Ambient mass spectrometry - Dipartimento di Scienze della vita

Ambient mass spectrometry
Ambient mass spectrometry is defined as mass
spectrometric analysis
with no or minimal effort for sample preparation,
preparation,
using direct sampling and ionization at ambient
conditions.
Ambient mass spectrometry
Ambient mass spectrometry
Ambient mass spectrometry
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)
Anal. Chem. 83, 1084–1092 (2011)
Esplosivi
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
DART:
“Direct Analysis in Real Time”
Commercial product introduced March 2005
First open-air, ambient ion source for MS
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.
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.
DART:
“Direct Analysis in Real Time”
ions, electrons, and
excited-state species in a plasma
kV
N2, He
Electronic or vibronic excited state species
(metastable helium atoms or nitrogen
molecules)
DART:
“Direct Analysis in Real Time”
Penning ionization
M* + S Æ S+• + M + electron
Sample ionized directly by energy
transfer from metastables (M*)
Proton transfer (positive ions)
M*
DART Source
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
MS API
Interface
Instant detection
of illicit drugs on
currency
Analysis of unknown pills or detection of counterfeit drugs
DART approach: seconds!
Conventional method: hours!!!
Immediate response and detection on surfaces or
in fluids
Rapid detection of explosives
1.5 torr
1.5×10-5 torr
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.
Matrix-assisted laser desorption
ionization
Premio Nobel 2002 per la Chimica
La commissione per i Nobel dell'Accademia Reale delle Scienze Svedese 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 citizen).
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
desorbimento blando per le analisi di spettrometria 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 Institute, La Jolla, USA
UV
UV
UV
UV
UV
UV
Mid-IR
Far-IR
Used for (atmospheric pressure) APMALDI,
absorbs O-H stretching mode of water
Matrix
-
assisted
laser
desorption
ionization
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]+ .
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
Hillenkamp, F. Adv. Mass Spectrom. 1989, 11A, 354.
Magnification of the Target
10 shots
100 shots
1000 shots
11,000 shots
Marvin Vestal, Virgin Instruments
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.
Atmospheric Pressure-Matrix Assisted Laser Desorption
Ionization (AP-MALDI)
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 preactivated “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
SALDI
surface-assisted laser desorption/ionization
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
MALDI-TOF
DESI
Olanzepine: atypical antipsychotic
Angew. Chem. Int. Ed. 2010, 49, 3834 –3838
MALDI Biotyper
MALDI TOF MS fingerprinting ‐ workflow
Select a Colony
Smear a Thin‐Layer onto
a MALDI Target Plate
Unknown
Microorganism
Identified
Species
BioTyper
Data Interpretation
Generate MALDI‐TOF
Profile Spectrum
MALDI Biotyper
Standard Operation Protocol (SOP) – Cell Smear
Direct Smear on Maldi Target
Intens. [a.u.]
8600.46
0
* DSM 20167T\0_G4\1\1 SLin, Sm oo thed, "Bas eline s ubt."
4263.21
6000
9631.46
6929.01
8239.00
7245.48
6460.15
6176.31
5673.61
4494.22
2000
8989.05
5164.55
6000
7513.30
Get Spectras
8000
4000
Apply 1 µL of matrix
* Arth ro b_s ulfureus _B571\0_F8\1\1SLin
4263.38
Intens. [a.u.]
Select a Colony
5000
4000
5000
6000
8000
9000
Insert Target
9631.12
8600.54
8989.24
7253.89
7000
m /z
MALDI Biotyper - Basic
6254.64
5380.64
5000
7157.65
7273.87
3000
6410.90
5096.01
6315.49
4000
7870.62
2000
ribosomal Protein
RL36
RS32
RL34
RL33meth.
RL32
RL30
RL35
RL29
RL31
RS21
1000
m/z
4364,33
5095,82
5380,39
6255,39
6315,19
6410,60
7157,74
7273,45
7871,06
8368,76
8368.99
4364.06
MALDI Biotyper is robust, as it relies on highly abundant proteins
Intens. [a.u.]
0
8238.88
1000
7513.41
6928.55
2000
6460.00
5673.45
3000
6175.92
4493.98
5164.04
4000
~ 1h for 96 Samples
0
4000
E.coli
4500
5000
5500
6000
6500
7000
7500
8000
Mass
Range:
m/z
2000 – 20000 Da
MALDI Biotyper - Basic
Low influence of culture conditions
Pseudomonas oleovorans grown on different media
Psdm. oleovorans B396_Medium 360
1000
0
Psdm. oleovorans B396_Medium 464
1000
0
Psdm. oleovorans B396_Medium 53
1000
0
Psdm. oleovorans B396_Medium 65
1000
0
Psdm. oleovorans B396_Medium 98
1000
500
0
Psdm. oleovorans B396_MRS10
2000
1000
0
Psdm. oleovorans B396_YPD
2000
1000
0
4000
5000
6000
7000
8000
9000
10000
11000
m/z
MALDI Biotyper - Basic
Broad applicability of MALDI TOF MS profiling
Intens. [a.u.]
Filamentous fungi, yeast, gram+ and gram‐ bacteria
Aspergillus fumigatus
3000
2000
Intens. [a.u.]
1000
0
Bacillus subtilis
8000
6000
4000
2000
0
Candida albicans ATCC 10231
1.0
0.8
0.6
0.4
0.2
0.0
Escherichia coli DH5alpha
2500
2000
1500
1000
500
0
3000
4000
5000
6000
7000
8000
9000
10000
m/z
Clinical Research Solution
The MALDI Biotyper Solution
Data Acquisition
• Benchtop instrument
• Automated system
• Unattended Operation
Sample Preparation
•
•
•
•
Inactivation
Optimized quality
Robust
5 min protocol
BioTyper Reference Library
• Ready‐to‐use library
• Open system, that can
be expanded by the user
• Real time analysis
BioTyper Data Analysis
• Automated data processing
• Signal identification
• Pattern matching
MS imaging by DESI
Analysis of lipids in a rat brain tissue sample
J. M. Wiseman, D.R. Ifa, Q. Song, R. G. Cooks, Angew. Chem. 45, 7188
DESI
DESI
P
Ambient, vacuum
Sp. Res.
10-50µm
Ambient
Vacuum
> 300µm
1µm
Spettrometria di massa di ioni secondari
(SIMS)
Ioni primari
(KeV)
Ioni secondari
campione
More recently polyatomic primary
ions, i.e. C60+ ions
P
Ambient, vacuum
Sp. Res.
10-50µm
Ambient
Vacuum
> 300µm
1µm
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.
Energia:
Ioni:
Fase:
HARD
+•
GAS
SOFT
+/n+/–/n–
CONDENSATA
Press.:
VUOTO
P. ATM.
EI
CI
MALDI
ESI
DESI
APCI
APPI
Sistema di
Introduzione
DI, MIMS, GC, HPLC,
CZE, CEC, ITP
Sorgente
EI, CI, PD, FD, FAB,
LSIMS, ESI, APCI,
MALDI
Separazione degli ioni
secondo il loro rapporto
massa/carica (m/z)
Analizzatore
Settori (EB; BE; EBE …)
Quadrupolo (Q, QqQ)
Trappola ionica, FT-ICR
Orbitrap, Tempo di volo (TOF)
Ibridi (BEqQ; QTOF ….)