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Ion Sources and Mass Analyzers in Protein Characterization Principles of MS and MS/MS Matrix Assisted Laser Desorption Ionization (MALDI) Electrospray Ionization (ESI), Nano-ESI Time of Flight Quadrupole Mass Filter Quadrupole Ion Trap Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Lenses and prisms focus and refract light. Analogous systems can focus and deflect ions in a vacuum. 1. Get molecules into the gas phase & ionize them. 2. Give the ions a defined energy or velocity. 3. Separate or sort the ions on the basis of that defined property. 4. Detect the ions & assign their masses. A very simple mass spectrum of Carbon Dioxide Copyright ASMS: http://www.asms.org/whatisms/p5.html Online Separations with MS Detection Sensitivity, Specificity, Transparency of Data Differentiation of Co-eluting analytes Looking at MS Data: LC/MS Data is Three Dimensional Mass spec data systems generate “total ion chromatograms” by integrating spectra and plotting intensity versus time. It is analogous to that generated using a diode array UV-detector on an HPLC system. The data is fundamentally 3-dimensional. A “selected ion chromatogram” is the same graph of intensity over time for a defined m/z. It is analogous to a UV chromatogram for a single wavelength. Looking at MS Data: Mass spectra show m/z, not mass Mass spectrometers separate molecules on the basis of their mass to charge ratio, not their mass. That means the x-axis is not necessarily reflective of M. Mass spectra are normalized to the abundance (intensity) of the highest peak in a given spectrum. The y-axis is always scaled from 0-100. Absolute intensity is also often shown in the corner of the spectrum as an arbitrary number unique to each data system. Mass Resolution M1 M2 dM Resolution is often defined as M/dM. FWHM 25% valley “Unit resolution” means that two adjacent peaks are resolved from one another. In low resolution, dM may be 1 mass unit. In high resolution, dM may be 0.010 mass unit. However, the actual resolution depends on how one defines the separation between the peaks (e.g. 50% vs 10% valley). Larger Peptides = More Complex Isotope Patterns As ions grow larger, the “12C” peak is not necessarily most abundant. The mass resolution of analyzers may not always be adequate to distinguish individual peaks. In this case, average masses are used. It is important to be aware of the capabilities of the mass analyzer one is using. Analyzer Resolution: Average vs. Monoisotopic Masses Average mass: The mass of an ion for a given empirical formula calculated using the relative average atomic mass of each element, e.g. C = 12.01115, H = 1.00797, O = 15.9994. Monoisotopic mass: The mass of an ion for a given empirical formula calculated using the exact mass of the most abundant isotope of each element, e.g., C = 12.000000, H = 1.007825,O = 15.994915. Tandem Mass Spectrometry (MS/MS) Mass Spectrometer Tandem MS permits selection and isolation of specific ions for subsequent analysis. Tandem Mass Spectrometer Tandem instruments have multiple mass analyzers. Mass Analyzers Magnetic Sector and Double Focusing Instruments Quadrupole Mass Filters Quadrupole Ion Traps Fourier Transform Ion Cyclotron Resonance Time of Flight Mass Analyzers: The Quadrupole Mass Filter A potential of ~100-1000 V is applied alternately to the opposing pairs of rods at a frequency of a few MHz. At a specific combination of DC & RF, an m/z has a stable trajectory through the rods, and all other m/z are lost. The mass range is scanned as the voltages are swept from min to max, but at constant DC/RF ratio. Faster Scanning than sector instruments (but not as fast as ion traps or TOF). Mass Range generally m/z 0-2000 or 0-4000. Facile MS/MS using Triple Quadrupole (Q-q-Q) analyzer. Exquisitely sensitive in selected ion monitoring (both analyzers parked at one m/z). Largely replaced by the ion trap and hybrid Q-q-TOF for biopolymer analysis. MS/MS in a Triple Quadrupole (Q-q-Q) Mass Spectrometer Mass Analyzers: The Quadrupole Ion Trap Facile MSn High resolution over narrow ranges Extremely Sensitive Fast Scanning Small Inexpensive Mass Analyzers: Fourier Transform Ion Cyclotron Resonance Ions in a magnetic field move in circular orbits characteristic of their m/z values. If energy is provided at a frequency equal to their precession frequency, and in a direction perpendicular to their plane of precession, the ions will absorb the energy, enabling them to be detected. Extremely High Resolution MSn capability Must Operate at very good vacuum Superconducting Magnet Difficult to operate Becoming increasingly reliable Mass Analyzers: Fourier Transform Ion Cyclotron Resonance Mass Analyzers: Time of Flight (TOF) Constant Kinetic Energy zeV = ½ mv2 v = (2zeV/m)½ Linear TOF Reflectron TOF Ion Sources Gas Phase Ionization: Electron Impact (EI) Chemical Ionization (CI) Desorption Ionization: 252Cf Plasma Desorption (PDMS) Fast Atom Bombardment (FAB) / Secondary Ion MS (SIMS) Laser Desorption (LDMS) Matrix Assisted Laser Desorption (MALDI) Spray Ionization: Thermospray (TSP) Atmospheric Pressure Chemical Ionization (APCI) Electrospray (atmospheric pressure ionization) (ESI, API) The Nobel Prize in Chemistry 2002 John B. Fenn electrospray ionization for MS Koichi Tanaka soft laser desorption ionization for MS Kurt Wuthrich solution NMR for protein structures http://www.nobel.se/chemistry/laureates/2002/index.html John B. Fenn – Nobel Lecture "Electrospray Wings for Molecular Elephants" http://www.nobel.se/chemistry/laureates/2002/fenn-lecture.html MALDI-TOFMS Analyte: 10 – 1000 fmol 1 – 500 kDa MALDI-TOFMS the three most commonly used matrices Some Characteristics of MALDI-TOFMS Ions are easy to generate Buffers, salts, some detergents easily tolerated Excellent sensitivity (< 20 fmol for digests) High resolution at low mass with time lag focusing Resolution drops off at higher mass (>20 kDa) Protein or peptide mixtures can show suppression effects Different matrices yield different results A MALDI Target with Digest Samples Spotted on Nitrocellulose Films R. G. Davis, GlaxoSmithKline MALDI-TOFMS Constant Kinetic Energy zeV = ½ mv2 v = (2zeV/m)½ Ion Sources: Electrospray Very gentle and efficient way of getting gas phase ions from solutions. A fine spray of charged droplets is generated in an electric field. Droplets evaporate - analyte molecules are left carrying charges. Multiply Charged Ions are the rule. Concentration dependent – High sensitivity at very low flow rates (<< 1 ul/min). Electrospray is a concentration-dependent technique. Lower flow rates are favored significantly. Smith et al, Acc. Chem. Res. 2004 Electrospray Mass Spectrum of Myoglobin m1 = (M+n)/n m2 = (M+n+1)/(n+1) +21 +12 Quasimolecular ions, [M+nH], from myoglobin, Mr= 16,951.5 Da. Using adjacent pairs of ions, the molecular mass of the myoglobin can be calculated very accurately. Tandem Mass Spectrometry (MS/MS) is the Method of Choice for Sequence Analysis of Peptides Speed Sensitivity Tolerance for Amino-terminal Blocking Groups High Specificity for Protein Identification Tandem Mass Spectrometry (MS/MS) Mass Spectrometer Tandem MS permits selection and isolation of specific ions for subsequent analysis. Tandem Mass Spectrometer Tandem instruments have multiple mass analyzers. Tandem Mass Spectrometry : Product Ion Scan 1. “Parent” Ions are selected and isolated 2. Collision-Induced-Dissociation Results in fragmentation 3. “Daughter” Ions are characterized with the second mass analyzer Q1 Q2 Q3 MASS FILTER RF ONLY MASS FILTER PRECURSOR ION NEUTRAL GAS PRODUCT ION SELECTION COLLISIONS DETECTION ION SOURCE DETECTOR Tandem Mass Spectrometry: Precursor Ion Scan 1. “Product” Ion is selected and Q3 is parked 2. Q1 is scanned normally 3. Only precursors which fragment to produce selected product ion are detected. Q1 Q2 Q3 MASS FILTER RF ONLY MASS FILTER PRECURSOR ION NEUTRAL GAS PRODUCT ION SELECTION COLLISIONS DETECTION ION SOURCE DETECTOR Tandem Mass Spectrometry: Neutral Loss Scan 1. The mass of a functional group whose loss is to be detected is selected. 2. Both Q1 and Q3 are scanned simultaneously, offset by the selected “neutral loss” mass. 3. Collision-Induced-Dissociation Results in fragmentation 4. Daughter” Ions are detected only when the specified loss occurs in Q2, indicating the presence of the moiety of interest. Q1 Q2 Q3 MASS FILTER RF ONLY MASS FILTER PRECURSOR ION NEUTRAL GAS PRODUCT ION SELECTION COLLISIONS DETECTION ION SOURCE DETECTOR MS/MS of Angiotensin III: selection and fragmentation of the (M+H)+ molecular ion at m/z932 Micromass “Back to Basics” http://www.micromass.co.uk/basics/index.html MS/MS of Angiotensin III: selection and fragmentation of the (M+H)+ molecular ion at m/z932 Another way to 532 669 784 label an MS/MS spectrum is to draw lines through the structure, with 400 pointers indicating which part of molecule is being detected following fragmentation. These markers may be labeled with masses. Micromass “Back to Basics” http://www.micromass.co.uk/basics/index.html MaxEnt-3TM for Sequencing y''11 1448.8 100 y''10 TM MaxEnt-3 1285.7 Y y''9 % 136.1 1156.7 a1 197.1 z11 L y''7 z10 a11 1672.9 y''8 1431.8 86.1 1268.69 1513.8 b2 y''4 914.6 1042.7 265.1 388.2 1655.8 505.3 643.3 781.4 mass 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 Y;136.1 100 y''10 2+ y''11 2+ 643.4 724.9 643.9 y''9 2+ a1 578.8 197.1 % L z5 86.1 558.3 644.4 y''7 2+ z7 457.8 Raw data 293.1 897.5 757.9 m/z 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 Links to Information on Mass Spectrometry Information on FTICR at the national high magnetic field lab http://www.nhmfl.gov/science/cimar/icr/ Introduction to mass spectrometry at SciMedia.com http://www.rmsb.u-bordeaux2.fr/rmsb/ms/IntroMS.html The Thermo Finnigan homepage http://www.thermo.com/eThermo/CDA/BU_Home/BU_Homepage/0,12482,113,00.html The Micromass homepage, Mass Spec Back to Basics course http://www.micromass.co.uk/basics/default.asp Mass Spec Glossary http://www.genomicglossaries.com/content/mass_spectrometry.asp The I-mass homepage http://www.i-mass.com/ I-mass tutorials http://www.i-mass.com/guide/tutorial.html American Society for Mass Spectrometry: What is Mass Spectrometry http://www.asms.org/whatisms/.
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  • Electron Ionization

    Electron Ionization

    Chapter 6 Chapter 6 Electron Ionization I. Introduction ......................................................................................................317 II. Ionization Process............................................................................................317 III. Strategy for Data Interpretation......................................................................321 1. Assumptions 2. The Ionization Process IV. Types of Fragmentation Pathways.................................................................328 1. Sigma-Bond Cleavage 2. Homolytic or Radical-Site-Driven Cleavage 3. Heterolytic or Charge-Site-Driven Cleavage 4. Rearrangements A. Hydrogen-Shift Rearrangements B. Hydride-Shift Rearrangements V. Representative Fragmentations (Spectra) of Classes of Compounds.......... 344 1. Hydrocarbons A. Saturated Hydrocarbons 1) Straight-Chain Hydrocarbons 2) Branched Hydrocarbons 3) Cyclic Hydrocarbons B. Unsaturated C. Aromatic 2. Alkyl Halides 3. Oxygen-Containing Compounds A. Aliphatic Alcohols B. Aliphatic Ethers C. Aromatic Alcohols D. Cyclic Ethers E. Ketones and Aldehydes F. Aliphatic Acids and Esters G. Aromatic Acids and Esters 4. Nitrogen-Containing Compounds A. Aliphatic Amines B. Aromatic Compounds Containing Atoms of Nitrogen C. Heterocyclic Nitrogen-Containing Compounds D. Nitro Compounds E. Concluding Remarks on the Mass Spectra of Nitrogen-Containing Compounds 5. Multiple Heteroatoms or Heteroatoms and a Double Bond 6. Trimethylsilyl Derivative 7. Determining the Location of Double Bonds VI. Library