8/13/2015

Mass Spectrometry Workshop

Árpád Somogyi

Associate Director CCIC, 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 Activation 12:00pm (Jeremy) (Vicki Wysocki)

12-1pm Lunch Break Lunch Break

1-2:30pm Lab visit (Group 1) Lab visit (Group 2)

Small (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 – (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 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 : 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- • • 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 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 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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