The Analysis of Solutions and SurfacesThe Analysis of ... · Arm ~ 0.2 psi TriVersa NanoMate for Higher FlowsTriVersa NanoMate for Higher Flows 320um columns and above. Set-up TriVersa-NanoMate

The Analysis of Solutions and SurfacesThe Analysis of Solutions and Surfaces using TriVersa NanoMate

Mark Allen, Mark Baumert, Reinaldo AlmeidaAdvion, Harlow, UK

OutlineOutline- How it works- SolutionSolution

-Infusion - Lipid and Non Covalent Binding-LC coupling and Fraction Collection - Plant Metabolites

- SurfacesLi id E t ti S f A l i (LESA)-Liquid Extraction Surface Analysis (LESA)

- Liver – G2 ion mobility - Patient PlaquesPatient Plaques - Lung- Food/Leaves pesticide screening p g-TLC Plate Reader for compound identification

TrIVersa NanoMateTrIVersa NanoMate

IMPROVING THE PERFROMANCE OF MASS SPECTROMETRYMASS SPECTROMETRY

More time = more sensitivity = more information to identify compounds

Over 500 systems WorldWide in LeadingOver 500 systems WorldWide in Leading Laboratories

Multi purpose systemIMPROVING THE PERFROMANCE OF MASS SPECTROMETRY

N C l

LESA – tissue analysis

Metabolomics

Non Covalent Interactions

In tact protein

Metabolite Identification

Post Translation Modifications

D I iti

HistonesPlants Metabolomics

A tib d I

Chinese Medicine

Organic Chemistry Support

Drug Impurities Glycosylation/Glycomics

Lipids

Flexiblechip-based ESI system

Fast change overI f i t LC

Antibody Igg

Petroleomics


Infusion to nanoLC to fraction collection

Petroleomics

Reaction MonitoringHydrogen Deuterium Exchange

Compatible with most Mass SSpectrometers

Co-Marketing AgreementsCo Marketing Agreements

Thermo FischerWaters

AB SciexBruker AB SciexBruker

and most recently..Agilent

Introducing The ESI Chip and TriVersa nanoMate:A f 400 i d d t l t lArray of 400 independent nanoelectrospray nozzles

The ESI ChipTM – A Stand Alone Source

V220 x 106 V/mIn the ESI Chip, the electric

field around the nozzle tip is formed from the potential pdifference between the microfabricated silicon substrate as an integrated counter electrode and the voltage applied to the fluid i th d ti i tt ti

• Incorporation of the ESI counter electrode into the spray nozzle

via the conductive pipette tip.

• Incorporation of the ESI counter electrode into the spray nozzle.This is very different from conventional electrospray devices, which define the electric field by the potential difference between the spray device (fluid potential) and the mass spectrometer inlet or atmospheric pressure ionization (API) interface.

• As the distance between the electrodes is only a few microns and t t t l t d t bl l t i fi ld iconstant, an extremely strong and stable electric field is

reproducibly generated, essentially decoupling the ESI process from the inlet of the mass spectrometer.

NanoMate Advantages Over Pulled Capillary NanoSprayNanoSpray

Conventional pulled capillary NanoMate Advantages• One analysis per capillary• Manual operation• Great expertise required• Avg throughput: 8 10

• Up to 400 analyses per chip• Fully automated• Minimal expertise required• Avg throughput: 100’s samples/day• Avg. throughput: 8-10

samples/day• Unpredictable spray stability may

compromise data quality

• Avg. throughput: 100 s samples/day• Better data quality and improved

quantitation over pulled capillaries• No microscope needed

Automated InfusionAutomated Infusion

Samples are Introduced to the Chip InletUsing Conductive Pipette TipsUsing Conductive Pipette Tips

Silicon chip bearing a20 x 20 array of 10 μm ID

nanospray nozzlesHigh Voltage850 1500 V

MS

850-1500 VSample

MSOrifice

10 μL conductive pipette tip

NanoESI flow rates approx 100 to 500 nL/min

TriVersa NanoMateTriVersa NanoMate

Why do our customers use the TVNM ?Why do our customers use the TVNM ?– Infusion

• Long stable spray from small sample volumes• Zero carry-over infusion analyses of simple samples

Protein Core Laboratories - protein structure/research Non covalent interactionsNon covalent interactionsIntact protein Identification Histone Characterization P l i difi i (PTM) Ch i iPost translation modifications (PTM) CharacterizationGlycosylation

Lipidomics (maybe in hospitals and pharma also)

Lipid Analysis, Novel High-Throughput Methodology for Lipidome Analysisfor Lipidome Analysis

“High throughput, reproducible and sensitive analysis of total lipid extracts much easier.”

Reference: Christer Ejsing PhD Thesis 2007, Molecular Characterization of the Lipidome by Mass Spectrometry Technische Universität Dresden http://deposit d nb de/cgi

Laboratory: Dr Andrej ShevchenkoSpectrometry, Technische Universität Dresden http://deposit.d-nb.de/cgi-

bin/dokserv?idn=983638306&dok_var=d1&dok_ext=pdf&filename=983638306.pdfDr. Andrej ShevchenkoMPI Mol Cell Biol & GeneticsDresdenGermany

Automated Shotgun Lipidomics – yeast cells

“We achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes.” “This analysis provided 95%This analysis provided 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches.”

The lipid metabolic network of S. cerevisiae. Enzymes are annotated by gene name (essential genes are indicated in red). The lipid metabolic networkwas compiled using the Saccharomyces Genome Database (www.yeastgenome.org) and references therein. Lipids monitored by absolute quantification are indicated by green circles. Lipids that were only identified are shown by gray circles.

Reference:Ejsing CS, Sampaio JL, Surendranath V, Duchoslav E, Ekroos K, Klemm RW, Simons, Shevchenko A (2009): Global analysis of the yeast lipidome by quantitative shotgun

Laboratory:Dr. Andrej ShevchenkoMPI Mol Cell Biol & GeneticsDresdenGlobal analysis of the yeast lipidome by quantitative shotgun

mass spectrometry. PNAS 106 (7), 2136-2141.DresdenGermany

Non-covalent Interaction, Compound Screening

“… high sample throughput.” Determination of the dissociationconstants (Kd) for the complexes of two different proteins with their ligands.

“… improving analysis reproducibility.”

“… eliminates carryover”

“… very small sample consumption”

“great potential for rapid screening of compound libraries in drug discovery programs.”

Reference:Zhang S, Van Pelt CK & Wilson DB (2003): Quantitative Determination of Noncovalent

Laboratory: Prof. David B. Wilson

Binding Interactions Using Automated Nanoelectrospray Mass Spectrometry. Anal. Chem. 75, 3010-3018.

Molecular Biology and Genetics Cornell University, New York USA

Non Covalent Interactions Receptor-Ligand-InteractionsPheromone Binding Protein (PBP)

Bombykol

ight

: MPI

Bio

phys

ical

ibpc

.mpg

.de/ Ligand-bound PBP

Unbound PBP

Bombyx moriPict

ures

Cop

yri

Che

mis

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http

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ww

.mp

Principle of the Assay:Ratio of ligand-bound PBP/ unbound PBP/ unbound PBP at increasing ESI-MS cone voltages

Reference:Hooper AM, Dufour S, He X, Muck A, Zhou J-J, Almeida R, Field LM, Svatos A, Pickett JA (2009): High-throughput ESI-MS analysis of binding between the Bombyxmori pheromone-binding protein BmorPBP1, its pheromone components and some

Laboratory:Dr. Aleš SvatošMax Planck Institutefor Chemical Ecologyp g p , p p

analogues. Chem. Commun. 5725–5727. for Chemical EcologyJena

NanoLC couplingNanoLC coupling

0.2 - 0.8 µL/min

nanoLC or Nanoaquity butt connected via teflon sheath

Move to next nozzle in 3 seconds so no sample loss if sprayer plugs mid run

Use your own nanoLC columnsUse your own nanoLC columns


Why Do our customers Use for NanoLC?Why Do our customers Use for NanoLC?– NanoLC coupling

• The most stable nanoESI for direct coupling to nanoLC.• Automated next nozzle in 3 seconds • Fast change from Infusion to nanoLC

Peak Area ComparisonPeak Area ComparisonRT: 3.20 - 3.40

NL 3 06E5

ESI ChipPeak Area ~2x60

70

80

90

100

ndan

ce

RT: 3.26AA: 415254

NL: 3.06E5TIC F: + c NSI SRM ms2 653.901 [906.399-906.401] MS ICIS 08apr10_003

Peak Area ~2x greater

10

20

30

40

50

Rel

ativ

e Abu

n

RT 3 26 NL: 8 40E4

Other Nanospray S

60

70

80

90

100

RT: 3.26AA: 232397

NL: 8.40E4TIC F: + c NSI SRM ms2 653.901 [906.395-906.405] MS ICIS

Source10

20

30

40

50

3.20 3.22 3.24 3.26 3.28 3.30 3.32 3.34 3.36 3.38Time (min)

18Nano-LC/MS in Routine Bioanalysis: Application to Human NGF Biomarker StudiesPresented by: Gary A. Schultz, PhDhttp://advion.com/events/webinars.php

TriVersa Spray Sensing Technology

High aqueous, higher spray current ~125nA

Re-equilibration of analytical column

Low aqueous, lower spray current ~40nA

19

TriVersa NanoMate – Lockmass capability

Fraction Collection

200µL/min

Fraction Collection

200µL/min

0,2 µL/min HPLC

5 - 1000 µL/min

199,8 µL/min

Robotic

Nanoelectrospray

ProbeBackpressure

20s / Fraction 67 µL / FractionInfusion

Conductive Pipette0 – 3 kV

ArmProbe

~ 0.2 psi

TriVersa NanoMate for Higher FlowsTriVersa NanoMate for Higher Flows 320um columns and above

Set-upSet up

TriVersa-NanoMate

Fraction collection into 384 plates (or 96 plates)

240 µL/min incoming flow

Robotic arm for fractioncollection and later Infusion

240 µL/min incoming flow rate

Low volume splitting ‘T’

300 nL/min to nESI chipand MS

≤ 0.2 % of the flow

22

The Power of Signal Averaging

TriVersa NanoMate Users – Fraction C ll ti ith i f iCollection with infusion

PharmaceuticalDrug Metabolism/DMPK – development

Radioactive metabolites

Drug Impurities

Chinese Medicine More information from complex samples.

AcademiaMetabolomicsPlant Metabolomics

p p

BiotechAntibody IGG

Traditional Chinese Medicine Sample Profiling with UPLC/oaTOF/NanoMateUPLC/oaTOF/NanoMate

John Shockor, Waters Corporation June, 2009

UPLCTriversa Nanomate

ACQUITY UPLC/Xevo Qtof MS with MSE

Maximum chromatographic resolution,sensitivity, and speedExact mass analysis with data-rich information

ACQUITY UPLC/Xevo Qtof MS with MSE

Maximum chromatographic resolution,sensitivity, and speedExact mass analysis with data-rich information

MarkerLynx XS data processingIdentify leading markers using extended statistical analysis tool

such as PCA/OPL

MarkerLynx XS data processingIdentify leading markers using extended statistical analysis tool

such as PCA/OPL

Synapt HDMSFraction Collection with NanoMate

Infuse fraction collected back into MS for MSMS analysis for structural elucidation

Fraction Collection with NanoMateInfuse fraction collected back into MS for MSMS

analysis for structural elucidation

NanoMate can quickly switch between LC, fraction collection and infusion mode Re-inject Fraction into UPLC/Qtof MS

for further separationRe-inject Fraction into UPLC/Qtof MS

for further separationLockmass compatible

for further separationSeparate isomers, coeluted peaks so that more detailed info about the sample can be revealed

for further separationSeparate isomers, coeluted peaks so that more detailed info about the sample can be revealed

Glycomics, high throughput brain ganglioside (GG) analysis

“… improved ionization NanoMate robot was coupled to a high-capacity ion trap (HCT) mass spectrometer to create a system merging automatic chip-based electrospray ionization (ESI) infusion, ultrafast ion detection, and multistage sequencing at superior sensitivity.

efficiency”“… improved signal stability”“… high run-to-run

NanoESI chip MS experiments enabled the identification of more than 50 glycoforms exhibiting a high degree of heterogeneity in the ceramide motifs and alterations such as O-acetylation.

25 distinct GG species … were identified for the first time.

reproducibility”„... >15-fold sensitivity increase”increase

… method of choice in modern glycomics”

Reference:Almeida R, Mosoarca C, Chirita M, Udrescu V, Dinca N, Vukelic Z, Allen M, Zamfir AD

Laboratory: PD Dr. Alina D. Zamfir

(2008):Coupling of fully automated chip-based electrospray ionization to high-capacityion trap mass spectrometer for ganglioside analysis. Analytical Biochemistry 378, 43-52.

Mass Spectrometry Laboratory, National Institute for Research and Development in Electrochemistry and Condensed Matter, 300224 Timisoara, Romania

Plant MetobolismUPLC-FTICR-MS coupling via TriVersa NanoMateSolution:

Source: Bettina Seiwert, DGMS, Konstanz, 2009, Advion Lunch Seminar.

Reference:Giavalisco P, Köhl K, Hummel J, Seiwert B, Willmitzer L (2009): 13C Isotope-labeled metabolomes allowing for improved compound annotation and relative quantification in liquid chromatography-mass spectrometry-based metabolomic

Laboratory:Prof. Dr. Lothar WillmitzerMPI Molecular Plant PhysiologyPotsdam-Golmq q g p y p y

research. Anal. Chem. 81 (15), 6546-6551.Potsdam GolmGermany

Antibody - Analysis of Glycosylation Structures.b

ioch

.ox.

ac.u

k/gl

ye/

igg1

.gif

Sour

ce:

http

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arch

ive

“CID-MS/MS analysis (was done) by static nanosprayy ( ) y p yinfusion using a Triversa Nanomate (Advion Biosciences, Ithaca, NY). CID-MSn was carried out on the glycosylatedand non-glycosylated chymotryptic peptide NSGAL at a collision energy of 25–30 V.”gy

Reference:Valliere-Douglass JF, Kodama P, Mujacic M, Brady LJ, Wang W, Wallace A, Yan B, Reddy P, Treuheit MJ, Balland A (2009): Asparagine-linked oligosaccharides present on a non-consensus amino acid sequence in the CH1

Laboratory:Dr. Alain BallandProcess and Product Developmentoligosaccharides present on a non-consensus amino acid sequence in the CH1

domain of human antibodies. JBC 284 (47), 32493-32506.Product DevelopmentAmgen Inc., USA

LESA – Liquid Extraction Surface Analysis

Simultaneously identify drugs and their phase I

y

Simultaneously identify drugs and their phase I & II metabolites directly from tissue samples

using chip-based NanoESIusing chip based NanoESI28

Normal Operation using the TriVersa NanoMate SystemSyste

Mass Spectrometer

Sampling tip

Nozzle

Aspirate sample

T f l

Sample

Transfer sample

Apply HV, spray samplewith 200nl/minSample with 200nl/min

Operation using the TriVersa NanoMate Systemfor LESA

Mass Spectrometer

Sampling tip

Nozzle

Sample Aspirate solvent

Solvent

Dispense solvent on sampleAspirate sample solutionTransfer sampleSolvent a s e sa p eSpray sample

LESA Workflow

Step 3 – Analyte IonizationRobot aspirates extracted analytes from target and

i i i l

Step 1 – Solvent DeliveryDisposable Tip picks up

extraction solvent at reservoir

Step 2 – Analyte ExtractionRobot places extraction

solvent on target and initiates aspirate/dispense cycles for

initiates electrospray at a 400 nozzle nESI chip

analyte extraction

Resolution of

K t V V B k l GJ F ll t t d li id t ti b d f li d i i ti i

Resolution of approx. 1mm

Kertesz V, VanBerkel GJ: Fully automated liquid extraction-based surface sampling and ionization using a chip-based robotic nanoelectrospray platform. Journal of Mass Spectrometry 2009 45(3) 252-60


Why do our customers use the TVNM ?Why do our customers use the TVNM ?• Liquid Extraction Surface Analysis for

direct analysis of tissuedirect analysis of tissue– NanoESI quality data directly from a surface– Complimentary to MALDIp y– See analytes that may not be visible using MALDI – Validate MALDI results or Prescreen prior to MALDI

Long stable infusion times enables signal averaging and ion– Long stable infusion times enables signal averaging and ion mobility investigations

• No matrix interference as no matrix used so Less suppression than MALDI (<500 Damatrix signal) due to liquid extraction

• Semi quantitative, but we need more proof here

Surfaces Analyzed using LESASurfaces Analyzed using LESA

-Whole TissueWhole Tissue- Liver- Patient Plaques - Lipids - Lung - Food/Leaves pesticide screening TLC Pl t R d f d id tifi ti-TLC Plate Reader for compound identification

Tissue: 7.5 mg/kg propranolol, IV dosed mouse, 60 min after doseEluent: 80/20/0.1 ACN/H2O/FA

Data collected: MRM mode, 1 min/sample

15 45 13

Dosed tissue Control tissue

12 3

45 6 5 632

nsity

, cps

10000

15000

20000

nsity

, cps

10000

15000

20000stomach

liverlung

kidneymuscle

brain

brain liver

Propranolol Propranolol

4 6 8 10 12 14

Inte

n

0

5000

14 16 18 20 22 24

Inte

n

0

5000liver

kidney muscle lung stomach

Hydroxypropranolol glucuronide Hydroxypropranolol glucuronide

nten

sity

, cps

5000

10000

15000

20000

Inte

nsity

, cps

5000

10000

15000

20000(Phase II) (Phase II)

4 6 8 10 12 14

I 014 16 18 20 22 24

I 0

t,min t,min

Analysis of Tissue using LESA + Synapt G2

Eleanor Blatherwick, Sue Slade and Jim Scrivens

School of Life Sciences

University of Warwick

Ion mobility cell

Ad iAdvionTriVersa

Nanomate

Liquid Extraction Surface Analysis

(LESA) ( )

Collision energy can either be applied in the trap or transfer regions, allowing mobility separation after or before y p

fragmentation respectively.


Selectivityy

LESA LESA LESA LESALESA LESA LESA LESA

MS MSMS MS

MS/MS

Ion Mobility

MS/MS

MS/MS Ion Mobility


MS/MS Ion Mobility

Fenclozic acid• Drug compound – Fenclozic acid• [M+H]+= 254 m/z• Contains one chlorine atom

• Characteristic isotope pattern in mass spectrum• 3:1 intensity ratio of Cl35:Cl37

Nano-ESIvoltage

1.55 kV

• Fenclozic acid administered to rats at 10 mg/kg• Livers harvested from both dosed and

Gas pressure 0. 5 psi

Extraction solvent

1 µl 50 % ACN + 0.1 % formic acid

control rats, sectioned to 30 µm• Frozen & stored at ‐80˚C

(aq)

Extraction time 5 seconds on tissue.

• Positive ion HDMS/MS mode• MS/MS transition of m/z 254 – 208• Ion mobility separation prior to MS/MS f t tifragmentation

Blatherwick, E.Q., Van Berkel, G.J. et al. (2011) XENOBIOTICA 41 (8) 720-734.

LESA –MS/MS spectra

Drug gstandard

Dosed tissue section

NegativeNegative control tissue section


LESA – arrival time distributions

Drug

• Extracted arrival time distribution through the mobility cell of m/z 254 ion

a)

b)

Drug standard

y /

• Same mobility profile for drug standard and dosed

Dosed tissue section

drug standard and dosed tissue

• Different mobility profile

Negative

Different mobility profile for control tissue

• Provides additionalNegative control tissue section

• Provides additional evidence that fenclozicacid is being detected from dosed tissue samplesdosed tissue samples


Diclofenac• Drug compound – Diclofenac•Common non‐steroidal anti‐inflammatory drug (NSAID)• [M‐H]‐= 294 m/z• Contains two chlorine atoms• Readily formed fragment ion at m/z 249

Nano-ESI -1.56 kV

• 14C‐Diclofenac administered to mice at 10 mg/kg• Whole‐body sections Nano ESI

voltage1.56 kV

Gas pressure

0. 6 psi

• Mounted on tape, dried & stored at room temperature

N ti i HDMS/MS dExtraction solvent

1 µl 80 % ACN (aq)

Extraction 5 seconds on

• Negative ion HDMS/MS mode• MS/MS transition of m/z 249 – 213• Ion mobility separation following MS/MS fragmentation Extraction

time5 seconds ontissue, 1second delaypost aspiration

fragmentation


LESA Sampling/ionisation

Diclofenac parent i I l iion

m/z 294Isolation

MS/MS fragmentation

Activation

Ion mobility Separation

MS/MS D t tispectra Detection


Improving selectivitya) 1 µg

Diclofenac

b) 100 ng

c) 10 ngLESA

sampling positions

Diclofenac spotted

• Control rat kidney sections

positions

• Three different concentrations of diclofenac spotted onto tissue:• a) 1 µg diclofenac dosed on tissue.• b) 100 ng diclofenac on tissue.• c) 10 ng diclofenac on tissue.

LESA MS used to sample across the kidney at each sampling positionLESA‐MS used to sample across the kidney at each sampling position indicated using the selective mobility MS/MS method.


Improving selectivity ‐ATDsa)

Kidney tissue only

b) c)1 µg 100 ng 10 ng

Diclofenac on tissue

The extracted arrival time distributions (ATDs) for m/z 249 (Red) and the resulting m/z 213 daughter ion (Black) through the mobility cell are displayed above for two

samplings from each tissue sectionsamplings from each tissue section.

These results indicate that with an IMS separation after MS/MS fragmentation, di l f b fid tl id tifi d ith j t 10 tt d tidiclofenac can be confidently identified with just 10 ng spotted on tissue.


Dosed whole‐body sections 3 hours post‐doseKidney Sampling points

Liver Brain

LESA-IMS-MS/MS sampling of wild type mouse whole body tissue section 3 hours post-dose. Arrival time distribution of m/z 249 (red) and m/z 213 (black) from a) kidney b) liver and c) brain. d) Optical image f ( ) ( ) f ) y ) ) ) p g

of WT whole-body tissue section, with sampling positions highlighted in yellow.


Dosed whole‐body sections 24 hours post‐dose

KidneySampling points

Liver Brain

LESA-IMS-MS/MS sampling of wild type mouse whole body tissue section 24 hours post-dose. Arrival time distribution of m/z 249 (red) and m/z 213 (black) from a) kidney b) liver and c) brain. d) Optical

image of WT whole-body tissue section, with sampling positions highlighted in yellow.


Lipid Analysis of Atherosclerotic Plaqueswith LESATM

“Lipids were directly analyzed from tissue sections with a Adviontissue sections with a AdvionTriVersa NanoMate … “

“Notably, LESA allowed a rapid analysis of plaque lipids directly from tissue sectionswithout time and labor-intensive sample preparation.”

„The solvent extraction volume was 1.5 μL (chloroform/methanol/isopropanol 1:2:4 containing 7.5 mM

i t t ) d th di d l 1 0 L

References:Stegemann C, Drozdov I, Shalhoub J, Humphries J, Ladroue C, Didangelos A, Baumert B, Allen M, Davies AH, Monaco C, Smith A, Xu Q, Mayr M (2011): Comparative Lipidomics Profiling of Human Atherosclerotic Plaques. Circ. Cardiovasc. Genet p blished online April 21

Laboratory:Prof. Dr. Manuel MayrKing‘s College London

ammonium acetate) and the dispensed volume was 1.0 μL.

Genet., published online April 21. UK


“The signals as well as the signal intensities were comparable to shotgun lipidomics of tissue extracts”





“To our knowledge, this study is the most

h icomprehensive MS analysis of the lipid content in human atherosclerotic plaque toatherosclerotic plaque to date.”




Guinea Pig lung model• Whole Guinea pig lung is extracted

from the animal, connected to aperfusator and mechanically ventedperfusator and mechanically ventedusing a cannula inserted into thetrachea and connected to a smallrodent ventilator (60 strokes/minuteat 2 mL/stroke)

• Lung Tissue model dosed intra-tracheally with 3 mg Fluticasonepropionate in 5 mL air using apropionate in 5 mL air using amicro-sprayer needle

• Lung Tissue was then frozen, slicedin 16 um thickness and placed onpglass slides for vacuum drying andfurther LESA processing

Tissue samples and pictures courtesy of Walter Korfmacher and Fangbiao Li from Merck Research Laboratories

MS data from location ‘B3’MS data from location B3Q1 scan, negative ion mode

MS/MS of m/z 499.1, negative ion modeQ , g

Fl ti i tFluticasone propionate(M-H)-

Analytical standard: negative ion modenegative ion mode MS/MS m/z 499.3

B3

Positive vs. negative ion mode SRM(9 mm lateral resolution)

B2 B4B3 B5 B6

1,000,000 1,000,000

B2 B4B3 B5 B6

100,000

ty cps)

ity cps)

363,00

0

±3

10,000

, ,

100,000

10,000

, ,

Ca. 10x signal improvement in negative ion

d

M signa

l inten

siative ion mod

e (

M signa

l inten

sitive ion mod

e (c

0

13,200

257

500

506

83

1,000

10,000

1,000

10,000mode

100SRne

ga SRpo

si

340

80

2

124 5

190 5

10

100

10

0 0

B2 B4B3 B5 B6

Pesticide Screening from Food Surfaces

GSpinach

L f

Grape-half

Leaf

Apple SkinSkin

54

Spray Set-up ‘Pump’AerosolSpray Set upUsing 4 mL Methanol spray

Pump Aerosol Sprayer

g p ytotal equaling 1/20 of tolerance level (8,000ng):-400 ng/g Malathion

500 ng/g Se in- 500 ng/g Sevin- 10 ng/g Simazin- 20 ng/g Carbofuran- 100 ng/g Diazinon (no known tolerance level)

Spray bottle rinsed with MeOH

‘Real World’ Sprayer

Spray bottle rinsed with MeOH before use

55

S.E. Edison, et al. Surface swabbing technique for rapid screening for pesticides using ambient pressure desorption ionization with high-resolution mass spectrometry, RCMS, 2011 25, 127-139

Structures of PesticidesMalathion CarbofuranDiazinon

MW 330.1(M+H)+: 331.0434

MW 304.1(M+H)+: 305.1083

MW 221.1(M+H)+: 222.1126

SimazineSevin

(Isobaric Compounds)

MW 201.1(M+H)+: 202.0864

MW 201.1(M+H)+: 202.0854

Infusion of 50 uM Equimolar Mixture in S S l t E tiSpray Solvent: Exactive

Di iDiazinon

SimazineSevin Carbofuran

Simazine

Malathion

57

58

Apple – Exactive MalathionMalathion

Sprayed Apple(20x below tolerance level – 400 ng/g Malathion)

Apple from Supermarket

Theoretical mass spectrum of Malathion

Malathion detected at 2.7 ppm deviation from

(M+H)+ = 331.0434

59

ppm deviation from theoretical value

Apple – Exactive CarbofuranCarbofuran

Sprayed Apple(20x below tolerance level – 20 ng/g Carbofuran)

A l f S k hApple from Supermarket: shows trace carbofuran present.

Apple from Supermarket, additionally washed

Theoretical mass of Carbofuran

Carbofuran detected at 3.1 ppm deviation from theoretical value

60

Also detected from purchased Apple, needed extra cleaning to wash off !

Summary of LESA for Pesticide S iScreening

• Rapid (<2 min/sample) screening for pesticides on food surfaces is feasiblesurfaces is feasible

• Detection limits easily see 20-fold below tolerance levels• Method is automated without conventional sample preparation

or chromatographyor chromatography– ‘preparation’ is placing a sample skin or leaf on a glass slide

• High mass resolution and MS/MS techniques are complimentarycomplimentary

• Trace evidence for carbofuran in purchased apple and diazinon in purchased grape and spinachp g p p

• ‘Liquid extraction surface analysis (LESA) of food surfaces employing chip‐based nano‐electrospray mass spectrometry’employing chip based nano electrospray mass spectrometry Henion et al RCM 2011

TLC-MS/MS: Integrated Liquid Surface Sampling-Nano Electrospray Determination of Small Drug Molecules

from Thin Layer Chromatography Platesfrom Thin Layer Chromatography Plates.

TriVersa NanoMate

6262

TriVersa NanoMate Pipette tip ‘Extracting’ spot from TLC Plate

Strychnine LESA Mass Spectra of TLC Spot from Dog Stomach Contents 335

(M+H)+LTQ Q1 Full-scan MS of dog 100(M+H)+LTQ Q1 Full scan MS of dog

stomach contents

30405060708090

elat

ive

Abu

ndan

ce

263.08156.00 189.00 217.08

Davidow developing solvent85/10/5 ETOAc/ MeOH/NH4OH

Strychnine

LTQ full-scan CID mass spectrum from dog stomach contents 100 150 200 250 300 350 400

m/z

0102030R

e

163.08 227.00 239.17371.08309.25294.92195.08 351.25 393.25258.00 330.92

Solvent Front

5060708090

100e

Abu

ndan

ce264.13264

(M+H)+

100 150 200 250 300 350 400m/z

010203040

Rel

ativ

222.10234.10184.06 272.13220.10 307.20248.16

194.11 335.25168.09156.08122.03106.03 353.32 370.98

Full scan CID mass spectrum of standard strychnine

5060708090

100

ve A

bund

ance

Full-scan CID mass spectrum of standard strychnine

264 (M+H)+

MW=334

264.11

g St

d

g St

d

xtra

ct

olve

nt

6363100 150 200 250 300 350 400

m/z

010203040

Rel

ativ

222.10234.11184.04 272.13220.08 307.20248.16194.09 335.21168.08156.08122.04108.01 353.06 371.04 388.97

200

ng

200

ng ExSo

AcknowledgementsAcknowledgements• Mark Baumert, Reinaldo Almeida, Simon Prosser, Daniel

Eikel and Jack Henion, Advion• Dr. Shaun MacMahon, FDA's Center for Food Safety

and Applied Nutrition (FDA- CFSAN)and Applied Nutrition (FDA CFSAN)• Dr. Tim Croley, FDA• Vilmos Kertesz and Gary Van Berkel of ORNL for first

d t ti LESA f ibilitdemonstrating LESA feasibility

Multi purpose systemMulti purpose system

N C l

LESA – tissue analysis

Metabolomics

Non Covalent Interactions

In tact protein

Metabolite Identification

Post Translation Modifications

D I iti

HistonesPlants Metabolomics

A tib d I

Chinese Medicine


Drug Impurities Glycosylation/Glycomics

Lipids

Flexiblechip-based ESI system

Fast change overI f i t LC

Antibody Igg

Petroleomics


Infusion to nanoLC to fraction collection

Petroleomics

Reaction MonitoringHydrogen Deuterium Exchange

Conclusion

TriVersa NanoMate® and

RePlayTM are integral partsRePlayTM are integral parts

of modern LC/MS-

applications, improving the

stability, information

quality and sample

throughput in many areas

of molecular analysis.

Examples from Published Studies

Customers from Central Europe using the TriVersa NanoMate include:

Selected Users - TriVersa NanoMateApplication Name Facility Country

Protein Core Lab Bruce Stanley Penn State University USA

Protein Core Lab Leslie Hicks Danforth Agricultural Center USA

Protein Core Lab Allis Chien Stamford University USA

Protein Core Lab Mark Skehel Cancer Research UK UK

Protein Core Lab Marta Vilaseca Casas University Barcelona Spain

Protein Core Lab Helen Cooper University of Birmingham UK

Protein Core Lab Kostas Petritius Translational Genomics Research Institute USA

Protein Core Lab Manuel Mayr Kings College London UL

Non Covalent Interactions Alison Ashcroft University of Leeds UK

Non Covalent Interactions Carol Robinson University of Cambridge UK

Non Covalent Interactions John Crosby University Of Bristol UK

Non Covalent Interactions Ales Svatos MPI Jena Germany

Non Covalent Interactions Alain Van Dorssellar University of Strasbourg France

In tact Protein ‐Top Down Stephen Master university of Pennsylvania USA

In tact Protein ‐Top Down Neil Kelleher University of Illinois USA

In tact Protein ‐Top Down Ying Ge University of Wisconsin USA

In tact Protein ‐Top Down Alan Marshall University of Florida USA

In tact Protein ‐Top Down Julia Chamot‐Rooke Ecole Polytechnic France

Histones Alex Imhof University of Munich Germany

Glycosylation Carlito Lebrillo UC Davis USAy y

Glycosylation Vern Reinhold University New Hampshire USA

Glycosylation Catherine Costello University of Boston USA

Glycosylation Igor Almeida University of Texas El Paso USA

Lipids Xianlin Han Washington University St. Louis USA

Lipids Andrej Schevchenko MPI Dresden GermanyLipids Andrej Schevchenko MPI Dresden Germany

Lipids Christer Ejsing University of Southern Denmark Denmark

Lipids Dr Taguchi University of Tokyo Japan

Lipids Dominic Schwudke Tata Research Intitute India

Organic Chemistry Support Bridget Stein University of Swansea UK

Selected Users - TriVersa NanoMateApplication Name Facility CountryApplication Name Facility Country

Metabolite Identification Jose Castro Perez Merck USA

Metabolite Identification Josh Rowe Allergan USA

Metabolite Identification Glen Dillow Alcon USA

Metabolite Identification Johnathan Josephs Bristol Myers Squib USA

Metabolite Identification Gary Bowers GSK USA

Metabolite Identification Gerard Hopfgartner University of Geneva Switzerland

Metabolomics Olaf Boernsen Novartis Switzerland

Metabolomics Thomas Hankemeier University of Leiden UK

Metabolomics Mark Viant University of Birmingham UK

Metabolomics Paul Skip University of Southampton UK

Plant Metabolomics Chris Vlahakis Pioneer USA

Plant Metabolomics Tim Snow DuPont USA

Plant Metabolomics Patrick Giavalisco MPI Golm Germany

Antibody IGG Anne Zeck Roche Penzberg Germany

Antibody IGG Joerg Thomas Regula Roche Penzberg GermanyAntibody IGG Joerg Thomas Regula Roche Penzberg Germany

Antibody IGG Amgen Colorado Steve Cockrill USA

Antibody IGG Amgen Seattle Allain Balland USA

LESA

Tissue Analysis Gary Van Berkel Oak Ridge National Laboratory USA

Software Control enables integration with external multiple plate fraction collection and radiochemical detection

This flow can be directed to an online flow scintillation detector or multiplate 96 well fractionscintillation detector or multiplate 96-well fraction collector for radioactive measurements on dried fractions.(Data exchange via External Collector Synchronization Software:Integration with external multiple plate fraction collection and radiochemical detection.) These fractions can be used for in-depth MS

analysis of metabolites (optionally) identifiedanalysis of metabolites (optionally) identified by radioactive detection.

Automated Shotgun Lipidomics

2-Step Lipid Extraction

Optimized Solvent Systems

“Settings of the ion source (NanoMate HD) must allow stable and reproducible spray with the flow injection rate of 200-300 nL/min.” (Ejsing et al 2006 Anal Chem 78 6202 14 )

Overview of the quantitative shotgun lipidomics approach. Yeast cell lysates were spiked with internal lipid standards. Samples were processed by 2-step lipid extraction for fractionation of apolarand polar lipids. The lipid extracts were analyzed by automated shotgun lipidomics analysis in negative and positive ion mode. Lipid species were detected by MPIS or MRM analysis on a QSTAR i b FT MS l i LTQ O bi hi Q ifi i f l hi d b h i l l i f ll d b MRM l i Id ifi i d ifi i f

Absolute Quantification (Ejsing et al. 2006, Anal Chem 78, 6202-14.)

Reference:Ejsing CS, Sampaio JL, Surendranath V, Duchoslav E, Ekroos K, Klemm RW, Simons, Shevchenko A (2009): Global analysis of the yeast lipidome by quantitative shotgun

Laboratory:Dr. Andrej ShevchenkoMPI Mol Cell Biol & GeneticsDresden

instrument, or by FT MS analysis on a LTQ Orbitrap machine. Quantification of ergosterol was achieved by chemical acetylation followed by MRM analysis. Identification and quantification of detected lipid species were performed by Lipid Profiler and ALEX.

Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. PNAS 106 (7), 2136-2141.

DresdenGermany

Automated Shotgun Lipidomics – human plasma

“Top-down shotgun lipidomics demonstrated h h t i i i d b

Plasma lipidome of 19 men with hypertension and 51 normotensive male controls was screened by top-down shotgun profiling on a LTQ Orbitrap hybrid mass spectrometer. The analysis encompassed 95 lipid species of 10 major lipid classes.

that hypertension is accompanied by specific reduction of the content of ether lipids and free cholesterol that occurred independently of lipidomic alterations induced by obesity and insulin resistance.”

“These results may form the basisThese results may form the basis for novel preventive and dietary strategies alleviating the severity of hypertension.“

Reference:Graessler J, Schwudke D, Schwarz PEH, Herzog R, Shevchenko A, Bornstein SR (2009): Top-down lipidomicsreveals ether lipid deficiency in blood plasma of hypertensive

Laboratory:Dr. Andrej ShevchenkoMPI Mol Cell Biol & GeneticsDresdenreveals ether lipid deficiency in blood plasma of hypertensive

patients. Plos One 4 (7), e6261.DresdenGermany

Plant MetobolismUPLC-FTICR-MS coupling via TriVersa NanoMateProblem with the Integration of the UPLC:UPLC produces narrow peaks with a width of 3-6 seconds, BUT the scanning rate of the FT-ICR MS is quiet low (~1 second per scan @ R100000)

Source: Bettina Seiwert, DGMS, Konstanz, 2009, Advion Lunch Seminar.

Reference:Giavalisco P, Köhl K, Hummel J, Seiwert B, Willmitzer L (2009): 13C Isotope-labeled metabolomes allowing for improved compound annotation and relative quantification in liquid chromatography-mass spectrometry-based metabolomic

Laboratory:Prof. Dr. Lothar WillmitzerMPI Molecular Plant PhysiologyPotsdam-Golmq q g p y p y

research. Anal. Chem. 81 (15), 6546-6551.Potsdam GolmGermany

MetabolomicsUPLC-FTICR-MS coupling via TriVersa NanoMateSince highly sensitive on-line coupling of UPLC with FTICR-MS is technically infeasible due to their different scan rates, at-line coupling of these techniques was developed for rapid analysis.

“Good reproducibility and high recovery was observed.”

“M li bl i t“More reliable assignments were achieved by use of at-line coupling of UPLC and FTICR-MS compared with off-line pmeasurements.”

Reference:Li X, Fekete A, Englmann M, Frommberger M, Lv S, Chen G, Schmitt-Kopplin P (2007): At-line coupling of UPLC to chip-electrospray-FTICR-MS Anal Bioanal Chem 389

Laboratory:PD Dr. Phillippe Schmitt-KopplinInstitute for Ecological ChemistryNeuherberg/Münchenchip-electrospray-FTICR-MS. Anal. Bioanal. Chem. 389,

1439–1446.Neuherberg/MünchenGermany

Documents

The Analysis of Solutions and SurfacesThe Analysis of ... · Arm ~ 0.2 psi TriVersa NanoMate for Higher FlowsTriVersa NanoMate for Higher Flows 320um columns and above. Set-up TriVersa-NanoMate