1 Ambient mass spectrometry
Ambient mass spectrometry is defined as mass spectrometric analysis
with no or minimal effort for sample preparation, using direct sampling and ionization at ambient
conditions.
2 Ambient mass spectrometry
Ambient mass spectrometry
3 Low-temperature plasma (LTP) probe for desorption and ionization
of samples in the ambient environment
G. R. Cooks et al. Anal. Chem. 80, 9097 (2008)
Ambient mass spectrometry
Anal. Chem. 83, 1084–1092 (2011)
Esplosivi
4 Ambient mass spectrometry
ELDI MALDESI LAESI LADESI CALDI
LA-APCI LA-FAPA
Chen, H.; Gamez, G.; Zenobi, R. J. Am. Soc. Mass Spectrom. 2009, 20, 1947
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DART:
“Direct Analysis in Real Time”
Commercial product introduced March 2005
1. Cody, R. B.; Laramee, J. A. “Method for atmospheric pressure ionization”, US Patent Number 6,949,741 issued September 27, 2005.
2. Laramee, J. A.; Cody, R. B. “Method for Atmospheric Pressure Analyte Ionization”, US Patent Number 7,112,785 issued September 26, 2006.
First open-air, ambient ion source for MS
DART:
“Direct Analysis in Real Time”
• Fast and easy way to introduce samples
• Minimal sample preparation for most samples
• Can tolerate “dirty” or high-concentration samples and without contamination
• Fast fingerprinting of materials
• Not useful for large biomolecules (no good for DNA analysis, proteins)
• DART does not ionize metals, minerals, etc.
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DART:
“Direct Analysis in Real Time”
N2, He
kV
ions, electrons, and
excited-state species in a plasma
Electronic or vibronic excited state species (metastable helium atoms or nitrogen molecules)
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Penning ionization
Sample ionized directly by energy transfer from metastables (M*) Proton transfer (positive ions) 1. He* ionizes atmospheric water 2. Ionized water clusters transfer
proton to sample
Electron capture (negative ions) 1. Penning electrons rapidly
thermalized
2. Oxygen captures electrons 3. O2- ionizes sample
DART Source
MS API Interface
M*
DART:
“Direct Analysis in Real Time”
M* + S S+• + M + electron
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Instant detection of illicit drugs on currency
Analysis of unknown pills or detection of counterfeit drugs
DART approach: seconds!
Conventional method: hours!!!
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Immediate response and detection on surfaces or in fluids
Rapid detection of explosives
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1.5 torr 1.5×10-5 torr
11 REIMS - Ion formation mechanism
The mechanism of electrosurgical tissue ablation involves 1) the Joule-heating of a conductive tissue by electric current
2) followed by the evaporation and the ionization of the water content 3) and finally the fragmentation of the tissue due to vigorous cavitation and
the explosion of the bubbles.
In the light of this scenario, the ion formation may follow two distinctively different pathways.
One mechanism involves the desorption of neutral molecules followed by gas phase ionization via proton transfer reaction with the ionized water molecules.
The presence of large amount of ionized water is considered to be essential for maintaining the ablation process, thus this mechanism certainly takes place during electrosurgical cutting.
The mechanism is highly similar to that of atmospheric pressure chemical ionization (APCI). The positive ions observed at m/z 369.3522 (assigned as [cholesterol+H]+-H2O ) are clearly products of this mechanism.
The alternative mechanism is based on the rapid thermal evaporation of the tissue material, which can be considered as an aqueous solution of molecular and ionic species.
Given that the rate of evaporation and the rate of thermal degradation are comparable, both the intact molecular ions and their primary thermal degradation products appear in the gas phase. The mechanism is similar to that of thermospray ionization in filament-off mode, where direct transfer of preformed ions from solution to gas phase was suggested as ionization mechanism.
Series of [ M-H]- ions of phosphoethanolamines (PE’s) and PE-NH3 molecules are tentatively associated with this latter mechanism.
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Mintacím szerkesztése
medimass
Glycerophospholipids
C
C C
C
H H
P X H H
H
O O
O O O
O
O R1
C O R2
Phosphatidylethanolamines (PE)
Phosphatidylserines (PS)
Phosphatidylinositols (PI)
Phosphatic acids (PA) Phosphatidylgycerols (PG)
O O R2 O R2
O O
O P O O 2
O O R2 O R2
O O
O P O O
NH3+
O O R2 O R2
O O
O P O O
NH3+ O O-
O O R2 O R2
O O
O P O
O OH
OH OH HO
OH O O R2 O R2
O O
O P O
O OH
OH
REIMS Tissue Data
REIMS_human_liver_cancer_030810_002 #2-31RT:0.05-1.24 AV:30NL:2.89E2 T:FTMS - p NSI Full ms [150.00-2000.00]
600 650 700 750 800 850 900
m/z 0
10 20 30 40 50 60 70 80 90 100
Relative Abundance
REIMS_human_liver_cancer_030810_001 #25-29RT:1.00-1.16AV:5NL:1.13E3 T:FTMS - p NSI Full ms [150.00-2000.00]
600 650 700 750 800 850 900
m/z 0
10 20 30 40 50 60 70 80 90 100
Relative Abundance
REIMS_human_liver_cancer_030810_002 #2-32RT:0.05-1.28AV:31NL:2.80E2 T:FTMS - p NSI Full ms [150.00-2000.00]
696.5 697.0 697.5 698.0 698.5 699.0 699.5 700.0 700.5
m/z 0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Relative Abundance
REIMS_human_liver_cancer_030810_001 #25-29RT:1.00-1.16AV:5NL:8.06E2 T:FTMS - p NSI Full ms [150.00-2000.00]
696.5 697.0 697.5 698.0 698.5 699.0 699.5 700.0 700.5
m/z 0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Relative Abundance
Human liver metastatic tumor, in-vivo Human healthy liver, in-vivo
Zoltan Takats
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Desorption Electrospray Ionization
(DESI)• Novel “gentle” ionization method for surface analysis
• Operates in atmosphere under ambient conditions
• Requires no sample preparation
• Effective for both organic and biological compounds
• Allows for in situ analysis of biological tissues
• Wide range of applications from clinical testing, environmental monitoring, forensics, homeland defense, process analytical testing (PAT), and surface imaging
G. Cooks et al., Science, 2004, 306, 471-473
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droplet-thin film collision simulations. incident angle: 55° ; speed: 120 m/s
α= 55°, 120 m/s, t = 0.8 μs γ= 0.5 (‐Y)
γ= 0.5 (‐Z)
22
New Ambient Ionization Method:
Paper Spray Ionization
•Method Developed for Blood Analysis of Drugs of Abuse
•NO Sample Preparation of Blood Matrix
•Sensitive Method Detecting Nanogram Levels in Blood Matrix
•Potential Applications for Micro-fluidics using Filter Paper
Liu, J.; Wang, H.; Cooks, R. G.; Ouyang, Z.
+ + +
++
++ +
+ ++ +
++ + ++ ++ + + +++ + +
High Voltage Applied to Filter or Chromatography Paper (~4.5 kV)
10 μL of Solvent Pipetted to the Backend or Over Sample
10 μL of Sample Pipetted to Center or Tip of Paper
Fine Droplets or Charged Ions Analyzed Introduced to MS Inlet
~5 mm MS
Inlet
Suitable to for Miniature MS and Portable Applications
Atenolol MW=266
m/z 267 Atenolol 10 ppm spiked in whole blood
0.4μl blood loaded for each sampling spot 10 μl methanol/H2O added for spraying
m/z 267 CID
MS spectrum of direct paper spray of blood spiked with atenolol (4ng/spot)
MS/MS spectrum to identify atenolol
Paper spray/mini MS (direct detection Atenolol in blood)
251
225 190 208 145
173
180 He Wang, Jiangjiang Liu, Guangming Huang
He Wang, Jiangjiang Liu, Guangming Huang, et al. unpublished
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Matrix-assisted laser desorption ionization
Fenner, N.C.; Daly, N.R. Laser Used for Mass Analysis. Rev. Sci. Instrum. 1966, 37, 1068-1070.
26 P
P
P r r r e e e m m m i i i o o o N N N o o o b b b e e e l l l 2 2 2 0 0 0 0 0 0 2 2 2 p p p e e e r r r l l l a a a C C C h h h i i i m m i m i i c c c a a a
La commissione per i Nobel dell' Accademia Reale delle Scienze Sved ese ha deciso di assegnare il Premio Nobel 2002 per la Chimica
Per lo sviluppo di metodi per l'identificazione e le analisi della struttura delle macromolecole biologiche
per metà congiuntamente a:
John B. Fenn , born 1917 in New York City, USA (US citize n).
Virginia Commonwealth University, Richmond, USA
ed a
Koichi Tanaka , born 1959 (43 years) in Toyama City, Japan Shimadzu Corp., Kyoto, Japan
Per il loro lavoro sullo sviluppo di metodi di ionizzazione per desorbimen t o blando per le analisi di spettro metria di massa
delle macromolecole biologiche
e per l'altra metà a
Kurt Wüthrich , born 1938 (64 years) in Aarberg, Switzerland
Eidgenössische Technische Hochschule (ETH), Swiss Federal Institute of Technology, Zürich, Switzerland The Scripps Research Ins titute, La Jolla, USA
Franz Hillenkamp
Institute of Medical Physics and Biophysics of the University of Münster
Michael Karas
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Used for (atmospheric pressure) APMALDI, absorbs O-H stretching mode of water
UV UV UV UV UV UV Mid-IR Far-IR
Matrix
-assisted laser desorption
ionization
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Matrix Wavelength Typical Applications
2,5-Dihydroxybenzoic acid (2,5-DHB) 337 / 355 nm Proteins and Oligosaccharides
Sinapinic acid (SA) 337 / 355 nm Proteins
α-cyano-4-hydroxycinnamic acid (HCCA) 337 / 355 nm Peptides and proteins
3-Hydroxycinnamic acid (3-HPA) 337 / 355 nm Oligonucleic acids
Picolinic acid ( PA) 266 nm Nucleic acids
2,4,6-Trihydroxyacetophenone (2,4,6-THAP) 337 / 355 nm Oligonucleic acids and Acidic oligosaccharides 6-Aza-2-thiothymine 266, 337, 355 nm Oligonucleic acids and
Acidic oligosaccharides 2-(4'-Hydroxybenzeneazo) benzoic acid (HABA) 337 / 355 nm Proteins & carbohydrates
2,6-Dihydroxyacetophenone (2,6-DHAP) 337 / 355 nm Oligonucleic acids
3-Aminoquinoline 337 nm Oligosaccharides
3-Hydroxy picolinic acid 337 nm Nucleic acids
Nicotinic acids 266 nm Proteins, peptides and
Adduct formation
Thiourea 266 nm Large protein
337 nm: Nitrogen laser; 355, 266 nm: Nd:YAG laser;
Funzioni della matrice
Solvente per le molecole di analita.
Le molecole di matrice assorbono l’energia della radiazione laser e la trasferiscono come energia di eccitazione al sistema solido.
Ruolo attivo nella ionizzazione delle molecole di analita.
Reazioni chimiche portano alla formazione di molecole protonate [M+H]+, a cluster molecolari del tipo [nM]+ ed a ioni del tipo [M+matrice]+ .
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Caratteristiche della matrice
• Solubilità: l’analita e la matrice devono essere solubili nello stesso solvente.
• Assorbimento: la matrice deve avere una banda di assorbimento in corrispondenza della lunghezza d’onda del laser usato, in modo che l’energia dell’impulso laser si depositi sulla matrice e non sull’analita
• Reattività: la matrice usata non deve modificare covalentemente l’analita
31
Hillenkamp, F. Adv. Mass Spectrom. 1989, 11A, 354.
32 The
Lucky Survivor model
postulates that analytesare incorporated in the matrix crystals with their respective charge states preserved from solution. At typical preparation conditions, peptides and proteins, the preferentially investigated analyte classes, are protonated and would therefore be incorporated as positively precharged analytes together with their counterions.
The
Gas Phase Protonation Model
predicts neutral analytes in the gas phase originating either fromincorporation in the matrix crystals as uncharged species or from quantitative charge recombination with the respective counterions in the case of precharged analytes.
Gas phase collisions of neutral analytes with protonated [ma + H]+ or deprotonated [ma – H]– matrix ions lead to proton transfer reactions and to protonated or deprotonated analytes, respectively
33 Both models, the refined Lucky Survivor model as well as the gas
phase protonation model, comprise the necessity of
protonated matrix ions [ma + H]
+for analyte protonation.
The surviving analyte charge originates from solution and is, therefore, initially localized on a basic
analyte functional group, which was protonated during sample preparation.
T. W. Jaskolla, M. Karas, J. Am. Soc. Mass Spectrom. 22, 976-988 (2011)
34
Magnification of the Target
35
-1.0000 -0.8000 -0.6000 -0.4000 -0.2000 0.0000 0.2000 0.4000 0.6000 0.8000 1.0000
-1.0000 -0.8000 -0.6000 -0.4000 -0.2000 0.0000 0.2000 0.4000 0.6000 0.8000 1.0000
10 shots
100 shots
1000 shots
11,000 shots
Marvin Vestal, Virgin Instruments
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Mass spectrum of Hg-Papain oligomerization
… mass spectrum of a mixture of ubiquitin, cytochrome C and equine myoglobin using 2,5-dihydroxybenzoic acid (DHB) as the matrix.
37 Atmospheric Pressure-Matrix Assisted Laser Desorption
Ionization (AP-MALDI)
38
The different types of ProteinChip Arrays. The chromatographic ProteinChip Arrays incorporate hydrophobic, cationic, anionic, metal ions or hydrophilic spots. These “chemical surfaces” are best suited for protein expression profiling studies. Another series of ProteinChip Arrays have pre-activated
“biological surfaces” designed for coupling of biomolecules with applications in antibody–antigen assays, receptor–ligand interaction studies, and DNA–protein binding experiments.
C. H. Borchers et al, J. Am. Soc. Mass Spectrom. 21, 1680–1686, 2010
39 matrix-free laser desorption/ionization MS approach
the absence of matrix interference in the low mass region of the mass spectra and thus SALDI (Sunner and Chen, 1995) permits rapid analysis of small molecules
A number of different materials (i.e. graphite, TiO2, HgTe nanotube layers, etc) that can serve as SALDI matrices
SALDI
surface-assisted
laser desorption/ionization40
SALDI
MALDI-TOF DESI
41
MATRIX DEPOSITION
Manual devices Airbrush
Automatic matrix depositors (MALDI spotter)
vibrational vaporization
piezoelectric technology
Acoustic droplet ejection
42
Manual spraying
1) droplet size is too large to make good crystal structures
2) gaps in the matrix coverage.
Automated spraying
homogenous coverage
43 Olanzepine: atypical antipsychotic
44
45
46
Angew. Chem. Int. Ed. 2010, 49, 3834 –3838
MALDI Biotyper
MALDI TOF MS fingerprinting - workflow
Unknown Microorganism
Identified Species
BioTyper
Data Interpretation Select a Colony
Smear a Thin-Layer onto a MALDI Target Plate
Generate MALDI-TOF Profile Spectrum
47 MALDI Biotyper
Standard Operation Protocol (SOP) – Cell Smear
4263.38 5164.55 6460.15 7245.48
4494.22 7513.30
6176.31
5673.61 8600.46 8989.05 9631.46
8239.00
6929.01
* Arthrob_s ulfureus _B571\0_F8\1\1SLin
0 2000 4000 6000 8000Intens. [a.u.] 4263.21 5164.04
4493.98 7253.89
6460.00
6175.92
5673.45 8989.24
8600.54 9631.12
7513.41
6928.55 8238.88
* DSM 20167T\0_G4\1\1SLin, Smoothed, "Bas eline s ubt."
0 1000 2000 3000 4000 5000 6000
Intens. [a.u.]
4000 5000 6000 7000 8000 9000
m/z
Select a Colony Direct Smear on Maldi Target Apply 1 mL of matrix
Insert Target Get Spectras
MALDI Biotyper - Basic
MALDI Biotyper is robust, as it relies on highly abundant proteins
4364.06 5380.64 6254.646315.49
5096.01 7157.65 7273.87
6410.90 7870.62 8368.99
0 1000 2000 3000 4000 5000
Intens. [a.u.]
4000 4500 5000 5500 6000 6500 7000 7500 8000
m/z ribosomal Protein m/z
RL36 4364,33
RS32 5095,82
RL34 5380,39
RL33meth. 6255,39
RL32 6315,19
RL30 6410,60
RL35 7157,74
RL29 7273,45
RL31 7871,06
RS21 8368,76
E.coli
~ 1h for 96 Samples Mass Range:
2000 – 20000 Da
48 MALDI Biotyper - Basic
Low influence of culture conditions
Psdm. oleovorans B396_Medium 360
0 1000
Psdm. oleovorans B396_Medium 464
0 1000
Psdm. oleovorans B396_Medium 53
0 1000
Psdm. oleovorans B396_Medium 65
0 1000
Psdm. oleovorans B396_Medium 98
0 500 1000
Psdm. oleovorans B396_MRS10
0 1000 2000
Psdm. oleovorans B396_YPD
0 1000 2000
4000 5000 6000 7000 8000 9000 10000 11000 m/z
Pseudomonas oleovorans grown on different media
MALDI Biotyper - Basic
Broad applicability of MALDI TOF MS profiling
Filamentous fungi, yeast, gram+ and gram- bacteria
Aspergillus fumigatus
0 1000 2000 3000
Intens.[a.u.]
Bacillus subtilis
0 2000 4000 6000 8000 In
tens.[a.u.]
Candida albicans ATCC 10231
0.0 0.2 0.4 0.6 0.8 1.0
Escherichia coli DH5alpha
0 500 1000 1500 2000 2500
3000 4000 5000 6000 7000 8000 9000 10000
m/z
49 Clinical Research Solution
The MALDI Biotyper Solution
Sample Preparation
• Inactivation
• Optimized quality
• Robust
• 5 min protocol
Data Acquisition
• Benchtop instrument
• Automated system
• Unattended Operation
BioTyper Data Analysis
• Automated data processing
• Signal identification
• Pattern matching
BioTyper Reference Library
• Ready-to-use library
• Open system, that can be expanded by the user
• Real time analysis
50
R.M. Caprioli et al, J. Mass Spectrom. 2012, 47, 1473–1481
Using a
transmission geometry
configuration, the laser beam in the ion source of a MALDI mass spectrometer can be focused to dimensions less than 1 μm, making possible the directimaging of heterogeneous tissue sections and single cells with sub-cellular resolution.
51
Analysis of lipids in a rat brain tissue sample
MS imaging by DESI
J. M. Wiseman, D.R. Ifa, Q. Song, R. G. Cooks, Angew. Chem. 45, 7188
52 DESI
53 DESI
100%
20%
Study of Latent Fingermarks by MALDI MS Imaging
Rosalind Wolstenholme, Robert Bradshaw, Malcolm R. Clench and Simona Francese, 2009, RCM
54
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Ioni secondari
Spettrometria di massa di ioni secondari (SIMS)
Ioni primari (KeV)
campione
More recently polyatomic primary ions, i.e. C60+ ions
56
Selected ion images from a 40 keV C60+ analysis of an area of rat brain tissue incorporating part of the corpus callosum.
Images (a–d) are of an 800 µm×800 µm area, the total primary ion fluence was 4.3×1011 cm-2, the maximum count in a pixel within the images are quoted. The images show the distribution of: (a) PC head group (m/z 184), (b) the collection of lipid peaks in the mass range 700–850, (c) cholesterol (m/z 369), and (d) a single phospholipid—PC 34:1 (m/z 760). Image (e) is from a 200 µm×200 µm field of view image showing the fine structure of the cholesterol (m/z 369) within this feature, using an accumulated fluence of 1.2×1011 ions/cm-2.
P Ambient, vacuum Ambient Vacuum Sp. Res. 10-50µm > 300µm 1µm
57 CI
ES I
MALDI
DESI APCI APPI
Energia: HARD SOFT
Fase: GAS CONDENSATA Ioni: +• +/n+/–/n–
EI
Press.: VUOTO P. ATM.
58
Sistema di Introduzione
DI, MIMS, GC, HPLC, CZE, CEC, ITP
Sorgente
EI, CI, PD, FD, FAB, LSIMS, ESI, APCI,
MALDI
Analizzatore
Settori (EB; BE; EBE …) Quadrupolo (Q, QqQ) Trappola ionica, FT-ICR Orbitrap, Tempo di volo (TOF)
Ibridi (BEqQ; QTOF ….)
Separazione degli ioni secondo il loro rapporto
massa/carica (m/z)