ROXY™ EC/MS System

ROXY™ EC/MS System

1

2

Service Training 2012

• Introduction to ROXY EC system

• basics & application review

• ReactorCell and µ-PrepCell maintenance

• filling the µ-PrepCell

• Dialogue training• program concept• practical workshop including event programming

• ROXY EC system Installation• establishing communication with RS232 cable• trigger cable• grounding kit

• Practical insights on application• test compounds • mass spectrometric analysis• optimization of conditions

Application Areas Electrochemistry/MS

3

ROXY EC System

ReactorCell™µ-PrepCell™

Electrochemistry up front MSInstrumental set-up

4

Electrochemistry (EC) upfront MSInstrumental set-up

ROXY™ EC System

ROXY™ EC/LC System

ROXY™ EC/LC System

5

6

Electrochemistry upfront MS

2-D MS Voltammogram

7

3-D MS Voltammogram

Electrochemistry upfront MSPrediction of drug/xenobiotic metabolism

ReactorCell (or µ-PrepCell)Drug

7

8

Electrochemistry upfront MSPrediction of drug/xenobiotic metabolism

Amodiaquin metabolic pathway:

Faber et al., Angew. Chem. Int. Ed. Engl. 5 (2011) A52-58

9


Amino acid Functional group Oxidized forms, with mass change

Tyrosine

phenol

Tryptophan

Cysteine

Methionine

thiol

indole

methylthioether

quinol, +16 Da quinone, +14Da

indolol, +16 Da indolone, +14Da

sulfenic acid, +16 Da sulfinic acid, +32Da sulfonic acid, +48 Da

methylsulfoxide, + 16 Da methylsulfone, + 32 Da10

Electrochemistry upfront MSProtein chemistry

11

Drug – protein adduct formation

12

Electrochemistry upfront MSProtein chemistry

Tyrosine containing peptides: 1000mV

Mechanism of cleavage after Tyrosine (Tyr; Y) & Tryptophan (Trp; W) residues

Oxidation and cleavage pathways are pH dependent:

• oxidation yield decreases with increasing pH• cleavage products formed only in acidic and neutral conditions

J. Roeser et al., Anal. Chem., 2010, 82 (18), 7556

Tryptophan containing peptides: 800mV

Cleavage of Angiotensin I (DRVYIHPFHL)

ADVANTAGES:

1) …alternative to enzymatic digestion by electro-chemical push button reaction in seconds!

2) clean, no enzymes, no non-specific cleavage, no auto-digestion, etc.

CURRENT STATUS:

1) cleavage of big proteins is under development,

2) optimization to increase the reaction yield.

14

Electrochemical Disulfide Bond Reduction

15

Tested compounds

Peptide/protein Nr of AA Nr of bonds

Somatostatin 14 1

Insuline 512 Inter

1 Intra

α - Lactalbumin 123 4

Comparison with MD

500 1000 1500 2000m/z

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

1147.75

956.75

1434.32850.98681.12 960.56 1152.22

1169.72 1439.61626.38

1213.92 1911.391480.74

851.07681.18

858.86

680.36866.11571.43 1134.04

1170.661369.11 1643.48 1812.84

NL: 5.26E5111214 Insulin MD pulse 01#7887-8260 RT: 23.22-24.32 AV: 374 T: ITMS + c ESI Full ms [360.00-2000.00]

NL: 3.15E5insulin 01#3203-3903 RT: 9.37-11.41 AV: 701 T: ITMS + c ESI Full ms [360.00-2000.00]

Insulin reduced on MD electrode

Insulin reduced on new electrode

Insulin

Reduced Insulin

No Insulin present

600 700 800 900 1000 1100 1200 1300m/z

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

1147.5278

956.4407

960.26941160.3113

680.7421 850.9267 1133.5199970.7545646.0303 944.4320751.2788 1361.73711255.4004

680.7420

850.6758

1147.7295956.7773858.1774780.3308666.9364 1211.20111080.6786964.4340 1316.5495917.9375537.0156

NL: 1.15E5

02#1069-1084 RT: 10.76-11.86 AV: 16 F: FTMS + c ESI Full ms [300.00-2000.00]

NL: 6.77E4 02#1017-1029 RT: 6.93-7.81 AV: 13 F: FTMS + c ESI Full ms [300.00-2000.00]

Insulin - new working electrode

Cell OFFNo reduction

Pulse ONComplete reduction

SomatostatinNew working electrode

300 400 500 600 700 800 900 1000 1100 1200m/z

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

546.5790

559.2278819.3646

571.8769419.6727 838.3381296.9704

615.5303385.9019 706.8891474.8328

517.1583

1008.4596 1241.1783749.4350

1140.0590

547.2509

820.3723

559.8999279.9241

320.9508439.8490 835.3762706.8571567.5920

510.2107

784.0946 926.6889 986.0088 1129.5707 1236.7040

NL: 5.19E4

111216 Somatostatipulse 02#152-163 RT: 4.78-5.29 AV: 12 F: FTMS + c ESI Full ms [100.00-2000.00]

NL: 2.43E4111216 Somatostatipulse 02#96-112 RT: 2.14-2.89 AV: 17 F: FTMS + c ESI Full ms [100.00-2000.00]



Z = 3

Z = 2

Z = 3Z = 2

α – LactalbuminNew working electrode

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

1576.32

1575.98

1773.101772.98

1580.531777.971418.691782.84

864.711792.081289.90

1593.07994.67 1420.38 1807.051182.571290.44878.72 1615.27998.66 1910.60

1183.161092.22

1182.91

1092.30

1014.28946.80

1290.631290.36 1412.87

1184.41

1094.53 1186.41 1576.99887.68 1017.061577.211419.39949.12 1293.35

1581.42 1935.861181.74 1526.191422.29

1783.731233.91 1839.651614.941302.63 1959.88

NL: 8.28E4111216 Lactalbuminpulse01#772-811 RT: 9.41-11.21 AV: 40 F: FTMS + c ESI Full ms [800.00-2000.00]

NL: 1.11E4111216 Lactalbumin pulse01#887-921 RT: 14.69-16.24 AV: 35 F: FTMS + c ESI Full ms [800.00-2000.00]



Electrochemical reduction of the protein results in shift of charge state distribution suggesting conformational change of protein (S-S bridges reduction).

Electrochemical disulfide bond reduction

• on-line, electrochemical disulfide bond reduction with DESI MS

• identification of disulfide containing peptides from enzymatic digestion mixture

• derivatization of thiols by selenamid

• charge state distribution in proteins (native vs. reduced)

Zhang et al., J. Proteome Res., 2011, 10, 1293

Electrochemical Desalting of Proteins

0 V 2.8 V

Deconvoluted MS at 0V and 2.8V showing protein desalting.

correspond to [Na+ + K+] combinationcorrespond to background formylation of the protein

Poster at BSPR, CambridgeMohamed Benama

Electrochemical Oxidation as a Surface MappingProbe of Higher Order Protein Structure

McClintock et al., Anal. Chem. 2008, 80, 330423

Cell OFF

Cell ON

24


Oxidative Damage of DNA

25

26

Laborious, time-consuming and hardly automatable

Stability of the (modified) nucleic acids during sample prep

Low specificity and sensitivity


27


Mass Voltammograms of Nucleosides

28Herbert Oberacher, Institute of Legal Medicine, Innsbruck, Austria; Electrochemical Simulation of Oxidation Processes Involving NucleicAcids On-line Monitored with Electrospray Ionization-Mass Spectrometry - poster IMSC 2009

Several studies on cell cultures and rodents have demonstrated

that acetaminophen can covalently bind to nucleic acids

after metabolic activation.

EC/MS of guanosine + APAP: EC/MS of guanosine:


Electrochemistry upfront MS provides new tool to asses the antioxidant potency of chemicals !

30


31


Anal. Bioanal. Chem. 378 (2004) 917– 925

LC–EC–UV/VIS and LC–EC–MS chromatograms for a mixtureof the sixteen priority pollutant PAH.

Positive-ion mode: naphthalene (1), acenaphthylene(2), acenaphthene (3), fluorene (4), phenanthrene (5),anthracene (6), fluoranthene (7), pyrene (8), benzo[a]anthracene(9), chrysene (10), benzo[b]fluoranthene (11), benzo[k]fluoranthene(12), benzo[a]pyrene (13), dibenzo[a,h]anthracene (14),benzo[ghi]perylene (15), indeno[1,2,3-cd]pyrene (16).

(a) UV chromatogram recorded at 254 nm;(b) MS chromatogram, scan mode m/z 150–400, electrochemical

flow cell off; (c) MS chromatogram, scan mode m/z 150–400, electrochemical

flow cell 1.6 V;(d) MS chromatogram, selected ion monitoring (SIM) mode,

electrochemical flow cell 1.6V

Signal enhancement

33


34


35


Compound Environmental persistence EC starting voltage (mV)

Sulfadizine Medium 1100

Metabenzthiazuron Stable 1800

Ethidimurin Medium -1420

Ibuprofen Medium 1200

17ß-estradiol Unstable 300

Clotrimazol Stable 1750

PCB31 Very Stable 4500

Tetracene Stable 1600

Persistence in environment, stability in EC?

=> QSAR modeling…

Compound Structure EC starting voltage (mV)

Naphthalene 340

Anthracene 930

Phenanthren 600

Benzo[a]anthracene 750

Tetracene 1600

Stability and structure

Chrysene 1300

38


39

Electrochemistry upfront MSOther applications

Joint Conference of German and PolishMass Spectrometry Society

Poznan, Poland

March 4 - 7, 2012

40

Electrochemistry upfront MSOther applications

This work demonstrates the hyphenation of an electrochemical reaction cell with

a continuous-flow bioaffinity assay and parallel LCHR-MS.

Summary

EC/MS represents a powerful technique for study of REDOX

reactions in life science

41

ROXY™ EC/MS System

Documents

Transcript of ROXY™ EC/MS System