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Mass Spectrometry WorkshopIonization Methods

Abraham Badu

Assistant ProfessorChemistry and Biochemistry

July 9, 2018OSU

Three major info from MS

• Molecular weight

• Structural information via fragmentation

• Isotope ratios

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Advances in Mass Spectrometry

Ion Source

Mass Analyzer

DetectorIon 

SourceMass 

AnalyzerDetector

vacuum vacuum

‐ Atmospheric Pressure Chemical Ionization (APCI)‐ Electrospray Ionization (ESI) 

→ For non‐volatile liquid samples

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

Mass Analyzer

Detector

Ambient IonizationMethods

Analyte Sampling from their Native Environment without Sample

Pre-treatment

vacuum

Some Sample TypesHuman Tissue

BanknotesWhole PlantDrug Tablet

‐ Electron Ionization (EI)‐ Chemical Ionization (CI)→ For vola le compounds  

(≈1% of all compound)

‐MALDI → For thermally 

unstable    solid samples 

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/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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EI

FAB

Fast atom bombardment MALDI

ESI

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

Developed in 1918 by Dempster

Still widely used in

Forensic EnvironmentalDrug Metabolism, etc.

Electron Impact (EI) Ionization

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

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Electron Ionization (EI) – hard ionization 

filament

Source cage

Repeller

Sample inlet Electron collector

To analyzer

(g) (g)

70 eV

Fragmentation in Electron Ionization

Excess energy is deposited into the ion, which causes fragmentation

Why 70 eV?It gives reproducible fragmentation patterns necessary for the creation of library

What electron is removed?

How much excess energy is unaccounted for?

Consider formaldehyde: H2C=O

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A Useful Theoretical Insight

Excess gained ≈ 4 eVStatistically Distributed

OH

PhenolMW 94 Da

- OH

m/z 77

- H2 O

m/z 76

Direct bond cleavage

Rearrangement

Critical Energy

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(g) (g)

70 eV

M(s)

heat

→ hard ionization

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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

– C2H5+ + M → [M-H]+ + C2H6

– C2H5+ + M → [M+ C2H5]+

Fill chamber with reagent gas

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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

(g) (g)

70 eV

M(s)

heat

(reagent)

(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

Desorption Ionization

ESI and MALDI for Biological Systems

Spray Ionization

Softworks well for

large molecules

ESI

MALDI

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Matrix-Assisted Laser Desorption\ Ionization

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 analytemolecules 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.

Cation (H+, Na+ etc)

Matrix

Analyte

Pulsed Laser

Sample Plate

Desorbed plume of matrix and analyte ions

Extraction Grid to TOF

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 instrumentMALDI is a vacuum-based ionization method

Often coupled to Time-of-Flight Instrument

MALDI Matrix Materials

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MALDI MatricesMatrix Abbrev Sample Type Condition

2,5-dihydroxybenzoic acid DHB Peptides < 5,000 polymers, dedrimers

Good universal matrix“cold matrix”

50% ACN in 0.1% TFA,THF, 2:1

chloroform:MeOH

3,5-dimethoxy-4-hydroxycinnamic acid (Sinapinic acid)

SA Peptides and Proteins > 10,000

“hot matrix”

50-70% ACN in 0.1% TFA

-cyano-4-hydroxycinnamic acid HCCA Excellent for peptides, digestion products

and proteins

50% ACN in 0.1% TFA

Dithranol Non-polar polymers THF, Methylene chloride

Indoleacrylic acid IAA Non-polar polymers THF, methylene chloride

3-hydroxypicolinic acid HPA DNA and negative ion samples

See me for more specific procedure

Trihydroxyacetophenone THAP DNA and negative ion samples

See me for more specific 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

0 25000 50000 75000 100000

2.00E+008

4.00E+008

6.00E+008

8.00E+008

1.00E+009

1.20E+009

Inte

nsity

m/z

[M+2H]2+

23445.54

[M+H]+

47155.22

[2M+H]+

94472.97

Alkaline Phosphatase

Resolution modest, no sequence info

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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0

1000

2000

3000

4000

Inte

ns.

[a.u

.]

500 1000 1500 2000 2500 3000 3500m/z

0

500

1000

1500

2000

Inte

ns.

[a.u

.]

1675 1700 1725 1750 1775 1800 1825 1850 1875m/z

Synthetic Polymer Analysis by MS (MALDI-TOF)

1684.205

1728.2311772.260

1816.2921660.310

4444

4444

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

Poly(ethylene terephtalate) (PET)C10H8O4 (192.042259)

Polystyrene (PS)C8H8(104.062600)

Poly(methyl methacrylate) (PET)C5H8O2 (100.052430)

Poly(vinyl alcohol) (PVA)

C2H4O (44.026215)

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

Repeating unit masses of polymers

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Molecular weight distribution defines polydispersity of polymers

Polydispersity (PD) = Mw/Mn

Polystyrenewith Ag+

Zhu, H.; Yalcin, T.; Li, L. JASMS, 1998, 9, 275-281.

829.987

991.986

1143.205

1313.231

1433.246

1553.257

1723.288

1123.212

2013.333

2303.4052593.467

2933.604

* NB_89_M4\0_M16\1\1SRef, "Baseline subt."

0.0

0.5

1.0

1.5

2.0

5x10

Inte

ns.

[a.u

.]

1000 1500 2000 2500 3000 3500 4000 4500m/z

Truncated Vectra Polymer (Somogyi et. al., Macromolecules,2005, 38, 4067-4071

PD > 1.05

PD≈1.01

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

NN

FF

F F

HOCOOH

M-1

NN

FF

F F

FF

FF

F FM-3

FF

F F

HOCOOCH3

M-2

NN

FF

F F

HOOH

FF

F FM-4

FF

F F

HOOH

FF

F F

M-5

FF

F F

FF

FF

F F

M-6

FF

F F

FCOOCH3

M-7

NN

FF

F F

H3COOCH3

FF

F FM-9

NN

FF

F F

HOCOOCH3

M-8

Somogyi, et al., Macromolecules, 2007, 40, 5311-5321.

Desorption Ionization

ESI and MALDI for Biological Systems

Spray Ionization

Softworks for large

molecules

ESI

MALDI

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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 voltagePneu. assit.

Electrosonic spray (ESSI) Atm =1 μL/min Pneumatic assistance

Thermospray 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

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

Solvent and Voltage Effects

Electrospray Ionization (ESI)

++ + + ++

+++

+ + + +++

+++

+

Evaporation ++++

+++++

+

+

+

Columbic Explosion

Critical RadiusCharge Repulsion > Surface Tension

++

++ +++

+

+++

Fused silica

Metal Capillary3-5 kV

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ESI-MS Example: Hen Egg Lysozyme

m/z

Re

lativ

e

Ab

un

danc

e

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

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Multiple Charging in Electrospray Ionization

Siuzdak “Mass Spectrometry in Biotechnology”

Interpretation of ESI-Mass Spectra

m

z

MW nH

n

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Interpretation of ESI-Mass Spectra

m

z

MW nH

n

m

z

MW nH

n

m

z

MW n H

n

1

1

ESI-MS Example: Hen Egg Lysozyme

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

m/z

Re

lativ

e

Ab

un

danc

e

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Interpretation of ESI-Mass Spectra (cont’d)

1431 6 1301 41

1

1300. 4130.2

10

1431 6

1431 6 10 10 1 00814,316 10.08

14,305 9 14,305 1438

0.005%)

. .

. :

. .

. : .

(

MW n Hn

MW n Hn

n

UseMW nH

nto solve for MW

x MW xMW

MW Da theoretica l Da

difference of

n (1431.6) – nH = (n + 1) 1301.4 – (n + 1) H

n (1431.6 - 1301.4) = 1301.4 – H

(1301.4 - H )(1431.6 – 1301.4)

+ +

+

+

ESI-MS of Myoglobin

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z0

100

%

A15A16

A17

A18

A19

A20

A21

A14A13

A12

A11

A10

10000 11000 12000 13000 14000 15000 16000 17000 18000 19000 20000mass74

100

%

A

m/z deconvolutesto16952

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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

Apollo™ II with a dual ESI/MALDI configuration

MALDIplate

UV laser

Ion funnels

Sources still being developedDESI- desorption ESI

(sample not in solution)

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DART

DARTDirect Analysis in Real Time

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