Mass Spectrometry Workshop Árpád Somogyi Associate Director CCIC, Mass Spectrometry and Proteomics Laboratory OSU August 17, 2015 1
Workshop schedule Time Monday, 8/17 Tuesday, 8/18 Introduction to Mass Spectrometry 9-10:20am Ionization methods (Arpad) Mass Analyzers (Arpad) T-ICR instrumentation and techniques (Mike reitas) 10:35-12:00pm GC-MS analysis, examples and EI spectra interpretation (Jeremy) Ion Activation (Vicki Wysocki) 12-1pm Lunch Break Lunch Break 1-2:30pm Lab visit (Group 1) Lab visit (Group 2) 2:40-4pm HPLC-MS/MS and metabolomics, Data processing programs (Yu Cao) Small molecule (ESI) spectrum interpretation (Arpad) Open Discussion/Users Presentations Lecture only: boring, limited active learning Goal: attentive class, active learning 2
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: 1-10 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 3
What can we provide by mass spec? MW determination nominal accurate (elemental composition) isotope pattern high resolution ragmentation fragmentation rules libraries ( fitting ) MS/MS (or MS n ) Thermodynamic parameters ionization energy (IE) appearance energy (AE) heats of formation ( H f ) activation enthalpy ( H # ), activation entropy ( S # ). 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) ast atom bombardment (AB) Electrostatic (ESA) Magnet (B) Time-of-flight (TO) Quadrupole (Q) Ion Traps (2D & 3D IT) Ion-Cyclotron Resonance (ICR) Orbitrap (OT) Electron Multiplier Photomultiplier araday cap Array Detectors Multichanel plate (MCP) 4
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 Surface Photons Electrons Heat MS2 5
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
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 V AC 95% N 2 :5% CH 4 with adjustable flow rate Vacuum pump Glass tube to collect tholins at 195 K 7
Intens. 2.5 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 Intens. 1.50 1.25 1.00 0.75 0.50 0.25 0.00 4 3 2 1 0 99.06649 96.88399 114.07742 114.07766 126.07743 141.08831 127.09793 141.08834 156.09921 155.10399 168.09922 169.09449 181.09448 195.11009 183.11014 195.11020 207.11004 207.11015 222.12071 222.12083 235.11570 234.12064 249.15641 248.13624 300.14296 UHVT_001_ESI_pos_000001.d: +MS UHVT_001_LDI_pos_0_H24_000002.d: +MS 100 150 200 250 300 350 m/z UHVT_001_ESI_neg_000001.d: -MS 265.14795 108.03146 119.03627 141.02065 141.02059 160.03772 165.02070 185.05814 192.03152 198.05341 209.03302 209.03285 224.06911 224.06904 283.26429 293.17921 306.09696 321.21053 347.12280 UHVT_001_LDI_neg_0_I24_000003.d: -MS 100 150 200 250 300 350 m/z 345.10840 8/13/2015 T-ICR of Complex Organic Mixtures (UHVT_001) x10 8 Positive Ions: ESI 259.11514 277.13673 288.14130 301.13655 312.14132 341.30364 x10 8 Positive Ions: LDI 260.13632 273.13169 287.14753 327.15441 341.17031 x10 9 Negative Ions: ESI 239.08001 251.08001 x10 9 Negative Ions: LDI 233.03311 251.05489 264.07533 275.05486 306.09696 318.09726 328.10639 Not the same protonated/deprotonated neutral species in +/- modes 8
Modified van Krevelen diagram for LDI ions 9
Example of ultrahigh resolution in an TICR J. Throck Watson Introduction to Mass Spectrometry p. 103 10
N 2 +. N 2 /CH 4 (5%) 2s,5x10-7 torr 15s EI ionization with 40eV C 2 H 5 + HCNH + CH 2 =CH 2 +. HN 2 + Mass Spectrometry Imaging 11
Selection of [M+2H] +2 at m/z 246.1 600 500 m/z 400 300 200 100 Relative Intenisty 100 80 60 40 20 0 GRGDS SDGRG 0 1 2 3 4 Drift Time (ms) 5 6 7 12
Transfer CID of [M+2H] +2 at m/z 246.1 Drift Time (ms) 3.0 2.5 2.0 100 y 3 y 4 Relative Abundance 50 0 100 50 0 S i S i y 1 R R b 2 b 3 b 4 SDGRG dt = 2.68-2.83 ms GRGDS dt = 2.49-2.68 ms 100 200 300 m/z 400 500 ragmentation of Big Protein Complexes 13
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 (AB) 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 orensic Environmental Drug Metabolism, etc. 14
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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
ilament: 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 17
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 ] + 18
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 19
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
Sources still being developed DESI- desorption ESI (sample not in solution) DART 21
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 enn 22
Yes, of course!!! Nobel Price in Chemistry, 2002 John B. enn Koichi Tanaka Electrospray Ionization ESI Soft Laser Desorption SLD Matrix Assited Laser Desorption/Ionization MALDI 23
Matrix-Assisted Laser Desorption\ Ionization Pulsed Laser Extraction Grid to TO Desorbed plume of matrix and analyte ions Analyte Sample Plate Matrix Cation (H +, Na + etc) 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. Excellent for intact molecular weight determination for both polar and non-polar molecules with mass > 500 amu. MALDI Matrices Matrix Abbrev Sample Type 2,5-dihydroxybenzoic acid DHB Peptides < 5,000 polymers, dedrimers Good universal matrix cold matrix 3,5-dimethoxy-4-hydroxycinnamic acid (Sinapinic acid) SA Peptides and Proteins > 10,000 hot matrix -cyano-4-hydroxycinnamic acid HCCA Excellent for peptides, digestion products and proteins 50% ACN in 0.1% TA, TH, 2:1 chloroform:meoh 50-70% ACN in 0.1% TA 50% ACN in 0.1% TA Dithranol Non-polar polymers TH, Methylene chloride Indoleacrylic acid IAA Non-polar polymers TH, methylene chloride 3-hydroxypicolinic acid HPA DNA and negative ion samples Trihydroxyacetophenone THAP DNA and negative ion samples See me for more specific procedure See me for more specific procedure Nor-harmane Universal 50% ACN, TH, chloroform 24
The MALDI Plate with Different Samples in Different Matrices 25
2 GHz time digital converter 1000 Hz laser (acquisition of 1000 spectra in 1 second MALDI-TO 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 26
a.i. Exercise 2 This is a MALDI-TO 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-TO 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 27
Molecular weight distribution defines polydispersity of polymers Zhu, H.; Yalcin, T.; Li, L. JASMS, 1998, 9, 275-281. Polystyrene with Ag + PD 1.01 Polydispersity (PD) = M w /M n Intens. [a.u.] x10 5 2.0 1.5 1.0 829.987 991.986 1123.212 1143.205 1313.231 1433.246 1553.257 1723.288 * NB_89_M4\0_M16\1\1SRef, "Baseline subt." Trancated Vectra Polymer (Somogyi et. al., unpublished) PD > 1.05 0.5 2013.333 2303.405 2593.467 2933.604 0.0 1000 1500 2000 2500 3000 3500 4000 4500 m/z MS is an absolute method for MW determination (but remember possible degradation!) Zhu, H.; Yalcin, T.; Li, L. JASMS, 1998, 9, 275-281. 28
Problems with polymer analysis Sample preparation Several polymers are not well soluble in conventional solvents Solventless technique Cationizing with Na +, K +, and Ag + by spiking Synthesis of tailor (home) made matrices Degradation during ionization (especially with MALDI) Photodissociation in MALDI (MALDI/ESI comparison desirable) End group analysis reliable but better to have high resolution/accurate mass (T-ICR) Some Examples for Tailor-Made Matrices HO N N M-1 COOH HO M-2 COOCH 3 N M-3 N HO N N M-4 OH HO M-5 OH M-6 M-7 COOCH 3 HO N N M-8 COOCH 3 H 3 CO N N M-9 OCH 3 Somogyi, et al., Macromolecules, 2007, 40, 5311-5321. 29
Repeating unit masses of polymers http://www.polymerprocessing.com/polymers/alpha.html Polystyrene (PS) C 8 H 8 (192.042259) Poly(ethylene terephtalate) (PET) C 10 H 8 O 4 (192.042259) Poly(methyl methacrylate) (PET) C 5 H 8 O 2 (100.052430) Poly(vinyl alcohol) (PVA) C 2 H 4 O (44.026215) - CH2-CH2-O- Poly(ethylene glycol) (PEG) Synthetic Polymer Analysis by MS (MALDI-TO) Intens. [a.u.] Intens. [a.u.] 4000 3000 2000 1500 1000 1816.292 1772.260 1660.310 1728.231 1684.205 44 44 44 44 500 2000 0 1675 1700 1725 1750 1775 1800 1825 1850 1875 m/z 1000 0 500 1000 1500 2000 2500 3000 3500 m/z 30
CH 3 OH exact mass: 32.0262 u Electrospray Ionization (ESI) + + + + Metal Capillary 3-5 kv + + used silica + + + + + + + + + + + + + + + + + + + Columbic Evaporation + ++ + + + Explosion + + + + + + Critical Radius Charge Repulsion > Surface Tension + + + An electric field on the capillary produces a spray of fine charged droplets The presence of a drying gas (Nitrogen) and heat evaporates off the solvent leaving a distribution of multiply charged de-solvated ions. Soft ionization In-line compatible with HPLC 31
or any peak j charges away Z 1 = j(p 2 M a ) p 1 p 2 Z 1 = 1(1030.8 1) (1124.6-1030.8) Z 1 = 10.978 round up to 11 Therefore, p 1 has 11 charges (protons) Pick as p 2 Pick as p 1 Calculate intact molecular weight M r = p 1 z 1 M a z 1 M r = (1124.6 * 11) (1 * 11) M r = 12359.6 (Actual for Cytochrome C is 12360.1 32
ESI-MS of Myoglobin 100 A17 A16 A15 A14 A13 A12 A18 A11 % A19 A20 A21 A10 0 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z 100 A % m/z deconvolutes to16952 74 10000 11000 12000 13000 14000 15000 16000 17000 18000 19000 20000 mass ESI vs MALDI Choose MALDI when: Peptides or protein with a MW < 5000 Da (MALDI can detect MW > 100KDa, but not very accurately) Complex mixtures (more than 5 compounds) Very little material with higher salt/buffer concentration Choose ESI when: MW > 5000 Da (proteins) Want better mass assignment Want good MS/MS data 33
Dual ionization mode on the apex-qe Apollo II with MALDI Option 34