Mass Spectrometry Course Árpád Somogyi Mass Spectrometry Laboratory, Department of Chemistry and Biochemistry University of Arizona, Tucson, AZ Eötvös University, Budapest April 11-20, 2012 1
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UA Chemistry 3
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Web addresses for protocols, etc.: www.chem.arizona.edu/facilities/msf/index.html www.proteomics.arizona.edu Lecture only: boring, limited active learning Goal: attentive class, active learning 5
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
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: 1-10 6 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
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
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
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
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Filament: tungsten or rhenium Current: 3-5 A U fil : a few Vs (1-3 V) T fil : ca. 2000-2300 K (ca. 150 0 C in the ion source) Source Block pressure: ca. 10-5 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
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 5 + + M [M-H] + + C 2 H 6 C 2 H 5 + + M [M+ C 2 H 5 ] + 14
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
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
Sources still being developed DESI- desorption ESI (sample not in solution) DART 17
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
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
H + H + 4/9/2012 For (very) high m/z 20
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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
AnchorChip TM, Bruker Daltonics, Germany MALDI-TOF spectrum of a pure protein (linear mode) 16000000 14000000 [M+H] + 14318.68 12000000 Intensity 10000000 8000000 6000000 [M+2H] 2+ 7157.18 4000000 2000000 [2M+H] + 14318.68 0 10000 20000 30000 40000 m/z 23
a.i. Exercise 2 This is a MALDI-TOF spectrum of two proteins. Identify the proteins by molecular weights and assign the labeled ions 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 5000 10000 15000 20000 25000 30000 35000 40000 m/z /export/home/tof/data/mslab/091705/pro_6/1lin/pdata/1 unknown Thu Oct 6 14:27:19 2005 Intens. [a.u.] 8000 5392.3 MALDI-TOF spectrum (protein profile) of a bacteria 9757.4 6267.7 6000 9080.3 4373.8 4000 4880.1 9508.3 8833.8 2000 3133.7 4620.1 7288.2 3645.1 3941.6 7884.4 8380.3 10314.3 0 3000 4000 5000 6000 7000 8000 9000 10000 11000 m/z 24
Glycated hemoglobin from a patient with diabetes Chace, D.H., Chem. Rev., 2001, 101, 445-477 25
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Identify Protein from Table 1 857 858 m/z 27
Determination of Charge States and MW of a protein from ESI spectra 100 +11 1301.53 100 75 Calculated Mass Spectrum 14306.0 Relative Intensity 75 50 25 +13 1101.40 +12 1193.20 +10 1431.47 Intensity +9 1590.33 50 25 0 +8 1789.00 5000 10000 15000 m/z 0 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z 28
Fragmentation of Big Protein Complexes 29
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
7- charge state [M-8H+K] 7- [M-9H+Na+K] 7-974.4564 977.5947 979.8747 [M-9H+2K] 7-974.6014 DNA mass calculator program: http://medlib.med.utah.edu/masspec/mongo.htm 31
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) 44 16 32
CH 3 OH exact mass: 32.0262 u 33
Interpretation of EI spectra Isotopes Isotope patterns Resolution Nitrogen rule Even/odd electron ion rule 34
Selected Isotope Ratios and 13C Contributions Use of Isotope Ratios to Distinguish Structures 35
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 1 0 0 8 0 6 0 4 0 2 0 0 1 0 0 8 0 6 0 4 0 2 0 0 375.2118 (exact mass) 375.4263 (average mass) 3 7 4 3 7 6 3 7 8 3 8 0 3 8 2 m / z (Ala) 5 C 15 H 29 O 6 N 5 1 9 7 6 1 9 7 8 1 9 8 0 1 9 8 2 1 9 8 4 m 1975.9541 (exact mass) 1977.1722 (average mass) / z (Ala) 25 C 90 H 129 O 26 N 25 (Ala) 25 C 90 H 129 O 26 N 25 3568 (nominal mass) R e la tiv e In te n s ity 1 0 0 8 0 6 0 4 0 2 0 3569.8663 (exact mass) 3572.0062 (average mass) (Ala) 50 C 150 H 252 O 51 N 50 3568 (nominal mass) 0 3 5 7 4 3 5 7 6 3 5 7 8 3 5 8 0 3 5 8 2 m / z 36
R e la tiv e In te n s ity 100 80 60 40 20 0 a 374.4183 (average mass) [Ala 5 +H] + 374.2040 (exact mass) 374 376 378 380 382 m /z D eviance from nom inal m ass (dalton) 8 7 6 5 4 3 2 1 0 c average mass exact mass 0 2000 4000 6000 8000 m /z 100 b 3572.9742 (average mass) R e la tiv e In te n s ity 80 60 40 20 0 3570 3572 3574 3576 3578 m /z [Ala 50 +H] + 3570.8741 (exact mass) 37
Characteristic Isotope Distribution of Selected Transition Metal Elements 38
190 255 Origin of m/z 190? Which transition metal element? 39
Transition Metal? Isotope Ratios to Identify Chemical Compositions No Accurate Mass! 40
Isotope Ratios to Identify Chemical Compositions No Accurate Mass! Transition Metal? 41
What Happened? 10 B: 25% 11 B: 100% Renormalized Isotope Distribution in the M +. Region 42
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
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 43 15 86 105 77 182 44
Identify Compounds 102 129 117 Halogen? Identify Compound Use Isotope Distribution in the M +. Region 45
Identify Compounds 46