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7/10/2016
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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
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Still widely used in
Forensic EnvironmentalDrug Metabolism, etc.
Electron Impact (EI) Ionization
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Direct bond cleavage
Rearrangement
RRK (simplified)
k(E) = (1 E0/E)s-1
Internal Energy Distribution P(E) and k(E) Curves
7
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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
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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DART
DARTDirect Analysis in Real Time
Penning Ionization M* + S S+. + M + electron (but also allows MH+, M-H-, etc)
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Can we teach elephants to fly?John Fenn
Yes, of course!!!Nobel Price in Chemistry, 2002
John B. Fenn Koichi Tanaka
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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
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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
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The MALDI Plate with Different Samples in Different Matrices
2 GHz time digital converter1000 Hz laser (acquisition of 1000 spectra in 1 second
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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
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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
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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)
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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.
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
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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
CH3OH exact mass: 32.0262 u
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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
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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
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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
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(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
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