Ambient mass spectrometry

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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.

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2 Ambient mass spectrometry

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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

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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)

Excited-state neutral atoms or molecules (“metastables”) are used to ionize the sample

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The first step: Penning Ionization

• Long-lived excited-state (metastable) atoms or molecules react with analytes that posses ionization potentials less than the

metastable energy,

He

^*

+ S  S

^+.

+ He + electron

Penning, F. M., Über Ionisation durch Metastabile Atome Naturwissenschaften 1927, 15, 818.

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

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Analysis of unknown pills or detection of counterfeit drugs

DART approach: seconds!

Conventional method: hours!!!

Immediate response and detection on surfaces or

in fluids

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Rapid detection of explosives

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1.5 torr 1.5×10^-5torr

REIMS - Ion formation mechanism

The mechanism of electrosurgical tissue ablation involves 1) the Joule-heatingof 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.

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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 PO O

2

O O R2 O R2

O O

O P OO

NH3+

O O R2 O R2

O O

O PO 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 PO

O OH

OH

REIMS Tissue Data

REIMS_human_liver_cancer_030810_002 #2-31 RT:0.05-1.24AV: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 #25-29 RT: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

droplet-thin film collision simulations. incident angle: 55° ; speed: 120 m/s

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22 α= 55°, 120 m/s, t = 0.8 μs

γ= 0.5 (‐Y)

γ= 0.5 (‐Z)

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.

Suitable to for Miniature MS and Portable Applications

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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

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Fenner, N.C.; Daly, N.R. Laser Used for Mass Analysis. Rev. Sci. Instrum. 1966, 37, 1068-1070.

P P

PrrreeemmmiiioooNNNooobbbeeelll 222000000222pppeeerrr lllaaaChCChhiiimmmiiicccaaa

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

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Franz Hillenkamp

Institute of Medical Physics and Biophysics of

the University of Münster Michael Karas

† 22 agosto 2014 (78 anni)

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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

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Matrix

^-

assisted laser desorption

ionization

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: Nitrogenlaser; 355, 266 nm: Nd:YAGlaser;

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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

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Hillenkamp, F. Adv. Mass Spectrom. 1989, 11A, 354.

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The

Lucky Survivor model

postulates that analytes are 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 from

incorporation 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

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.

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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)

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Magnification of the Target

-1.0000 -0.8000 -0.6000 -0.4000 -0.2000 0.0000 0.2000 0.4000 0.6000 0.8000 1.0000

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10 shots

100 shots

1000 shots

11,000 shots

Marvin Vestal, Virgin Instruments

Mass spectrum of Hg-Papain oligomerization

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… 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)

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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.

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C. H. Borchers et al, J. Am. Soc. Mass Spectrom. 21, 1680–1686, 2010

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/ionization

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SALDI

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MALDI-TOF DESI

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MATRIX DEPOSITION

Manual devices Airbrush

Automatic matrix depositors (MALDI spotter)

vibrational vaporization

piezoelectric technology

Acoustic droplet ejection

Manual spraying

1) droplet size is too large to make good crystal structures

2) gapsin the matrix coverage.

Automated spraying

homogenouscoverage

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44 Olanzepine: atypical antipsychotic

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Angew. Chem. Int. Ed. 2010, 49, 3834 –3838

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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

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

* Arth ro b_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\1 SLin, Sm oo thed, "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 L of matrix

Insert Target Get Spectras

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48 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 Intens. [a.u.]5000

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

MALDI Biotyper - Basic

Low influence of culture conditions

Psdm. oleovorans B396_Medium 360

0 1000

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

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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

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

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R.M. Caprioli et al, J. Mass Spectrom. 2012, 47, 1473–1481

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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 direct

imaging of heterogeneous tissue sections and single cells with sub-cellular resolution.

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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

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DESI

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100%

20%

Study of Latent Fingermarks by MALDI MS Imaging

Rosalind Wolstenholme, Robert Bradshaw, Malcolm R. Clench and Simona Francese, 2009, RCM

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Ioni secondari

Spettrometria di massa di ioni secondari (SIMS)

Ioni primari (KeV)

campione

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56 More recently polyatomic primary

ions, i.e. C₆₀⁺ions

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 4.3×10¹¹cm^-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 1.2×10¹¹ions/cm^-2.

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P Ambient, vacuum Ambient Vacuum Sp. Res. 10-50µm > 300µm 1µm

CI

ES

I MALDI

DESI APCI APPI

Energia: HARD SOFT

Fase: GAS CONDENSATA Ioni: +• +/n+/–/n–

EI

Press.: VUOTO P. ATM.

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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)