED) Array Detector e.g. Microchannel Plate (MCP) Charge (or Inductive) Detector e.g. FTMS (detect image current)
Mass Detectors - Electron Multiplier Gary Siuzdak, Scripps Center for Mass Spectrometry Electron multiplier - the most commonly used detector, made up of a series (12 to 24) of aluminum oxide (Al2O3) dynodes maintained at ever increasing potentials. Ions strike the first dynode surface causing an emission of electrons => These electrons are attracted to the next dynode held at a higher potential and generate more secondary electrons => A series of dynodes at increasing potential produce a cascade of electrons an overall current gain on the order of one million or higher
Mass Detectors - Faraday Cup Gary Siuzdak, Scripps Center for Mass Spectrometry Ions striking the dynode (BeO, GaP, or CsSb) surface causing secondary electrons to be ejected. This temporary electron emission induces a positive charge on the detector = > a current of electrons flowing toward the detector. Relatively high pressure tolerance Offering limited amplification of signal, not particularly sensitive
Mass Detectors - Array Detector An array detector - a group of individual detectors aligned in an array format, spatially detects ions according to m/z. Spatially differentiated ions can be detected simultaneously by an array detector. Microchannel Plate (MCP) http://hea-www.harvard.edu/hrc/mcp/mcp.html MCP consist of an array of miniature electron multiplier channels (~10µm diameter, ~15 µm spacing between channels). The channels are parallel to each other and often enter the plate at a small angle to the surface (~8 from normal).
General Comparison of Mass Detectors Detector Advantages Disadvantages Faraday Cup Photomultiplier Conversion Dynode (Scintillation Counting) Electron Multiplier High Energy Dynodes with electron multiplier Table 2.3. General Good comparison for checking ion of detectors. Low amplification ( 10) transmission and low sensitivity measurements Robust Long lifetime (>5 years) Sensitive ( gains of 10 6 ) Robust Fast response Sensitive ( gains of 10 6 ) Increases high mass sensitivity Cannot be exposed to light while in operation Shorter lifetime than scintillation counting (~3 years) May shorten lifetime of electron multiplier Array Fast and sensitive Reduces resolution Expensive Charge Detection Detects ions independent of mass and velocity Limited compatibility with most existing instruments
Mass Resolution (Resolving Power) Mass Resolution: the ability to discriminate between adjacent ions in a spectrum. Greater resolution corresponds directly to the increased ability to differentiate/separate ions. Resolution = M/ M M: m/z M: the full width at half maximum (FWHM) Gary Siuzdak,Scripps Center for Mass Spectrometry
Isotope Effect Natural Isotopic Abundances of Common Elements Isotope: are forms of an element containing same number of protons but different number of neutrons in the nucleus. Element M, % M+1, % M+2, % H 1, 100 2, 0.015 C 12, 100 13, 1.1 N 14, 100 15, 0.37 O 16, 100 17, 0.04 18, 0.20 P 30, 100 S 32, 100 33, 0.79 34, 4.4 Cl 35, 100 37, 32 Br 79, 100 81, 97.3 I 127, 100
Theoretic Isotopic Distribution Myoglobin (C 769 H 1215 N 209 O 221 S 4 ) Resolution: 10k Resolution: 100k Resolution: 1M
Mass Accuracy Mass accuracy - the ability with which the analyzer can accurately provide m/z information; a function of an instrument s stability and resolution. The accuracy varies dramatically from analyzer to analyzer depending on the analyzer type and resolution. An instrument with 0.01% accuracy can provide information on a 1000 Da peptide to ±0.1 Da or a 10,000 Da protein to ±1.0 Da. An alternative means of describing accuracy is using part per million (ppm) terminology, where 1000 Da peptide to ±0.1 Da could also be described as 1000.00 Da peptide to ± 100 ppm. Gary Siuzdak,Scripps Center for Mass Spectrometry
The Effect of Resolution on Mass Accuracy Mass Accuracy is reduced by the uncertainty associated with identifying the center of the peak with the lower resolution. Gary Siuzdak,Scripps Center for Mass Spectrometry
Mass Range & Scan Speed Mass Range: the m/z range of the mass analyzer. Quadrupole analyzers typically scan up to m/z 3000 A magnetic sector analyzer typically scans up to m/z 10,000 Time-of-flight analyzers have virtually unlimited m/z range Scan Speed: the rate at which the analyzer scans over a particular mass range. Scan speed varies with different analyzers. Most instruments require seconds to perform a full scan. Time-of-flight analyzers, e.g. complete analyses in milliseconds or less.
Tandem Mass Spectrometry (MS/MS or MS n ) Tandem mass spectrometry is the ability to isolate different molecular ions in the analyzer, generate fragment ions from the selected ion (parent ion or precursor ion), and then analyze the fragmented ions (product ion or daughter ion) spectrum. Either tandem in space or tandem in time. The fragmented ions are used for structural determination of original molecular ions. Gas/Heat/Light/Electron + + + Fragmentation chamber + + Product ion/ Daughter ion + MS1 + Ion Selection (parent ion/ precursor ion) + MS2
MS/MS Techniques in ESI/FTMS Electron Capture Dissociation (ECD) Laser IR multiphoton Dissociation (IRMPD) Blackbody Infrared Radiative Dissociation (BIRD) Superconducting Magnet Collisionally activated Dissociation (CAD) Turbo pumps skimmer ESI source Capillary
Post-Source Decay (PSD) in MALDI-TOF MS/MS is possible with MALDI TOF reflectron mass analyzers. MALDI fragmentation occurs following ionization, or post-source decay (PSD).
Mass Spectrum - A Bar Graph Format Molecular ion: An ion formed by the removal of one or more electrons to form a positive ion or the addition off one or more electrons to form a negative ion, also called parent ion or precursor ion. Fragment ion: A product ion (or daughter ion) resulting from the dissociation of a precursor ion. Relative abundance 100 50 fragment ion (or daughter ion) molecular ion (or parent ion) 13 C isotope peak 13 C 2 isotope peak 0 m/z (mass to charge ratio)
References 1.Grayson, M.A. Measuring mass, from positive rays to proteins, Chemical Heritage Press, Philadelphia, PA, 2002 2. Siuzdak, G. http://masspec.scripps.edu/mshistory/whatisms.php#esi 3. http://www-methods.ch.cam.ac.uk/meth/ms/theory/index.html 4. Gates, P. http://www.chm.bris.ac.uk/ms/theory/sector-massspec.html 5. Makarov, A. Electrostatic Harmonic orbital Trapping: A high-performance Technique of Mass Analysis, Anal. Chem. 2000, 72, 1156-1162 6.Busch K.L., Glish G.L., McLuckey S.A. Mass Spectrometry/Mass Spectrometry: Techniques and Applications of Tandem. John Wiley & Sons, 1989. 7. Cotter R. Time-Of-Flight Mass Spectrometry: Instrumentation and Applications in Biological Research. Washington, D.C.: ACS, 1997. 8. McCloskey J.A. & Simon M.I. Methods in Enzymology: Mass Spectrometry. Academic Press, 1997. 9. Kinter M. & Sherman NE. Protein Sequencing and Identification Using Tandem Mass Spectrometry. Wiley-Interscience, 2000. 10. Siuzdak, G. The Expanding Role of Mass Spectrometry in Biotechnology, MCC Press, 2003. 11. Borman, S.; Russel, H.; Siuzdak, G. A Mass Spec Timeline, Today s Chemist at Work, 2003 AMERICAN CHEMICAL SOCIETY 12. Chiu C. M.; Muddiman, D. C. What is mass spectrometry, American Society for Mass Spectrometry: Education http://www.asms.org/whatisms/index.html
Acknowledgements The author would like to thank Dr. Gary Siuzdak for kindly granting the permission to use the materials from his website: http://masspec.scripps.edu/mshistory/ 2005 Scripps Center for Mass Spectrometry. The author thank Dr. Jeff Walker, Dr. Lingjun Li and Alice Puchalski for helpful discussions. The financial support was provided by Wisconsin Partnership Fund for a Healthy Future.