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Home , Fast atom bombardment

7/9/2018

Mass Spectrometry Workshop Methods

Abraham Badu

Assistant Professor Chemistry and Biochemistry

July 9, 2018 OSU

Three major info from MS

• Molecular weight

• Structural information via fragmentation

• Isotope ratios

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Advances in Spectrometry

vacuum vacuum Mass Ion Mass Detector Detector Source Analyzer Source Analyzer

‐ (EI) ‐ Atmospheric Pressure (APCI) ‐ Chemical Ionization (CI) ‐ (ESI) → For volale compounds → For non‐volatile liquid samples (≈1% of all compound)

‐ MALDI Ambient vacuum → For thermally Mass Detector unstable Ionization Analyte Sampling from their Native Analyzer solid samples Environment without Sample Methods Pre-treatment Some Sample Types Human Tissue Banknotes Whole Plant Drug Tablet

Cooks et. al. Science 2004, 306, 471-473; Science 2006, 311, 1566 Badu-Tawiah et. al. Annu. Rev. Phys. Chem. 2013, 64, 481–505

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 bombardment (FAB) • Electrospray/nanospray ionization (nESI) • Matrix assisted laser desorption/ionization (MALDI) • Desorption electrospray ionization (DESI) • Direct analysis in real time (DART) • Native spray

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Ion Sources: volatiles; non-volatile liquids; non-volatile solids

EI ESI

FAB MALDI Fast atom bombardment

Electron Impact (EI) Ionization

Developed in 1918 by Dempster

Still widely used in

Forensic Environmental Drug Metabolism, etc.

EI is a gas‐phase process, and requires the analyte to be volatile

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Electron Ionization (EI) – hard ionization

(g) (g)

70 eV

Sample inlet Electron collector

Repeller To analyzer

Source cage filament

Fragmentation in Electron Ionization

Consider formaldehyde: H2C=O

Excess energy is deposited into the ion, which causes fragmentation Why 70 eV? It gives reproducible fragmentation What electron is removed? patterns necessary for the creation of library How much excess energy is unaccounted for?

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A Useful Theoretical Insight

Excess gained ≈ 4 eV Statistically Distributed

OH m/z 77 Direct bond cleavage H - O - H 2 O m/z 76 Rearrangement Phenol MW 94 Da

Critical Energy

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→ hard ionization

(g) (g)

70 eV heat

M(s)

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Chemical Ionization – Soft Ionization

• Ion-molecule reaction(s) between a reagent gas and a gas- phase analyte at a relatively high pressure

• Most common reagent gases – methane, isobutane, ammonia

• Mechanisms - +. + +. –CH4 + e (70 eV) → CH4 , CH3 , CH2 , … + + –CH4 + CH4 → CH5 + CH3 + + –CH3 + CH4 → C2H5 + H2

+ + –CH5 + M → [M+H] + CH4 + + Fill chamber with reagent gas –C2H5 + M → [M-H] + C2H6 + + –C2H5 + M → [M+ C2H5]

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Types of Reactions Used for CI

Protonation is one type of ionization

M + AH+ → MH+ + A

+ + CH3CH2NH2 + NH4 → CH3CH2NH3 + NH3

Reaction proceeds only if it is

Thermoneutral or exothermic (i.e., ∆Hr ≤ 0) ΔHr = ∆PA = PA (reagent) – PA (analyte)

For negative ∆PA, PA (analyte) > PA (reagent) i.e., energy will be released! --

Proton affinity (PA): M + H+ → MH+

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Proton affinity (PA):

PAs of common CI reagents (kcal/mol) methane (131) < water (173) < methanol (185)

< CH2=C(CH3)2 (197) < ammonia (205)

→ energy released is controllable via proper selection of reagents

→ energy released (≈∆PA) is absorbed by analyte

→ extend of fragmentation is also controllable

CIMS: Two different reagent gases

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Major Limitation of EI and CI

Requires volatility, good for thermally stable compound (<1% of all compounds)

Solution: develop desorption and spray ionization

(reagent) (g) (g)

70 eV heat

M(s) (not applicable to large molecules)

Can we teach elephants to fly?

John Fenn

How important will this be?

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How important? Nobel Price in Chemistry, 2002

John B. Fenn Koichi Tanaka

ESI and MALDI for Biological Systems

ESI

Spray Ionization

Soft works well for large molecules MALDI

Desorption Ionization

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Matrix-Assisted Laser Desorption\ Ionization Pulsed Laser

Extraction Grid to TOF

Desorbed plume of matrix and analyte 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.

Hypothesis/speculations about Ionization

• Disintegration of solid: – Statistical charging of clusters in a net neutral plume – Total charge due to • Charge separation of “salts” • Photoionization of matrix • Chemical Ionization – Charged clusters generated in selvedge evaporate and undergo • Ion-neutral reactions, • Ion-ion reactions • Ion-electron reactions

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Sample and matrix are spotted on MALDI plate

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MALDI instrument MALDI is a vacuum-based ionization method Often coupled to Time-of-Flight Instrument

MALDI Matrix Materials

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MALDI Matrices

Matrix Abbrev Sample Type Condition 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

MALDI Matrices: common features?

Alpha-cyano-4-hydroxy cinnamic acid

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MALDI Matrices: aromatic to absorb UV

Alpha-cyano-4-hydroxy cinnamic acid

Protein Analysis by MALDI-TOF MS

Why are singly charged ions predominantly formed in MALDI? “Lucky survivor model”

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High m/z can be observed with MALDI - TOF Resolution modest, no sequence info 1.20E+009

Alkaline Phosphatase 1.00E+009 + [M+H] 47155.22 8.00E+008 2+ [M+2H]

23445.54 + 6.00E+008 [2M+H] 94472.97 Intensity

4.00E+008

2.00E+008

0 25000 50000 75000 100000 m/z

MALDI Spectrum from Nicotinic Acid Matrix of Monoclonal Antibody (IgG)

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MALDI of peptide mixture (Also works for these “lower” MW)

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MALDI works well for polymers

http://www.psrc.usm.edu/mauritz/maldi.html

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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

Repeating unit masses of polymers

http://www.polymerprocessing.com/polymers/alpha.html

Polystyrene (PS) Poly(ethylene terephtalate) (PET)

C8H8(104.062600) C10H8O4 (192.042259)

Poly(methyl methacrylate) (PET)

C5H8O2 (100.052430)

Poly(vinyl alcohol) (PVA)

C2H4O (44.026215)

- CH2-CH2-O- Poly(ethylene glycol) (PEG)

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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 Truncated Vectra Polymer 2.0 1123.212 (Somogyi et. al., Macromolecules,

1143.205 1.5 2005, 38, 4067-4071 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

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)

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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.

ESI and MALDI for Biological Systems

ESI

Spray Ionization

Soft works for large molecules MALDI

Desorption Ionization

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Spray Ionization Techniques

Technique Pressure Flow Rate Comment Electrospray (ESI) Atm =1 μL/min No assistance Ion spray Atm >1 μL/min Pneumatic assistance Nano spray (nESI) Atm nL/min No assistance Sonic spray (SSI) Atm <1 μL/min No voltage Pneu. assit. Electrosonic spray (ESSI) Atm =1 μL/min Pneumatic assistance 1-10 Torr

Electrohydrodynamic Ionization (EHI) <10-3 Torr

Electrospray Ionization

• Formation of spray • Desolvation and breakup of droplets • Emergence of ions from charged droplets

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Generation of Stable Spray

Solvent and Voltage Effects

low voltage ~1-2 kV onset voltage

solvent CH3OH CH3CN (CH3)2SO H2O γ (N/m) 0.0226 0.030 0.043 0.073

Eon (Volt) 2200 2500 3000 4000 *calculated for a spray tip of 0.1 mm radius at D = 4 cm

Electrospray Ionization (ESI)

+++ + + Metal Capillary 3-5 kV ++ Columbic + Evaporation + + +++ Explosion + + + + + + + + + + Fused silica + + + + + + + + ++ + + ++ + + Critical Radius ++ Charge Repulsion > Surface Tension +

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ESI-MS Example: Hen Egg Lysozyme

1:1 (v/v) acetonitrile : 0.1% aqueous formic acid Relative Abundance

m/z

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Multiple Charging in Electrospray Ionization

Siuzdak “ in Biotechnology”

Interpretation of ESI-Mass Spectra

m MW nH  z n

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Interpretation of ESI-Mass Spectra

m MW nH  z n

 m MW nH m MW n1 H   z n z n  1

ESI-MS Example: Hen Egg Lysozyme

1:1 (v/v) acetonitrile : 0.1% aqueous formic acid Relative Abundance

m/z

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Interpretation of ESI-Mass Spectra (cont’d)

 MW n H  MW n1 H 1431.. 6 1301 4  n n  1 n (1431.6) – nH++ = (n + 1) 1301.4 – (n + 1) H n (1431.6 - 1301.4) = 1301.4 – H +

(1301.4 - H+ ) 1300. 4 n  10 (1431.6 – 1301.4) 130.2

MW n H  Use 1431.: 6  to solve for MW n 1431.. 6xMWx 10 10 1 008 MW 14,316 10.08

MW14,305.:. 9 Da theoretica l 14,305 1438 Da (difference of 0.005%)

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

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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 Solarix XR

UV laser

MALDI plate

Ion funnels

Apollo™ II with a dual ESI/MALDI configuration

Sources still being developed DESI- desorption ESI (sample not in solution)

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DART

DART Direct Analysis in Real Time

Penning Ionization M* + S  S+. + M + electron (but also allows MH+, M-H-, etc)

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