7/10/2016

1

Ionization Methods

Árpád Somogyi

Associate DirectorCCIC, Mass Spectrometry and Proteomics Laboratory

OSUAugust 17, 2015

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

7/10/2016

2

Still widely used in

Forensic EnvironmentalDrug Metabolism, etc.

Electron Impact (EI) Ionization

7/10/2016

3

7/10/2016

4

Direct bond cleavage

Rearrangement

RRK (simplified)

k(E) = (1 E0/E)s-1

Internal Energy Distribution P(E) and k(E) Curves

7

7/10/2016

5

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]+

7/10/2016

6

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

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”)

7/10/2016

7

Sources still being developedDESI- desorption ESI

(sample not in solution)

7/10/2016

8

DART

DARTDirect Analysis in Real Time

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

7/10/2016

9

Can we teach elephants to fly?John Fenn

Yes, of course!!!Nobel Price in Chemistry, 2002

John B. Fenn Koichi Tanaka

7/10/2016

10

Electrospray IonizationESI

Soft Laser DesorptionSLD

Matrix Assited LaserDesorption/IonizationMALDI

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

7/10/2016

11

MALDI MatricesMatrix Abbrev Sample Type

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

7/10/2016

12

The MALDI Plate with Different Samples in Different Matrices

2 GHz time digital converter1000 Hz laser (acquisition of 1000 spectra in 1 second

7/10/2016

13

10000 20000 30000 40000

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

[2M+H]+

14318.68

[M+2H]2+

7157.18

[M+H]+

14318.68

Inte

nsity

m/z

MALDI-TOF spectrum of a pure protein (linear mode)

5000 10000 15000 20000 25000 30000 35000 40000 m/z

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

a.i.

/export/home/tof/data/mslab/091705/pro_6/1Lin/pdata/1 unknown Thu Oct 6 14:27:19 2005

Exercise 2This is a MALDI-TOF spectrum of two proteins. Identify the proteins by

molecular weights and assign the labeled ions

7/10/2016

14

5392.3

9757.4

6267.7

9080.3

4373.8

4880.1

3133.7

7288.2

3645.17884.4

3941.6

4620.1

8380.3 10314.3

9508.3

8833.8

0

2000

4000

6000

8000

Inte

ns.

[a.u

.]

3000 4000 5000 6000 7000 8000 9000 10000 11000m/z

MALDI-TOF spectrum (protein profile) of a bacteria

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

Trancated Vectra Polymer (Somogyi et. al., unpublished)

PD > 1.05

PD≈1.01

7/10/2016

15

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

MS is an absolute method for MW determination(but remember possible degradation!)

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)

7/10/2016

16

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.

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

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

Polystyrene (PS)C8H8(192.042259)

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

7/10/2016

17

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

CH3OH exact mass: 32.0262 u

7/10/2016

18

Electrospray Ionization (ESI)

++ + + ++

+++

+ + + +++

+++

+

Evaporation ++++

+++++

+

+

+

Columbic Explosion

Critical RadiusCharge Repulsion > Surface Tension

++

++ +++

+

+++

Fused silica

Metal Capillary3-5 kV

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

7/10/2016

19

Z1 = j(p2 – Ma)p1 – p2

Z1 = 1(1030.8 – 1)(1124.6 - 1030.8)

Z1 = 10.978 round up to 11Therefore, p1 has 11 charges (protons)

Pick as p1

Pick as p2

Calculate intact molecular weight Mr = p1 z1 – Maz1Mr = (1124.6 * 11) – (1 * 11)Mr = 12359.6 (Actual for Cytochrome C is 12360.1

For any peak j charges away

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

7/10/2016

20

E. Nikolaev et al., Initial Characterization of a New Ultrahigh ResolutionFTICR Cell with Dynamic Harmonization, JASMS, 2011, 22, 1125-1133.

E. Nikolaev and coworkers, Twelve Million Resolving Power on 4.7T FT-ICRInstrument with Dynamically Harmonized Cell – Observation of Fine StructureIn Peptide Mass Spectra, JASMS, 2014, 25, 790-799.

Isotope distribution/charge state by ultrahigh resolution MS

Native (nano)spray

- mimic biological environments- prevent unfolding- keep protein complexes intact- narrower charge distributions than with ESI

7/10/2016

21

(a) Denatured urease was electrosprayed from an aqueous 50% (vol/vol) acetonitrile containing 0.1 % (vol/vol) formic acid solution revealing individual charge distributions from the multiply charged (26.6 kDa, orange) and (61.7 kDa, magenta) monomers of urease. (b) A mass spectrum of native urease electrosprayed from an aqueous ammonium acetate solution (bottom) displaying multiple ion signals that originate from multiple charged species of the α 12 β 12 intact urease machinery with a measured mass of 1,063.4 1.0 kDa. Insets are close-ups of the indicated regions. The cartoons are adapted from the X-ray structure of the intact α 12 β 12 urease.

Pinkse, M.W., Maier, C.S., Kim, J.I., Oh, B.H. & Heck, A.J. Macromolecular assembly of Helicobacter pylori urease investigated by mass spectrometry. J. Mass Spectrom. 38, 315–320 (2003). | Article | PubMed | ChemPort |

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

7/10/2016

22

Dual ionization mode on the apex-Qe

Apollo™ II with MALDI Option