Transcript Slide 1

ROXY™ EC/MS System
1
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
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Application Areas Electrochemistry/MS
Oxidative
tagging of
proteins
Disulfide bond
reduction
Peptide
bond
cleavage
Drug ̶ protein
binding
Desalting
Drug
metabolism
Electrochemistry
upfront MS
Metabolite
synthesis
Oxidative
damage of
DNA
Signal
enhancement
in MS
Pollutants
Skin
sensitization
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Electrochemistry up front MS
Instrumental set-up
ROXY EC System
µ-PrepCell™
ReactorCell™
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Electrochemistry (EC) upfront MS
Instrumental set-up
ROXY™ EC System
ROXY™ EC/LC System
ROXY™ EC/LC System
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Electrochemistry upfront MS
Disulfide bond
reduction
Oxidative
tagging of
proteins
Peptide
bond
cleavage
Drug ̶ protein
binding
Drug
metabolism
Drug/xenobiotic
Electrochemistry
metabolism
upfront MS
Metabolite
synthesis
Desalting
Oxidative
damage of
DNA
Signal
enhancement
in MS
Pollutants
Skin
sensitization
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Electrochemistry upfront MS
Prediction of drug/xenobiotic metabolism
ReactorCell (or µ-PrepCell)
Drug
3-D
2-DMS
MSVoltammogram
Voltammogram
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Electrochemistry upfront MS
Prediction of drug/xenobiotic metabolism
Amodiaquin metabolic pathway:
Faber et al., Angew. Chem. Int. Ed. Engl. 5 (2011) A52-58
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Electrochemistry upfront MS
Disulfide bond
reduction
Oxidative
tagging of
proteins
Peptide
bond
cleavage
Drug ̶ protein
binding
Desalting
Drug
metabolism
Protein
chemistry
Electrochemistry
upfront MS
Metabolite
synthesis
Oxidative
damage of
DNA
Signal
enhancement
in MS
Pollutants
Skin
sensitization
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Electrochemistry upfront MS
Protein chemistry
Amino acid
Functional group
Oxidized forms, with mass change
Tyrosine
phenol
quinol, +16 Da
quinone, +14Da
indole
indolol, +16 Da
indolone, +14Da
Tryptophan
Cysteine
thiol
sulfenic acid, +16 Da sulfinic acid, +32Da sulfonic acid, +48 Da
Methionine
methylthioether
methylsulfoxide, + 16 Da methylsulfone, + 32 Da
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Drug – protein adduct formation
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Electrochemistry upfront MS
Protein chemistry
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Mechanism of cleavage after Tyrosine (Tyr; Y)
& Tryptophan (Trp; W) residues
Tyrosine containing peptides: 1000mV
Tryptophan containing peptides: 800mV
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), 7
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.
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Electrochemical Disulfide Bond Reduction
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Tested compounds
Peptide/protein
Nr of AA
Nr of bonds
Somatostatin
14
1
Insuline
51
2 Inter
1 Intra
α - Lactalbumin
123
4
Comparison with MD
1147.75
100
Relative Abundance
90
80
Insulin
70
NL: 5.26E5
111214 Insulin MD pulse
01#7887-8260 RT:
23.22-24.32 AV: 374 T:
ITMS + c ESI Full ms
[360.00-2000.00]
956.75
60
50
Insulin reduced on MD electrode
40
30
850.98
681.12
20
10
0
100
1434.32
960.56
1152.22
1169.72
1213.92
626.38
681.18
1439.61
1480.74
1911.39
NL: 3.15E5
insulin
01#3203-3903 RT:
9.37-11.41 AV: 701 T:
ITMS + c ESI Full ms
[360.00-2000.00]
851.07
90
Reduced
Insulin
80
70
60
50
40
Insulin reduced on new electrode
30
858.86
20
10
680.36
571.43
866.11
No Insulin present
1134.04
1170.66
1369.11
0
500
1000
m/z
1500
1643.48
1812.84
2000
Insulin - new working electrode
1147.5278
100
Relative Abundance
90
80
70
NL: 1.15E5
Cell OFF
No reduction
02#1069-1084 RT:
10.76-11.86 AV: 16 F:
FTMS + c ESI Full ms
[300.00-2000.00]
60
956.4407
50
40
30
20
10
0
100
960.2694
646.0303680.7421 751.2788
850.9267 944.4320 970.7545
1133.5199
1160.3113
1255.4004
1361.7371
680.7420
NL: 6.77E4
90
80
850.6758
70
Pulse ON
Complete reduction
60
50
40
30
20
780.3308
10
0
537.0156
666.9364
600
700
800
858.1774 956.7773
917.9375 964.4340
900
1000
m/z
1147.7295
1080.6786
1100
1211.2011
1200
1316.5495
1300
02#1017-1029 RT:
6.93-7.81 AV: 13 F: FTMS
+ c ESI Full ms
[300.00-2000.00]
Somatostatin
New working electrode
Z=3
546.5790
Relative Abundance
90
80
70
111216 Somatostati
pulse 02#152-163 RT:
4.78-5.29 AV: 12 F: FTMS
+ c ESI Full ms
[100.00-2000.00]
Z=2
559.2278
60
NL: 5.19E4
Cell OFF
No reduction
100
819.3646
50
40
517.1583
30
571.8769
20
10
296.9704
419.6727
385.9019
474.8328
0
100
615.5303
838.3381
706.8891 749.4350
1008.4596
547.2509
Z=3
90
70
1241.1783
Pulse ON
Complete reduction
Z=2
820.3723
80
1140.0590
60
50
40
30
20
10
0
559.8999
279.9241
510.2107
439.8490
567.5920
320.9508
300
400
500
600
706.8571 784.0946
700
800
m/z
835.3762
926.6889 986.0088
900
1000
1129.5707
1100
1236.7040
1200
NL: 2.43E4
111216 Somatostati
pulse 02#96-112 RT:
2.14-2.89 AV: 17 F: FTMS
+ c ESI Full ms
[100.00-2000.00]
α – Lactalbumin
New working electrode
100
Relative Abundance
90
80
1576.32
Cell OFF
No reduction
70
1575.98
60
1773.10
50
1772.98
40
1580.53
1418.69
30
1777.97
1782.84
20
864.71
10
878.72
0
100
1289.90
1182.57 1290.44
994.67
998.66
1092.30
1184.41
1014.28
946.80
60
1290.63
50
1290.36
40
1094.53
887.68
30
1017.06
949.12
1293.35
1181.74
10
800
900
1000
1100
1233.91
1200
1910.60
NL: 1.11E4
111216 Lactalbumin
pulse01#887-921 RT:
14.69-16.24 AV: 35 F:
FTMS + c ESI Full ms
[800.00-2000.00]
1412.87
1186.41
20
1792.08
1807.05
Pulse ON
Complete reduction
1182.91
80
70
1593.07
1615.27
1420.38
1183.16
1092.22
90
0
NL: 8.28E4
111216 Lactalbumin
pulse01#772-811 RT:
9.41-11.21 AV: 40 F:
FTMS + c ESI Full ms
[800.00-2000.00]
1419.39
1576.99
1577.21
1422.29
1526.19
1302.63
1300
1581.42
1935.86
1614.94
1400
m/z
1500
1600
1700
1783.73 1839.65
1800
1900
1959.88
2000
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
Poster at BSPR, Cambridge
Mohamed Benama
0V
2.8 V
Deconvoluted MS at 0V and 2.8V showing protein desalting.
correspond to [Na+ + K+] combination
correspond to background formylation of the protein
Electrochemical Oxidation as a Surface Mapping
Probe of Higher Order Protein Structure
Cell
OFF
Cell
ON
McClintock et al., Anal. Chem. 2008, 80, 3304
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Electrochemistry upfront MS
Disulfide bond
reduction
Oxidative
tagging of
proteins
Peptide
bond
cleavage
Drug ̶ protein
binding
Desalting
Drug
metabolism
Oxidative
damage of DNA
Electrochemistry
upfront MS
Metabolite
synthesis
Oxidative
damage of
DNA
Signal
enhancement
in MS
Pollutants
Skin
sensitization
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Oxidative Damage of DNA
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Oxidative Damage of DNA
Laborious, time-consuming and hardly automatable
Stability of the (modified) nucleic acids during sample prep
Low specificity and sensitivity
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Oxidative Damage of DNA
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Mass Voltammograms of Nucleosides
Herbert 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
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Oxidative Damage of DNA
EC/MS of guanosine:
EC/MS of guanosine + APAP:
Several studies on cell cultures and rodents have demonstrated
that acetaminophen can covalently bind to nucleic acids
after metabolic activation.
Oxidative Damage of DNA
Electrochemistry upfront MS provides new
tool to asses the antioxidant potency of
chemicals !
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Electrochemistry upfront MS
Oxidative
tagging of
proteins
Disulfide bond
reduction
Peptide
bond
cleavage
Drug ̶ protein
binding
Desalting
Drug
metabolism
Electrochemistry
upfront MS
Metabolite
synthesis
Oxidative
damage of
DNA
Signal
enhancement
in MS
Pollutants
Skin
sensitization
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Signal enhancement
LC–EC–UV/VIS and LC–EC–MS chromatograms for a mixture
of 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
Anal. Bioanal. Chem. 378 (2004) 917– 925
Electrochemistry upfront MS
Disulfide bond
reduction
Oxidative
tagging of
proteins
Peptide
bond
cleavage
Drug ̶ protein
binding
Desalting
Drug
metabolism
Skin
sensitization
Electrochemistry
upfront MS
Metabolite
synthesis
Oxidative
damage of
DNA
Signal
enhancement
in MS
Pollutants
Skin
sensitization
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Electrochemistry upfront MS
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Electrochemistry upfront MS
Disulfide bond
reduction
Oxidative
tagging of
proteins
Peptide
bond
cleavage
Drug ̶ protein
binding
Desalting
Drug
metabolism
Environmental
analysis
Electrochemistry
upfront MS
Metabolite
synthesis
Oxidative
damage of
DNA
Signal
enhancement
in MS
Pollutants
Skin
sensitization
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Persistence in environment, stability in EC?
Compound
Environmental persistence
EC starting voltage (mV)
Sulfadizine
Medium
1100
Stable
1800
Ethidimurin
Medium
-1420
Ibuprofen
Medium
1200
17ß-estradiol
Unstable
300
Stable
1750
Very Stable
4500
Stable
1600
Metabenzthiazuron
Clotrimazol
PCB31
Tetracene
=> QSAR modeling…
Stability and structure
Compound
Structure
EC starting voltage (mV)
Naphthalene
340
Anthracene
930
Phenanthren
600
Benzo[a]anthracene
750
Tetracene
1600
Chrysene
1300
Electrochemistry upfront MS
Oxidative
tagging of
proteins
Disulfide bond
reduction
Peptide
bond
cleavage
Drug ̶ protein
binding
Desalting
Drug
metabolism
Electrochemistry
upfront MS
Metabolite
synthesis
Oxidative
damage of
DNA
Signal
enhancement
in MS
Pollutants
Skin
sensitization
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Electrochemistry upfront MS
Other applications
Joint Conference of German and Polish
Mass Spectrometry Society
Poznan, Poland
March 4 - 7, 2012
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Electrochemistry upfront MS
Other applications
This work demonstrates the hyphenation
of an electrochemical reaction cell with
a continuous-flow bioaffinity assay
and parallel LCHR-MS.
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Summary
EC/MS represents a powerful
technique for study of REDOX
reactions in life science
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