8/13/2015
Mass Spectrometry Workshop
Árpád Somogyi
Associate Director CCIC, Mass Spectrometry and Proteomics Laboratory
OSU August 17, 2015
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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
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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
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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)
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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
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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
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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
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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
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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
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Modified van Krevelen diagram for LDI ions
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Example of ultrahigh resolution in an FTICR
J. Throck Watson “Introduction to Mass Spectrometry” p. 103
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+ C2H5 N2/CH4 (5%) +. -7 N2 2s,5x10 torr 15s EI ionization with 40eV
HCNH+ +. CH2=CH2 + HN2
Mass Spectrometry Imaging
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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)
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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
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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.
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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?
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• 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
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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]
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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
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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”)
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Sources still being developed DESI- desorption ESI (sample not in solution)
DART
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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
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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
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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. 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 50% ACN in 0.1% TFA, polymers, dedrimers THF, 2:1 Good universal matrix chloroform:MeOH “cold matrix” 3,5-dimethoxy-4-hydroxycinnamic SA Peptides and Proteins 50-70% ACN in 0.1% acid (Sinapinic acid) > 10,000 TFA “hot matrix” -cyano-4-hydroxycinnamic acid HCCA Excellent for peptides, 50% ACN in 0.1% TFA digestion products and proteins Dithranol Non-polar polymers THF, Methylene chloride Indoleacrylic acid IAA Non-polar polymers THF, methylene chloride 3-hydroxypicolinic acid HPA DNA and negative ion See me for more specific samples procedure Trihydroxyacetophenone THAP DNA and negative ion See me for more specific samples procedure Nor-harmane Universal 50% ACN, THF, chloroform
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The MALDI Plate with Different Samples in Different Matrices
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2 GHz time digital converter 1000 Hz laser (acquisition of 1000 spectra in 1 second
MALDI-TOF spectrum of a pure protein (linear mode)
16000000 [M+H]+ 14000000 14318.68
12000000
2+ 10000000 [M+2H] 7157.18
8000000 Intensity 6000000
4000000 [2M+H]+ 14318.68 2000000
0 10000 20000 30000 40000 m/z
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Exercise 2 This is a MALDI-TOF spectrum of two proteins. Identify the proteins by a.i. 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
5392.3
Intens. [a.u.] Intens. MALDI-TOF spectrum (protein profile) of a bacteria 8000 9757.4
6267.7
6000 9080.3
4373.8
4000
4880.1
9508.3 8833.8 3133.7 4620.1 2000 7288.2
3645.1 7884.4 3941.6 8380.3 10314.3
0 3000 4000 5000 6000 7000 8000 9000 10000 11000 m/z
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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) = Mw/Mn
x105 829.987 * NB_89_M4\0_M16\1\1SRef, "Baseline subt."
Intens. [a.u.] 991.986 2.0 Trancated Vectra Polymer 1123.212 (Somogyi et. al., unpublished) 1143.205 1.5 1313.231 1433.246 PD > 1.05 1.0 1553.257
1723.288
2013.333 0.5 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.
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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 (FT-ICR)
Some Examples for Tailor-Made Matrices
F F F F F F F F HO N HO F N COOCH N F N COOH 3 F F F F F F F F M-2 M-3 M-1
F F F F F F F F F F F F F HO N HO F N OH OH F F F F F F F F F F F F M-6 M-4 M-5
F F F F F F F F H CO N F HO N 3 N OCH COOCH3 N COOCH 3 3 F F F F F F F F M-7 M-8 M-9
Somogyi, et al., Macromolecules, 2007, 40, 5311-5321.
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Repeating unit masses of polymers
http://www.polymerprocessing.com/polymers/alpha.html
Polystyrene (PS) Poly(ethylene terephtalate) (PET)
C8H8(192.042259) C10H8O4 (192.042259)
Poly(methyl methacrylate) (PET)
C5H8O2 (100.052430)
Poly(vinyl alcohol) (PVA)
C2H4O (44.026215)
- CH2-CH2-O- Poly(ethylene glycol) (PEG)
Synthetic Polymer Analysis by MS (MALDI-TOF) Intens. [a.u.] Intens. [a.u.] 2000 4000 1816.292 1772.260 1660.310 1500 1728.231 1684.205 44
3000 1000 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
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CH3OH exact mass: 32.0262 u
Electrospray Ionization (ESI)
+++ + + Metal Capillary 3-5 kV ++ Columbic + Evaporation + + +++ Explosion + + + + + + + + + + Fused silica + + + + + + + + ++ + + ++ + + 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
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For any peak j charges away Z1 = j(p2 –Ma) p1 –p2
Z1 = 1(1030.8 – 1) (1124.6 - 1030.8)
Z1 = 10.978 round up to 11 Therefore, p1 has 11 charges (protons)
Pick as p2
Pick as p1
Calculate intact molecular weight Mr = p1 z1 –Maz1 Mr = (1124.6 * 11) – (1 * 11) Mr = 12359.6 (Actual for Cytochrome C is 12360.1
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ESI-MS of Myoglobin
A15 100 A16 A14 A13 A17 A12
A18
A11 % A19 A10 A20 A21
0 m/z 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
A 100
m/z deconvolutes to16952 %
74 mass 10000 11000 12000 13000 14000 15000 16000 17000 18000 19000 20000
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
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Dual ionization mode on the apex-Qe
Apollo™ II with MALDI Option
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