1. Special thanks to: Jim Scrivens, Roman Zubarev, Claudia Blindauer, Ann Dixon, Scott Mcluckey, Fred W. McLafferty, Frank Turecek, Ron Heeren, and many others for helpful slides. Also thanks to my Warwick research group: Anna Pashkova, Andrea Clavijo- Lopez, Pilar Perez-Hurtado, Rebecca Wills, Huilin Li, Terry Lin, Yulin Qi, Andrew Soulby for help with the course. CH908: Mass Spectrometry Spring 2011 Professor Peter B. O’Connor

2. 15, 1 hour lectures - slides will be posted online prior to the lecture. 10-15 problem sets, about one for each lecture – these will not be marked - problem sets will be posted online prior to the lecture. - solutions will be posted online as well, sometime thereafter. 4 workshops for helping with the problem sets – expected to start with a lecture interpreting an example spectrum. 2 marked homework sets (25% of final mark, each) 1 Exam. Exam questions will be derived from the problem sets. (50% of final mark)

3. Fred W. McLafferty and Frantisek Turecek (1993), Interpretation of Mass Spectra – Fourth Edition, University Science Books, ISBN 0-935702-25-3 (Electron Impact spectra) For this lecture, read chapters 2-3. Edmond de Hoffmann and Vincent Stroobant (1999), Mass Spectrometry: Principles and Applications 2 nd Edition, Wiley, ISBN 0-471-48566-7 (General text) Richard B.Cole (1997), Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Applications, Wiley, ISBN 0-471-14564-5 (ESI / Instrumentation / Applications / LC-MS)

4. Objectives for this lecture: Introduce the following concepts: –masses of elements and molecules –isotopes and isotope distributions –mass accuracy and its limitations –mass resolving power –Electron Impact (odd electron ions) –Chemical Ionization (even electron ions) –Tandem mass spectrometry

5. Essentials of a Mass Spectrometer  Sample inlet system (possibly including chromatography)  Ion source  Mass analyzer (determines mass range, accuracy, and resolving power)  Ion detection system  Data system controls instrument and usually has tools to assist processing data  Connection to on-line database in some cases (-omics)

6. Ion Source Mass Analyzer Ion Detector Inlet Data System Vacuum Pumps Sample Introduction Data Output Mass Spectrometer

7. What does a Mass Spectrometer Do?  Generates ions (positive or negative) from samples introduced directly or from a GC or HPLC  Separates ions according to their mass-to-charge ratios, m/z. Often, z=1, but electrospray ionisation gives multiply charged ions.  Collects ions at each m/z and records relative abundances  A data system then can be used to process the recorded data (normalisation, background subtraction, mass range displayed, etc.)  Plots normalised relative abundance against m/z - this is the mass spectrum.

8. Advantages of Mass Spectrometry  NEAR UNIVERSAL APPLICABILITY  Almost all substances give a mass spectrum  SELECTIVITY  High Resolving Power allows selection of one component from a complex mixture  SPECIFICITY  Exact molecular weight is often specific to the compound under investigation; observation of a chosen fragmentation in MS often identifies a given component in a mixture  SENSITIVITY  Detection levels as low as 1 femtomole (10-15 moles). Complete structure from less than 100 femtomoles  SPEED

9. Limitations of Mass Spectrometry  Can sometimes have difficulty in distinguishing isomers from each other.  It can distinguish isobaric compounds, e.g. CO and N2, only if the resolving power is sufficiently high  May decompose or isomerize compounds during ionisation process. Secondary or higher structure observed in solution may be lost during ionisation process  Usually needs very, very, very pure samples – so sample preparation is THE KEY to obtaining good spectra.

10. Limitations of Mass Spectrometry  Detailed mechanism of ionisation and fragmentation processes are not fully understood; sometimes difficult to predict the mass spectrum of a particular molecule from first principles  Getting detailed structural information from the spectra requires a solid understanding of fragmentation mechanisms.  Quantitation requires use of an internal standard such as a deuterated analog

11. 1. Gravitational mass. Newton’s law of gravitation: F g = G  m  M/r 2 - gravitational force G – gravitational constant; m, M – masses of spherical bodies; r – center-of-mass distance. 2. Inertial mass. Newton’s second law of mechanics: F i = m  d v/dt, - external force v - velocity. Mass equivalency principle. Gravitational and inertial masses are equivalent. Confirmed experimentally to the accuracy of 10 -12 (1971). Mass and energy equivalency. Einstein’s law: E = m  c 2, - total energy c – speed of light, 3  10 8 m/s. Example: 1 eV = 1.07  10 -9 u (Da). r m M What is mass?

12. Who Was John Dalton? John Dalton (1766-1844) of Manchester, England was the first to discover the empirical Law of Multiple Proportions (around 1804): When any two elements are observed to form more than one compound between them, the mass ratios in one compound will be related to the mass ratios in the other in the proportions of whole numbers. Since hydrogen was the lightest element known, Dalton assumed that hydrogen should have an atomic mass of 1. Source: www.daltonics.bruker.com/about/johndalton.htm Mass Units Since the IUPAC meeting of 1968, the Dalton (Da) is defined as 1/12 th of the mass of the 12 C isotope of carbon. Note: the atomic mass unit (amu) was a previous unit based on 1/16 th of the mass of 16 O – it’s usage was officially discontinued in 1968, but it lingers in the literature causing confusion.

13. Mass: M = Σm e  n e, IsotopeMassAbundanceChemical Deviation from the masswhole number 1 H1.0078251099.9852%1.00794+0.0079 2 H (D)2.014102220.0148% 12 C12.0(0)98.892%12.011+0.011 13 C13.00335441.108% 14 N14.0030743999.635%14.00674+0.007 15 N15.00010770.365% 16 O15.9949150299.759%15.9994-0.0006 17 O16.99913290.037% 18 O17.999160020.204% 31 P30.9737647100%30.9737647-0.0262 32 S31.972073795.0%32.066+0.066 33 S32.97146190.76% 34 S33.96786464.22% 36 S35.9670900.014% What is Molecular Mass?

14. What is a mass spectrum?

15. “A+1” elements (carbon, nitrogen, hydrogen ) (fluorine, phosphorus, cesium, sodium, iodine) Monoisotopic “A” elements

16. “A+2” elements (oxygen, chlorine, bromine, silicon, sulfur)

17. Elemental Compositions of Metals CRC Handbook of Chemistry and Physics, 48 th Edition, 1967

18. monoisotopic mass dominates up to MW ~1100 - 1500 above MW ~7000, the monoisotopic peak is vanishingly small becomes more symmetric the width grows sublinearly. For <3 kDa, MW/FWHM ~1100, for 10 kDa, MW/FWHM ~2000 the most abundant mass is 0 to 1 Da below the average mass fine structure for all peaks but monoisotopic. Yergey J, Heller D, Hansen G, Cotter RJ, Fenselau C. Anal. Chem. 1983, 55, 353-356. MW 1000 MW 2000MW 3000MW 4000 FWHM How do isotopic distributions change with mass?

19. Nominal mass: m e is the integer mass value for the most abundant isotope (H=1, etc.). Monoisotopic mass: m e is the exact mass value for the most abundant isotope (H=1.00782510, etc.). Average mass: m e is the chemical (average) atomic mass value (H=1.00794, etc.). Isotopic cluster (distribution): a group of isotopic peaks representing the same molecule. Most abundant mass: such in the isotopic cluster. Mass quantities: Yergey J, Heller D, Hansen G, Cotter RJ, Fenselau C. Anal. Chem. 1983, 55, 353-356. Molecular mass is the isotopic distribution!

20. How to calculate the isotopic distribution? N= number of atoms i = i th isotope p = probability of being heavy isotope (e.g. 13 C) Note: the total isotopic distribution is the convolution of the individual isotopic distributions for each possible isotope. Yergey, J. A. Int. J. Mass Spectrom. Ion Physics, 1983, 52, 337-349. Rockwood, A. L.; Van Orden, S. L.; Smith, R. D. Anal. Chem. 1995, 67, 2699-2704.

21. Yergey, J. A. Int. J. Mass Spectrom. Ion Physics, 1983, 52, 337-349. Rockwood, A. L.; Van Orden, S. L.; Smith, R. D. Anal. Chem. 1995, 67, 2699-2704. Fine structure of isotopic peaks RP = 300k RP = 600k RP = 1500k RP = 2.7M

22. Is the Average Mass Reliable? Inherent uncertainty of average mass is ca. 10 ppm.

23. Is average mass reliable? Underestimation by 0.45±0.10 Da. Zubarev RA, Demirev PA, Håkansson P, Sundqvist BUR. Anal. Chem. 1995, 67, 3793-3798. 0.1 Da! Minimal

24. Senko MW, Beu SC, McLafferty FW, JASMS, 1995, 6, 229-233.

25. Effect of poor statistics Hundreds to thousands of ions are needed to identify reliably the monoisotopic mass of a peptide with MW >1 kDa! M mono Theory M mono Experiment

26. Statistical scatter in isotopic abundances Kaur, P.; O'Connor, P. B. Use of statistical methods for quantitative determination of the number of trapped ions Anal Chem 2003, 76, 2756-2762. 100 ions 1000 ions 10000 ions ∞ ions 100 ions 300 scans 5000 ions 300 scans Myoglobin C 60

27. Peak position determination

28. Peak position determination (Centroiding) Apex fitting 1012.989+/-0.025 Curve fitting 1012.996+/-0.010 1012.989 ± 0.012 Center of mass determination Error = FWHM/k k = f(Statistics, S/N)

29. Known: there are ☻and ☻inside. ☻? Extracting Information from Mass A priori: M(empty box) = 1.0000 g M(☻) = 3.141593 g M(☻) = 2.718282 g Measured: M(box) = 10.000±0.002 g Black Box ☻ ☻ ☻ ☻ ☻ 11 5.860 12 8.578 1311.296 20 6.283 21 9.002 2211.720 2314.438 30 9.425 3112.143 3214.861

30. Using “Exact Mass” for Elemental Composition Suppose you have a peak at 128.0454? What elemental compositions are possible for such a peak?

31. Limits of Mass Accuracy Dougherty RC, Marshall AG, Eyler JR, Richardson DE, Smaller RE JASMS, 1994,5, 120-123. Current mass standard: based on 12 C: 12 C solid 12 C gas 12 C + + e - 7.42 eV 11.26 eV 18.7 eV = 20.7 nDa = 1.7 ppb for 12 C

32. Resolving power and peak separation R = (m/z) 0 /FWHM(m/z) FWHM: full width at half maximum For isotopic resolution at MW = MW 0, one needs R FWHM >1.4MW 0 FWHM

33. Resolving power in FTMS Instrumentation: 4.7 Tesla FTMS

34. Close-mass separation in FTMS Fourier Transform Mass Spectrometry

35. Shi SD-H, Hendrickson CL, Marshall AG, PNAS 1998, 95, 11532-11537. Close-mass separation in FTMS

36. Close mass separation in FTMS Shi SD-H, Hendrickson CL, Marshall AG, PNAS 1998, 95, 11532-11537.

37. Electron Impact : Mass spectrometry of volatile materials  Ionization by electron impact (EI)  Radical ion formed, thus...  Significant fragmentation  Can use libraries (250K compounds) or interpret spectra  Very commonly used with gas chromatography  Chemical ionisation (CI) for softer ionization  Limited to volatile, stable compounds < 1000 Da

38. Ion Source Mass Analyzer Ion Detector Inlet Data System Vacuum Pumps Sample Introduction Data Output Mass Spectrometer

39. 194 67 109 55 82 42 165 136 94 40 60 80 100 120 140 160 180 200 Abundance Mass (amu) Mass Spectrum N C C N H C O C O N N CH C 3 H C 3 H Mass Spectrometer Typical sample: isolated compound (~1 nanogram) Mass Spectrometry

40. Electron Impact M + e - → M +. + 2e - Many Fragments….

41. Electron impact ionisation (EI)  The ionization potential is the electron energy that will produce a molecular ion. The appearance potential for a given fragment ion is the electron energy that will produce that fragment ion.  Most mass spectrometers use electrons with an energy of 70 electron volts (eV) for EI.  This is (usually) the most sensitive and stable value.  Decreasing the electron energy can reduce fragmentation, but it also reduces the number of ions formed.

42. ~70 Volts + _ + _ e-e- e-e- e-e- + +++ + + _ Electron Collector (Trap) Repeller Extraction Plate Filament to Analyzer Inlet Electrons Neutral Molecules Positive Ions Electron Impact ionisation source

43. Electron impact ionisation (EI)  Sample introduction  Heated batch inlet  Heated direct insertion probe  Gas chromatography  Liquid chromatography (particle-beam interface)  Benefits  Well-understood  Can be applied to virtually all volatile compounds  Very reproducible mass spectra  Fragmentation provides structural information  Libraries of mass spectra can be searched for EI mass spectral "fingerprint"  Limitations  Sample must be thermally volatile and stable  The molecular ion may be weak or absent for many compounds.

44. Chemical ionisation Methane: CH 4 + e -----> CH 4 +. + 2e ------> CH 3 + + H. CH 4 +. + CH 4 -----> CH 5 + +CH 3. CH 4 +. + CH 4 -----> C 2 H 5 + + H 2 + H. Isobutane: i-C 4 H 10 + e -----> i-C 4 H 10 +. + 2e i-C 4 H 10 +. + i-C 4 H 10 ------> i-C 4 H 9 + + C 4 H 9 +H 2 Ammonia: NH 3 + e -----> NH 3 +. + 2e NH 3 +. + NH 3 ------> NH 4 + + NH 2. NH 4 + + NH 3 --------->N 2 H 7 +

45. Even-Electron Ions  Under EI conditions, M +. ions are formed and a major fragmentation process is the loss of a radical, R., producing an even-electron ion.  Once a radical has been lost, all subsequent fragmentations involve the loss of a molecule to form further even-electron ions.  Under CI conditions, an even-electron ion, such as MH +, is formed; subsequent fragmentations involve the loss of a molecule to form further even-electron ions.

46. Sites of Protonation  In order to rationalise the fragmentation of MH + ions, one must consider at which sites in the sample molecule the proton is attached. The spectrum may then be understood in terms of the fragmentation of the different types of MH + ions.  In general, protonation occurs on heteroatoms having lone pairs of electrons, such as O, N, and Cl. This frequently followed by elimination of a molecule containing the hetero- atom. Other common protonation sites are aromatic rings and regions of unsaturation.

47. Even Electron Ions  Ephedrine ionised by methane CI may protonate for example on the O atom of the OH group:  Protonation on the N atom leads to the loss of CH 3 NH 2 by a similar mechanism, yielding an ion of m/z 135. Both m/z 148 and 135 are observed in the CI spectrum, indicating the presence of OH and HNCH 3 groups in the molecule.

48. Ephedrine EI and CI Spectra Ephedrine, 165 Da, gives an EI spectrum dominated by the m/z 58 fragment ion and no observable M +. ion. Methane CI gives an MH + ion at m/z 166 and fragments at m/z 148, 135 and 58 due to protonation on the OH and NHCH 3 groups or on the aromatic ring respectively

49. Chemical ionisation  Chemical ionization mass spectrometry is the first so-called `soft' ionization technique, to produce information about the molecular weight in many cases where electron impact mass spectrometry fails to do so.  One reason for this difference is the fact that whereas in electron impact ionization, the energy transfer distribution may include a small fraction with energies more than 10 eV above the ionization potential, the energy transfer in chemical ionization processes other than charge exchange does not exceed 5 eV with the more energetic protonating reagents. - this value can be controlled somewhat by selection of different reagent gases.

50. Chemical ionisation  Sample introduction  Heated batch inlet  Heated direct insertion probe  Gas chromatography  Liquid chromatography (particle-beam interface)  Benefits  Often gives molecular weight information through molecular-like ions such as [M+H] +,  Even when EI would not produce a molecular ion.  Simple mass spectra, fragmentation reduced compared to EI  Limitations  Sample must be thermally volatile and stable  Less fragmentation than EI, fragment pattern not informative or reproducible enough for  Library search  Results depend on reagent gas type, reagent gas pressure or reaction time, and nature of sample.

51. Tandem Mass Spectrometry or MS/MS MS/MSMS/MS/MS, or MS 3 Benefits: 1.Extremely high specificity 2.More structural information Limitations: 1.Isolation window 2.Fragmentation efficiency 3.Ion Losses

52. Self assessment What is the exact mass (to 0.1 mDa) of dihydroxy benzoic acid? Its M + ion? Its M+H + ion? A series of peaks spaced 2 Da apart indicate what? A 10 kDa protein ion has isotope peaks which are 0.01 Da wide (FWHM). What’s the resolution? What is the minimum resolution needed to separate two adjacent isotopes to the half height? In a mass spectrum, are fragments good?

53. Fini… CH908: Mass spectrometry Lecture 1

54. Missing: 1. Nitrogen rule slide and examples 2. Even electron ion fragmentation rule 3. R+DB slide and examples 4. The concept of MS/MS, Msn, isolation bleedthrough, specificity improvement 5. Energetics of ionization, proton affinity (CI)

55. Chemical ionisation  Chemical ionization uses ion-molecule reactions to produce ions from the analyte. The chemical ionization process begins when a reagent gas such as methane, isobutane, or ammonia is ionized by electron impact.  A high reagent gas pressure (or long reaction time) results in ion-molecule reactions between the reagent gas ions and reagent gas neutrals.  Some of the products of these ion-molecule reactions can react with the analyte molecules to produce analyte ions.

56. Chemical ionisation  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  In contrast to EI, an analyte is more likely to provide a molecular ion with reduced fragmentation using CI. However, similar to EI, samples must be thermally stable since vaporization within the CI source occurs through heating.

57. Electron impact ionisation (EI)  A beam of electrons passes through the gas- phase sample. An electron that collides with a neutral analyte molecule can knock off another electron, resulting in a positively charged ion. The ionization process can either produce a molecular ion which will have the same molecular weight and elemental composition of the starting analyte, or it can produce a fragment ion which corresponds to a smaller piece of the analyte molecule.

58. Schematic of electron impact source

59. Chemical ionisation  Chemical Ionization (CI) is applied to samples similar to those analyzed by EI and is primarily used to enhance the abundance of the molecular ion.  Chemical ionization uses gas phase ion- molecule reactions within the vacuum of the mass spectrometer to produce ions from the sample molecule.

60. Schematic of chemical ionisation source

61. Chemical ionisation  The chemical ionization process is initiated with a reagent gas such as methane, isobutane, or ammonia, which is ionized by electron impact.  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.

62. Chemical ionisation  Another reason is the greater stability of even-electron protonated ions (MH + ) compared with radical molecular ions (M +. ).  Much of the additional power of chemical ionization mass spectrometry arises from the fact that the characteristics of the CI mass spectrum produced are highly dependent on the nature of the reagent gas used to ionize the sample. As a consequence, it is possible to control the structural information observed by varying the nature of the reagent gas used.