Mass Spectrometry Course

Transcription

1 Mass Spectrometry Course Árpád Somogyi Mass Spectrometry Laboratory, Department of Chemistry and Biochemistry University of Arizona, Tucson, AZ Eötvös University, Budapest April 11-20,

2 2

3 UA Chemistry 3

4 4

5 Web addresses for protocols, etc.: Lecture only: boring, limited active learning Goal: attentive class, active learning 5

6 Please interrupt!! (With questions and comments) - not with ringing cell phones Some interactive learning checks to keep you engaged will be presented! Selected Thematic Introduction Ionization Methods Mass Analyzers MS/MS scan modes Ion Activation Methods Small Molecule Analysis: MS and MS/MS Spectrum Interpretation Interpretation of EI and CI mass spectra Sample Preparation, Separation Techniques Biological/biochemical/astrobioligical Applications Proteomics, Peptide Sequencing, Protein Identification, Library Search and Spectrum Interpretation 6

7 Mass Spectrometry Introduction, and Ionization Methods Eötvös University, Budapest April 11, 2012 What is Mass Spectrometry? Mass spectrometry is a powerful tool in analytical chemistry that provides detailed structural information for a wide variety of compounds (MW: daltons) by using a small amount of sample (μg-ng, picomol, femtomol) easily coupled with separation techniques (GC, HPLC) Ideal for analysis of complex mixtures 7

8 What can we provide by mass spec? MW determination nominal accurate (elemental composition) isotope pattern high resolution Fragmentation fragmentation rules libraries ( fitting ) MS/MS (or MS n ) Thermodynamic parameters ionization energy (IE) appearance energy (AE) heats of formation (ΔH f ) activation enthalpy (ΔH # ), activation entropy (ΔS # ). Sample Preparation Sample Introduction Direct probe/infusion GC HPLC Rough, Turbomolecular, and Cryo pumps Vacuum System Ionization Source Mass Analyzer Detector Computer Electron impact (EI) Chemical ionization (CI) Atmospheric pressure (API) Electrospray (ESI) Matrix assisted laser Desorption/ionization (MALDI) Surface enhanced LDI (SELDI) Fast atom bombardment (FAB) Electrostatic (ESA) Magnet (B) Time-of-flight (TOF) Quadrupole (Q) Ion Traps (2D & 3D IT) Ion-Cyclotron Resonance (ICR) Orbitrap (OT) Electron Multiplier Photomultiplier Faraday cap Array Detectors Multichanel plate (MCP) 8

9 Difference between single stage MS and tandem MS/MS Ionization Analysis MS: Collide with target to produce fragments MS/MS: Ionization Selection Activation Analysis The importance of sample preparation 9

10 Ionization Methods Neutral species Charged species Removal/addition of electron(s) M + e - (M +. )* + 2e - electron ionization Removal/addition of proton(s) M + (Matrix)-H MH + + (Matrix) - chemical ionization (CI) atmospheric pressure CI (APCI) fast atom bombardment (FAB) electrospray ionization (ESI) matrix assisted laser desorption/ionization (MALDI) desorption electrospray ionization (DESI) direct analysis in real time (DART) Electron Impact (EI) Ionization Still widely used in Forensic Environmental Drug Metabolism, etc. 10

11 11

12 12

13 Filament: tungsten or rhenium Current: 3-5 A U fil : a few Vs (1-3 V) T fil : ca K (ca C in the ion source) Source Block pressure: ca torrs (mean free path ca. 100 m) repeller: 1 V, m/z 100 u, distance 0.1 cm t res = 1.4x10-6 s 13

14 Chemical Ionization Ion-molecule reaction(s) between a reagent gas and the sample at a relatively high pressure Most common reagent gases methane, isobutane, ammonia Mechanisms CH 4 + e - CH 4 +., CH 3+, CH 2 +., CH 4+ + CH 4 CH 5+ + CH 3 CH 3+ + CH 4 C 2 H 5+ + H 2 CH 5+ + M [M+H] + + CH 4 C 2 H M [M-H] + + C 2 H 6 C 2 H M [M+ C 2 H 5 ] + 14

15 Protonation is one type of ionization M + AH+ MH+ + A CH3CH2NH2 + (NH3)nNH4+ CH3CH2NH3+ + (n-1) NH3 The extent of fragmentation depends on the exothermicity of the reaction Proton affinity (PA): M + H + MH + - ΔH r = PA 15

16 Proton affinity (PA): M + H+ MH+ - ΔHr = PA PAs of common CI reagents (kcal/mol) methane (131) < water (173) < methanol (185) < CH2=C(CH3)2 (197) < ammonia (205) If the analyte has a much higher PA than that of the unprotonated reagent, the protonation of the analyte will be (very) energetic (fragment rich CI spectra, semi-ci ) 16

17 Sources still being developed DESI- desorption ESI (sample not in solution) DART 17

18 DART Direct Analysis in Real Time Penning Ionization M* + S S+. + M + electron (but also allows MH+, M-H-, etc) Can we teach elephants to fly? John Fenn 18

19 Yes, of course!!! Nobel Price in Chemistry, 2002 John B. Fenn Koichi Tanaka Electrospray Ionization ESI Soft Laser Desorption SLD Matrix Assited Laser Desorption/Ionization MALDI 19

20 H + H + 4/9/2012 For (very) high m/z 20

21 21

22 The MALDI Plate with Different Samples in Different Matrices Manual Zip-Tip Purification (on dry aliquots of HPA matrix) High throughtput With MiniTrack V (Packard Bioscience Germany) 22

23 AnchorChip TM, Bruker Daltonics, Germany MALDI-TOF spectrum of a pure protein (linear mode) [M+H] Intensity [M+2H] [2M+H] m/z 23

24 a.i. Exercise 2 This is a MALDI-TOF spectrum of two proteins. Identify the proteins by molecular weights and assign the labeled ions m/z /export/home/tof/data/mslab/091705/pro_6/1lin/pdata/1 unknown Thu Oct 6 14:27: Intens. [a.u.] MALDI-TOF spectrum (protein profile) of a bacteria m/z 24

25 Glycated hemoglobin from a patient with diabetes Chace, D.H., Chem. Rev., 2001, 101,

26 26

27 Identify Protein from Table m/z 27

28 Determination of Charge States and MW of a protein from ESI spectra Calculated Mass Spectrum Relative Intensity Intensity m/z m/z 28

29 Fragmentation of Big Protein Complexes 29

30 7- charge state [M-8H+K] 7- [M-9H+Na+K] 7- ESI negative mass sepctrum of an oligonucleotide FT-ICR Instrument [M-7H] 7- [M-8H+Na] 7- [M-9H+2K] 7-30

31 7- charge state [M-8H+K] 7- [M-9H+Na+K] [M-9H+2K] DNA mass calculator program: 31

32 Partial Sequencing of Different Products of the Human Cytochrome P450 cyp2d6 Gene of Different Individuals Enzymes bovine spleen phosphodiesterase and snake venom phosphodiasterase Synthetic Polymer Analysis by MS (MALDI-TOF)

33 CH 3 OH exact mass: u 33

34 Interpretation of EI spectra Isotopes Isotope patterns Resolution Nitrogen rule Even/odd electron ion rule 34

35 Selected Isotope Ratios and 13C Contributions Use of Isotope Ratios to Distinguish Structures 35

36 Increasing 13C contribution in polyalanines R e la tiv e In te n s ity R e la tiv e In te n s ity (exact mass) (average mass) m / z (Ala) 5 C 15 H 29 O 6 N m (exact mass) (average mass) / z (Ala) 25 C 90 H 129 O 26 N 25 (Ala) 25 C 90 H 129 O 26 N (nominal mass) R e la tiv e In te n s ity (exact mass) (average mass) (Ala) 50 C 150 H 252 O 51 N (nominal mass) m / z 36

37 R e la tiv e In te n s ity a (average mass) [Ala 5 +H] (exact mass) m /z D eviance from nom inal m ass (dalton) c average mass exact mass m /z 100 b (average mass) R e la tiv e In te n s ity m /z [Ala 50 +H] (exact mass) 37

38 Characteristic Isotope Distribution of Selected Transition Metal Elements 38

39 Origin of m/z 190? Which transition metal element? 39

40 Transition Metal? Isotope Ratios to Identify Chemical Compositions No Accurate Mass! 40

41 Isotope Ratios to Identify Chemical Compositions No Accurate Mass! Transition Metal? 41

42 What Happened? 10 B: 25% 11 B: 100% Renormalized Isotope Distribution in the M +. Region 42

43 Charges, dots, full arrows, and fishhooks The Nitrogen Rule Compounds* that contain even number of N atoms have even number of nominal molecular weight Compounds* that contain odd number of N atoms have odd number of nominal molecular weight But what about singly protonated molecules and accurate molecular weights?? * Common organic compounds 43

44 Ion Stabilities Even electron ions are more stable than odd electron (radical) ions How about protonated molecules: even electron or not? And how about ions formed by electron impact (EI) ionization? Identify Compounds

45 Identify Compounds Halogen? Identify Compound Use Isotope Distribution in the M +. Region 45

46 Identify Compounds 46