8/13/2015 Mass Spectrometry Workshop Árpád Somogyi Associate Director CCIC, Mass Spectrometry and Proteomics Laboratory OSU August 17, 2015 1 8/13/2015 Workshop schedule Time Monday, 8/17 Tuesday, 8/18 Mass Analyzers Introduction to Mass Spectrometry (Arpad) 9-10:20am Ionization methods (Arpad) FT-ICR instrumentation and techniques (Mike Freitas) 10:35- GC-MS analysis, examples and EI spectra interpretation Ion Activation 12:00pm (Jeremy) (Vicki Wysocki) 12-1pm Lunch Break Lunch Break 1-2:30pm Lab visit (Group 1) Lab visit (Group 2) Small molecule (ESI) spectrum interpretation HPLC-MS/MS and metabolomics, Data processing programs (Arpad) 2:40-4pm (Yu Cao) Open Discussion/Users Presentations Lecture only: Goal: boring, limited attentive class, active learning active learning 2 8/13/2015 Please interrupt!! (With questions and comments) - not with ringing cell phones Some interactive “learning checks” to keep you engaged will be presented! 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: 1-106 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 3 8/13/2015 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 MSn) • Thermodynamic parameters – ionization energy (IE) – appearance energy (AE) – heats of formation (∆Hf) – activation enthalpy (∆H#), activation entropy (∆S#). Sample Preparation Rough, Turbomolecular, and Cryo pumps Sample Introduction Direct probe/infusion GC Vacuum System HPLC Ionization Source Mass Analyzer Detector Computer Electron impact (EI) Electrostatic (ESA) Electron Multiplier Chemical ionization (CI) Magnet (B) Photomultiplier Atmospheric pressure (API) Time-of-flight (TOF) Faraday cap Electrospray (ESI) Quadrupole (Q) Array Detectors Matrix assisted laser Ion Traps (2D & 3D IT) Multichanel plate (MCP) Desorption/ionization (MALDI) Ion-Cyclotron Surface enhanced LDI (SELDI) Resonance (ICR) Fast atom bombardment (FAB) Orbitrap (OT) 4 8/13/2015 Difference between single stage MS and tandem MS/MS Ionization Analysis MS: Collide with target to produce fragments MS/MS: Ionization Selection Activation Analysis What is Tandem Mass Spectrometry? (MS/MS) Activate MS1 Gas MS2 Surface Photons Electrons Heat 5 8/13/2015 Why Sample Preparation ? Analytes of interest found in variety of matrices (e.g., biofluids, urine, blood. tissues) Extraction/purification/cleanup Analysis What is Mass Spec good for? Detect and identify the use of steroids in athletes Monitor the breath of patients by anesthesiologists during surgery Determine the composition of molecular species found in space Determine whether honey is adulterated with corn syrup Locate oil deposits by measuring petroleum precursors in rock Monitor fermentation processes for the biotechnology industry Detect dioxins in contaminated fish Determine gene damage from environmental causes Establish the elemental composition of semiconductor materials Identify structures of biomolecules, such as carbohydrates, nucleic acids and steroids Sequence biopolymers such as proteins and oligosaccharides Determine how drugs are used by the body Perform forensic analyses such as conformation and quantitation of drugs of abuse Analyze for environmental pollutants Determine the age and origins of specimens in geochemistry and archaeology Identify and quantitate compounds of complex organic mixtures Perform ultra sensitive multielement inorganic analyses http://www.asms.org/whatisms/p1.html 6 8/13/2015 Mass Spec in Space Science and Astrobiology The Cassini-Huygens mission is a great success! “Titan is an Organic Paradise” Why? Origin of Color – Origin of Life???? Pre-biotic Earth: pre-biotic chemistry? The Role of Tholin? Ultrahigh vacuum (UHV) plasma generator to synthesize “tholins” (CxHyNz) in oxygen free environment VAC 95% N2:5% CH4 with adjustable Vacuum pump flow rate Glass tube to collect tholins at 195 K 7 8/13/2015 FT-ICR of Complex Organic Mixtures (UHVT_001) Intens. UHVT_001_ESI_pos_000001.d: +MS x108 181.09448 195.11009 2.5 2.0 1.5 Positive Ions: ESI 156.09921 207.11004 235.11570 114.07742 141.08831 168.09922 1.0 222.12071 249.15641 126.07743 0.5 259.11514 277.13673 99.06649 288.14130 301.13655 312.14132 341.30364 0.0 x108 UHVT_001_LDI_pos_0_H24_000002.d: +MS 169.09449 2.0 127.09793 183.11014 141.08834 207.11015 155.10399 1.5 195.11020 Positive Ions: LDI 222.12083 234.12064 1.0 248.13624 114.07766 260.13632 0.5 273.13169 287.14753 300.14296 96.88399 327.15441 341.17031 0.0 100 150 200 250 300 350 m/z Intens. UHVT_001_ESI_neg_000001.d: -MS x109 265.14795 1.50 185.05814 1.25 283.26429 293.17921 1.00 Negative Ions: ESI 224.06911 0.75 239.08001 251.08001 198.05341 0.50 160.03772 306.09696 209.03302 141.02065 0.25 108.03146 321.21053 119.03627 347.12280 0.00 x109 UHVT_001_LDI_neg_0_I24_000003.d: -MS 209.03285 4 3 Negative Ions: LDI 192.03152 2 224.06904 1 141.02059 233.03311 251.05489 328.10639 165.02070 264.07533275.05486 306.09696 318.09726 345.10840 0 100 150 200 250 300 350 m/z Not the same protonated/deprotonated neutral species in +/- modes 8 8/13/2015 Modified van Krevelen diagram for LDI ions 9 8/13/2015 Example of ultrahigh resolution in an FTICR J. Throck Watson “Introduction to Mass Spectrometry” p. 103 10 8/13/2015 + C2H5 N2/CH4 (5%) +. -7 N2 2s,5x10 torr 15s EI ionization with 40eV HCNH+ +. CH2=CH2 + HN2 Mass Spectrometry Imaging 11 8/13/2015 Selection of [M+2H]+2 at m/z 246.1 600 500 400 m/z 300 200 100 100 80 GRGDS SDGRG 60 40 20 0 Relative Intenisty 0 1 2 3 4 5 6 7 Drift Time (ms) 12 8/13/2015 Transfer CID of [M+2H]+2 at m/z 246.1 3.0 2.5 Drift Time (ms) Time Drift 2.0 y 100 4 y3 50 S SDGRG i R dt = 2.68-2.83 ms 0 b 100 4 50 y1 GRGDS Si Relative Abundance b R 2 b3 dt = 2.49-2.68 ms 0 100 200 300 400 500 m/z Fragmentation of Big Protein Complexes 13 8/13/2015 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. 14 8/13/2015 15 8/13/2015 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 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? 16 8/13/2015 • Filament: tungsten or rhenium • Current: 3-5 A •Ufil: a few Vs (1-3 V) 0 •Tfil: ca. 2000-2300 K (ca. 150 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 -6 –tres = 1.4x10 s 17 8/13/2015 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 - +. + +. –CH4 + e → CH4 , CH3 , CH2 , … + + –CH4 + CH4 → CH5 + CH3 + + –CH3 + CH4 → C2H5 + H2 + + –CH5 + M → [M+H] + CH4 + + –C2H5 + M → [M-H] + C2H6 + + –C2H5 + M → [M+ C2H5] 18 8/13/2015 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 - ΔHr = PA 19 8/13/2015 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”) 20 8/13/2015 Sources still being developed DESI- desorption ESI (sample not in solution) DART 21 8/13/2015 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 22 8/13/2015 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 23 8/13/2015 Matrix-Assisted Laser Desorption\ Ionization Pulsed Laser Extraction Grid to TOF Desorbed plume of matrix and analyte ions Analyte Matrix Cation (H+, Na+ etc) Sample Plate MATRIX - A small acidic organic molecule (matrix) is mixed with a low concentration of analyte in a common solvent and allowed to co-crystallize on a sample plate to form a “solid solution” by which the analyte molecules are isolated from each other. ASSISTED –matrix “assists” the desorption and ionization of the analyte(s) LASER – Typically a Nitrogen laser (351 nm) or Yag/Nd laser (334 nm) DESORPTION – Energy from the laser desorbs the matrix into the gas-phase and “carries” the analyte with it. IONIZATON - Detect [M+H]+ by transferring a proton from the matrix to the analyte Choose a matrix based on the molecular weight, solubility and chemical structure of the analyte.