Mass Spectrometry 2 pot

1 Lecture Date: February 27 th , 2008 Mass Spectrometry and Related Techniques 2 Ion and Particle Spectrometry 2 - Outline  Atomic and Molecular Mass Spectrometry – Skoog et al. Ch 11 and 20.  Please read this additional reference: – R. Aebersold and D. R. Goodlett, “Mass Spectrometry in Proteomics”, Chem. Rev., 2001, 101, 269-295.  Ion Mobility Spectrometry – If interested, see:  G. W. Eiceman, Critical Rev. Anal. Chem., 1991, 22, 471-489.  D. C. Collins and M. L. Lee, “Developments in ion mobility spectrometry – mass spectrometry”, Anal. Bioanal. Chem., 2002, 372, 66-73. 2 Homework Problems  If you read March’s paper on ion traps: – What is resonant excitation? Summarize how resonant excitation is used in typical ion trap MS experiments.  If you read the Russell and Edmondson paper on MALDI- TOF and accurate mass: – Summarize the advantages and disadvantages of MALDI-TOF (with DE and reflection) versus FTICR (including ESI-FTICR), especially in biochemical applications.  If you read the Aeberold and Goodlett proteomics paper: – Why is MS used so heavily in the study of post-translational modifications? Briefly describe an application to phosphopeptide sequence determinations.  If you read the Sleno and Volmer ion activation methods paper: – Pick any two of the ion activation processes described in the paper (e.g. in Table 1), describe how it works and the approximate energies involved, and list one advantage Applications of Mass Spectrometry  Interpretation of mass spectra is the key to most applications of the technique  Information contained in a mass spectrum: – Molecular weight (via exact or mono-isotopic mass). Usually obtained though a suitably accurate measurement of:  M + • (the molecular ion, an odd-electron species)  [M+H] + and [M-H] - (the protonated/de-protonated molecule, an even-electron species)  In some techniques, can be confirmed by [M+Na] + , [M+K] + , [M+NH 4 ] + , dimers, trimers, and other adducts, etc… – Molecular formula – Ionization energies – Isotopic incorporation (ex. 13 C, 14 C, 2 H, 3 H…) – Fragmentation and ion stability 3 Quasi-equilibrium Theory  Once we make an ion, what happens to it?  In EI, and similar techniques: the ionizing electron has little mass and high KE, so it barely moves the molecule that it hits but leaves it in a higher rotational/vibrational state.  Ionization energies can sometimes be determined from ion intensities. Diagram from Strobel and Heineman, Chemical Instrumentation, A Systematic Approach, Wiley, 1989. Molecular Structural Analysis: Fragmentation  Fragmentation can also be used to determine structure – common fragmentation pathways and rearrangements can be predicted in many cases  General rules: - More stable carbocations are more stable fragments (ex. tertiary carbocations are more stable than primary) - Resonance can stabilize fragments, ex. allylic carbocations and benzyl/tropylium ions - Loss of small, neutral, stable molecules is favored Figure from R. M. Silverstein, Spectrometric Identification of Organic Compounds, 6 th Ed., Wiley, 1998. O p-chloro-benzophenone Cl 4 Molecular Structural Analysis: Isotope Patterns  Isotope patterns can be used to determine molecular structure – Example: the well-known methods of calculating (M+1) and (M+2) intensities – Especially useful for detecting chlorine, bromine, sulfur, silicon and many other elements with characteristic profiles  Isotope patterns can also be used to extract out “isotope incorporation profiles” for labeled compounds – Examples: 13 C, 14 C, 2 H, 3 H-labeled molecules for metabolism studies – Applications in isotope chemistry include the detection of stable and radioactive isotopes in synthetic products and in nuclear chemistry. M (100%) M+1 (19.28%) M+2 (33.99%) M+3 (6.21%) m/z 215 220 O p-chloro-benzophenone Cl Molecular Structural Analysis: Accurate Mass  Nuclide masses are not integers. Example: Four things that weigh “28” amu: – CO, 27.9949 – 14 N 2 , 28.0062 – CH 2 N, 28.0187 – C 2 H 4 , 28.0312  m/z measurements to four decimal places or higher are needed  Accurate mass analysis is often used as a final confirmation of structure, or for unravelling complex fragmentation 5 Molecular Structural Analysis: Mass Defects -0.04 -0.03 -0.02 -0.01 0 0.01 0.02 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 1 H 2 H 13 C 14 N 15 N 16 O 31 P 32 S 12 C Atomic Mass Defects (All Different) Mass Defect (Da) Mass Defect = Atom Mass – Nearest Integer Every C c H h N n O o S s mass is unique! Mass (Dalton) Picture courtesy Prof. Alan Marshall, FSU/NHMFL Molecular Structural Analysis: MS-MS, and MS n  Step 1 – mass selection of an ion formed in the source  Step 2 – dissociation of the parent ion via collisions  Step 3 – mass analysis of the dissociated “daughter” ions  Step 4 – repeat… + + + + + + + + Collisions Mass Analyzer 1 Mass Analyzer 2 + + + + + + + + Mass Analyzer and Collision Chamber 6 More About MS n Systems  Tandem-in-space – Means that the mass selection and fragmentation occur in different physical locations within the spectrometer. – Examples: Triple-quad (QQQ), in which…  Tandem-in-time – Means that the mass selection and fragmentation occur in the same part of the MS but at different times – Example: ion traps + + + + + + + + Collisions Mass Analyzer 1 Mass Analyzer 2 Dissociation and Controlled Fragmentation in MS n  Collisionally-Induced Dissociation (CID) – also known as collisionally-activated dissociation (CAD) – CID is the principal ion-dissociation method for MS n . In CID, stable ions are fragmented by collisions with neutral gas atoms/molecules  CID uses low-pressure He or Ar gas – Ion traps typically use 10 -3 torr of He – Triple-quadrupole systems typically use 10 -6 torr of Ar – Also can use N 2 , Xe, etc…  Other methods: – Photo-induced dissociation  IRMPD (IR multiphoton dissociation) – via IR lasers  BIRD (blackbody infrared radiative dissociation) – Surface-induced dissociation (SID) – Electron-capture dissociation (ECD) L. Sleno and D. A. Volmer, “Ion activation methods for tandem mass spectrometry”, J. Mass Spectrom., 2004,39, 1091-1112. 7 Collisionally-Induced Dissociation  Low-energy CID – ions traveling with typical KE of <100 eV. – Ions excited to a higher vibrational state, ion-target complex has a lifetime  High-energy CID – ions travelling with typical KE > 1 keV – Ions excited to higher electronic states, no detectable ion-target complex  CID occurs via a two-step mechanism: Step 1. An endothermic activation step to form an M + ion that is internally excited (usually to a higher vibrational state) Step 2. An exothermic unimolecular decomposition to a fragment ion and a neutral. For more information about CID, see: L. Sleno and D. A. Volmer, “Ion activation methods for tandem mass spectrometry”, J. Mass Spectrom., 2004,39, 1091-1112. K. R. Jennings, Int. J. Mass Spectrom. Ion Phys., 1968,1, 227. F. W. McLafferty, et al., “Collisional Activation Spectra of Organic Ions”, J. Am. Chem. Soc., 1973,95, 2120-2129. K. Levsen and H. Schwarz, “Gas-phase Chemistry of Collisionally-activated Ions”, Mass Spectrom. Rev., 1983, 2, 77-148. S. A. McLuckey, “Principles of Collisional Activation in Analytical Mass Spectrometry”, J. Am. Soc. Mass Spectrom., 1992,3, 599-614. Molecular Structural Analysis with MS n  CID and MS n opens up a range of possiblities for MS Scan Modes – Precursor ion scans: keep MS2 constant, scan MS1 – Product ion scans: keep MS1 constant, scan MS2 – Neutral loss scans: scan MS1 and MS2 “in sync”, offset by the difference (neutral) of interest (ex. set MS2 to follow MS1 by 32 Da). – Selected reaction monitoring: hold MS1 and MS2 constant (observe a selected fragmentation) Collisions Mass Analyzer 1 Mass Analyzer 2 8 Applications of MS n Experiments  A short list of applications: MS n studies of drug metabolism, environmental samples,  Especially useful in drug metabolism because key “pieces” of drugs can be selected via their product (daughter) ions or their neutral loss characteristics  MS n is applicable to any analytical situation where complex, overlapping spectra are detected and need to be interpreted For more information about MS applications in drug metabolism, see: R. J. Perchalski, R. A. Yost and B. J. Wilder, Anal. Chem., 1982, 54, 1466-1471. M. S. Lee and R. A. Yost, Biomed. Environ. Mass Spectrom.,1988, 15, 193-204. Applications of MS n Experiments  Example: Structural analysis of linear alkylbenzylsulfonates - a common anionic surfactant that can be a soil pollutant  Can be monitored in soil by LC-ESI-MS n  Samples extracted with methanol, concentrated with SPE  Bruker Esquire 3000 ITMS, negative ion mode (compounds are negatively charged) in this mobile phase: water/methanol/tributylamine/NH 4 COOCH 3  m/z = 183 obtained from CID MS-MS of all chain lengths as a characteristic ion  m/z = 119 obtained from CID MS-MS-MS of m/z = 183 by loss of SO 2 V. Andreu and Y. Pico, Anal. Chem., 2004, 76, 2878-2885 9 Molecular MS Applications: Environmental Science  A compound was discovered in smoke derived from burning plant material that increases germination of a range of plant species that typically follow forest fires.  The compound is 3-methyl-2H-furo[2,3-c]pyran- 2-one, and it was synthesized after being isolated and analyzed by MS and NMR  GC-MS was able to detect this butenolide at low levels in “smoke waters”  The compound is stable at higher temperatures, and is active at 1 ppm to 100 ppt levels. It is derived from the combustion of cellulose. G. R. Flemmatti, Science., 305, 977 (2004) GC-MS (EI) Data: m/z = 150 (100%, M+) m/z = 122 (25%, loss of CO) m/z = 121 (71%) m/z = 66 (14%) m/z = 65 (16%) O O O CH 3 C 8 H 6 O 3 Exact Mass: 150.03 Mol. Wt.: 150.13 m/e: 150.03 (100.0%), 151.04 (8.8%) C, 64.00; H, 4.03; O, 31.97 Molecular MS Applications: Proteomics  Proteome: The group of proteins related to a cell type (with a certain genome) under certain conditions (often forced on the cell)  Genome: The complete DNA sequence of a set of chromosomes.  Proteomics: The analysis of native and post-translationally modified proteins to characterize complex biological systems. There are at least three “types” of proteomics: – Profiling Proteomics: Identify the proteins in a biological sample (or differences between proteins in multiple samples) – Functional Proteomics: Determine protein functions by finding specific functional groups or interactions – Structural Proteomics: Determine the tertiary structure of proteins and their complexes. D. Figeys, Anal. Chem., 75, 2891-2905 (2003) 10 Molecular MS Applications: Proteomics  MS is primarily used for profiling proteomics but has applications to other areas.  MS is often used in conjunction with gel electrophoresis techniques (2D GE, SDS-PAGE, etc…)  MS can be used to study post-translational modifications of proteins R. Aebersold and D. R. Goodlett, “Mass Spectrometry in Proteomics”, Chem. Rev., 2001, 101, 269-295. Molecular MS Applications: Proteomics  “Peptide mass mapping”: used to ID proteins by comparison to a database.  Accurate mass methods (single MS stage) are usually used, following digestion by an enzyme (e.g. trypsin) that “chews up” the peptide into fragments.  The better the mass accuracy, the less chance of isobaric (same mass) interferences. R. Aebersold and D. R. Goodlett, “Mass Spectrometry in Proteomics”, Chem. Rev., 2001, 101, 269-295. [...]... against DB 2 Used to ID “new” peptides (de novo sequencing), using chemical tools to ID fragments: 1 Edman degradation 2 H2O trypsin proteolysis 3 Methyl esterification R Aebersold and D R Goodlett, Mass Spectrometry in Proteomics”, Chem Rev., 20 01, 101, 26 9 -29 5 MS Methods for Surface Analysis  Secondary-ion MS (SIMS) in surface analysis Ions: Ar+, Cs+, N2+, O2+ – Secondary analyte ions are 5 -20 keV... temperature (K)  is the reduced mass of the ion-drift gas pair D is the ion-neutral cross-section area (=d2 for rigid-sphere collisions where d is the sum of the ion and drift-gas radii) G W Eiceman, Critical Rev Anal Chem., 22 , 471-489 (1991) D C Collins and M L Lee, “Developments in ion mobility spectrometry – mass spectrometry , Anal Bioanal Chem., 3 72, 66-73 (20 02) IMS: Ion Mobility Spectrometer... Chem., 20 04, 76, 21 87 -21 95 20 Hyphenated Ion Methods  MALDI-IM-oTOF enabling technology: medium-pressure IM cells that do not lose ions in the differential pumping region  Mobility differences for  different biomolecule classes can differ by ~15% 2D resolution! A S Woods, et al Today’s Chemist at Work, May 20 04, 32- 36 Further Reading Mass Spectrometry: 1 F W McLafferty, “Interpretation of Mass Spectra”,... Sensitivity (years) (parts per 1015) 3H 12. 3 0.1 Beryllium 10Be 1.6 x 106 5 Carbon 14C 5730 2 Aluminum 26 Al 720 ,000 3 Chlorine 36Cl 300,000 5 Calcium 41Ca 105,000 2 Iodine 129 I 106 10 Element Isotope Hydrogen 16 x K W Turteltaub and J S Vogel, “Bioanalytical Applications of AMS for Pharmaceutical Research”, Cur Pharm Design, 20 00, 6, 991-1007 J S Vogel, et al., “Accelerator Mass Spectrometry , Anal Chem., 1995,... ms, typical time resolution +/- 0.040 ms Diagram from G W Eiceman and J A Stone, Anal Chem., 76, 390A-397A (20 04) G W Eiceman, Critical Rev Anal Chem., 22 , 471-489 (1991) D C Collins and M L Lee, “Developments in ion mobility spectrometry – mass spectrometry , Anal Bioanal Chem., 3 72, 66-73 (20 02) 16 IMS: Theory • In IMS, larger ions have longer drift times because of their larger cross-sections • The... Applications of Accelerator Mass Spectrometry for Pharmaceutical Research”, Current Pharmaceutical Design, 20 00, 6, 991-1007 J S Vogel, et al., “Accelerator Mass Spectrometry , Anal Chem., 1995, 353A-359A 14 AMS: Basic Instrument Design and Operation • • • Negative ions are created (usually from a solid sample) These ions are accelerated (MeV) by ever increasing positive potentials The ions are rammed... 1995, 353A-359A See also C&E News, July 11, 20 05, pg 28 IMS: Ion Mobility Spectrometry • In IMS: • Sample vapor introduced by thermal desorption or other techniques • The vapors from the above are ionized using 63Ni (~10 mCi sample) to produce molecular ions or clusters of molecular ions • An electronic shutter gates ions into a drift tube with a ~20 0 V/cm potential • Ions drift down the tube, colliding... from Z Yang, et al Langmuir 20 00, 16, 74 82- 74 92 Mass Spectrometers as GC/LC Detectors • MS is increasingly finding use as a routine chromatography detector (especially in GC and LC) • Two modes: • Single-ion monitoring (SIM): observe 1-4 ions selectively – improved signal-to-noise for ions of interest • Total ion current (TIC): sum of all ions – can be noisy but also captures potential unknown m/z ratios... used in several recent terrorist attacks in Russia (August 20 04) - see C&E News, 6-Sep -20 04, pg 15 • Military/Defense • IMS can be used to detect common chemical weapons more than 50,000 systems (many handheld) are deployed with military units worldwide, as of 20 04 Picture and Data from G W Eiceman and J A Stone, Anal Chem., 76, 390A-397A (20 04) IMS: Applications • Handheld units • Early units weighed... Chem., 76, 390A-397A (20 04) 18 IMS: Dopants and Reactant Ions • Proton affinity determines ionization (especially in 63Ni sources) • Reactant ions are used to achieve selectivity • The analyte ion actually forms a pair with whatever suitable reactant ion is in the drift gas • Examples: • • • • Water (in air)  the hydrated proton [H2O]nH+ Acetone (Ac)  AcH+ and Ac2H+ Ammonia  [H2O]nNH4+ Methylene chloride . Analysis: Mass Defects -0.04 -0.03 -0. 02 -0.01 0 0.01 0. 02 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 1 H 2 H 13 C 14 N 15 N 16 O 31 P 32 S 12 C Atomic Mass. that weigh 28 ” amu: – CO, 27 .9949 – 14 N 2 , 28 .00 62 – CH 2 N, 28 .0187 – C 2 H 4 , 28 .03 12  m/z measurements to four decimal places or higher are needed  Accurate mass analysis is often used. Date: February 27 th , 20 08 Mass Spectrometry and Related Techniques 2 Ion and Particle Spectrometry 2 - Outline  Atomic and Molecular Mass Spectrometry – Skoog et al. Ch 11 and 20 .  Please