Matrix Assisted Laser Desorption Ionization (Mass spectrometry)
Mass Spectrometry Workshop Ionization MethodsMass Spectrometry Workshop Ionization Methods Abraham...
Transcript of Mass Spectrometry Workshop Ionization MethodsMass Spectrometry Workshop Ionization Methods Abraham...
7/9/2018
1
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
7/9/2018
2
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
7/9/2018
3
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
7/9/2018
4
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
7/9/2018
5
7/9/2018
6
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
7/9/2018
7
(g) (g)
70 eV
M(s)
heat
→ hard ionization
7/9/2018
8
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
7/9/2018
9
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+
7/9/2018
10
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
7/9/2018
11
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?
7/9/2018
12
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
7/9/2018
13
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
7/9/2018
14
Sample and matrix are spotted on MALDI plate
7/9/2018
15
MALDI instrumentMALDI is a vacuum-based ionization method
Often coupled to Time-of-Flight Instrument
MALDI Matrix Materials
7/9/2018
16
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
7/9/2018
17
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”
7/9/2018
18
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)
7/9/2018
19
MALDI of peptide mixture (Also works for these “lower” MW)
7/9/2018
20
MALDI works well for polymers
http://www.psrc.usm.edu/mauritz/maldi.html
7/9/2018
21
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
7/9/2018
22
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)
7/9/2018
23
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
7/9/2018
24
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
7/9/2018
25
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
7/9/2018
26
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
7/9/2018
27
Multiple Charging in Electrospray Ionization
Siuzdak “Mass Spectrometry in Biotechnology”
Interpretation of ESI-Mass Spectra
m
z
MW nH
n
7/9/2018
28
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
7/9/2018
29
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
7/9/2018
30
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
7/9/2018
31
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)
7/9/2018
32
DART
DARTDirect Analysis in Real Time
Penning Ionization M* + S S+. + M + electron (but also allows MH+, M-H-, etc)