Electrochemical disulfide bond reduction
Electrochemical disulfide bond reduction
Electrochemical disulfide bond reduction
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Protein Cleavage, Disulfide Bond Reduction,<br />
DNA Adduct Formation<br />
Using Electrochemistry/MS<br />
BSPR/EBI Conference 2011<br />
12th – 14th July<br />
Agnieszka Kraj<br />
Antec, The Netherlands
Outline<br />
• Applications overview<br />
• Principle of Electrochemistry<br />
• Reactions<br />
• Instrumentation<br />
• Electrochemistry in Proteomics<br />
• Conclusions
Application Areas Electrochemistry/MS<br />
Oxidative<br />
tagging of<br />
proteins<br />
Disulfide <strong>bond</strong><br />
<strong>reduction</strong><br />
Peptide <strong>bond</strong><br />
cleavage<br />
Drug ̶ ̶ protein<br />
binding<br />
Electrochemistry<br />
Proteomics<br />
upfront MS<br />
Desalting<br />
Drug<br />
metabolism<br />
Metabolite<br />
synthesis<br />
Signal<br />
enhancement<br />
in MS<br />
Oxidative<br />
damage of<br />
DNA
Principle of Electrochemistry (EC) upfront MS<br />
Reduction<br />
Oxidation
<strong>Electrochemical</strong>ly Oxidizable Amino Acid<br />
Amino acid Functional group Oxidized forms, with mass change<br />
Tyrosine<br />
phenol<br />
quinol, +16 Da<br />
quinone, +14Da<br />
Tryptophan<br />
indole<br />
indolol, +16 Da<br />
indolone, +14Da<br />
Cysteine<br />
Methionine<br />
thiol<br />
sulfenic acid, +16 Da sulfinic acid, +32Da sulfonic acid, +48 Da<br />
methylthioether<br />
methylsulfoxide, + 16 Da<br />
methylsulfone, + 32 Da
Electrochemistry (EC) upfront MS<br />
Instrumental set-up
Electrochemistry in Proteomics<br />
• peptide <strong>bond</strong> cleavage<br />
• <strong>disulfide</strong> <strong>bond</strong> <strong>reduction</strong><br />
• surface oxidation<br />
• desalting
Mechanism of cleavage after Y and W<br />
Tyrosine containing peptides: 1000mV<br />
Tryptophan containing peptides: 800mV<br />
Oxidation and cleavage pathways are pH dependent:<br />
• oxidation yield decreases with increasing pH<br />
• cleavage products formed only in acidic and neutral conditions<br />
J. Roeser et al., Anal. Chem., 2010, 82 (18), 7556
Cleavage of Angiotensin I (DRVYIHPFHL)<br />
ADVANTAGES:<br />
1) …alternative to enzymatic digestion<br />
by electro-chemical push button<br />
reaction in seconds!<br />
2) clean, no enzymes, no non-specific<br />
cleavage, no auto-digestion, etc.<br />
CURRENT STATUS:<br />
1) cleavage of big proteins is under<br />
development,<br />
2) optimization to increase the<br />
reaction yield.
<strong>Electrochemical</strong> Disulfide Bond Reduction
<strong>Electrochemical</strong> <strong>disulfide</strong> <strong>bond</strong> <strong>reduction</strong><br />
Insulin<br />
Chain B<br />
Non reduced<br />
Cell OFF<br />
Chain B<br />
Reduced<br />
Cell ON<br />
Chain A
<strong>Electrochemical</strong> Reduction of Lactalbumin<br />
<strong>Electrochemical</strong> <strong>reduction</strong> of the protein results in shift of charge state<br />
distribution suggesting conformational change of protein (S-S bridges <strong>reduction</strong>).
<strong>Electrochemical</strong> <strong>disulfide</strong> <strong>bond</strong> <strong>reduction</strong><br />
• on-line, electrochemical <strong>disulfide</strong><br />
<strong>bond</strong> <strong>reduction</strong> with DESI MS<br />
• identification of <strong>disulfide</strong> containing<br />
peptides from enzymatic digestion<br />
mixture<br />
• derivatization of thiols by selenamid<br />
• charge state distribution in proteins<br />
(native vs. reduced)<br />
Zhang et al., J. Proteome Res., 2011, 10, 1293
<strong>Electrochemical</strong> Desalting of Proteins<br />
Poster 42,<br />
Online <strong>Electrochemical</strong> Desalting of Proteins<br />
Mohamed Benama<br />
0 V 2.8 V<br />
Deconvoluted MS at 0V and 2.8V showing protein desalting.<br />
correspond to [Na + + K + ] combination<br />
correspond to background formylation of the protein
Relative Abundance (%)<br />
<strong>Electrochemical</strong> Oxidation as a Surface Mapping<br />
Probe of Higher Order Protein Structure<br />
McClintock et al., Anal. Chem. 2008, 80, 3304<br />
Lysozyme NMR structure (1E8L, model 6) showing surface with<br />
underlying secondary structure, <strong>disulfide</strong>s, and substrate binding<br />
site<br />
Lysozyme FT-MS spectra showing slight over-oxidation at +2.1V.<br />
Satellite peaks present in spectra may be due to sulfate adducts.<br />
Mass/Charge
Electrochemistry in Genomics<br />
DNA, nucleosides, etc.
Electrochemistry in Genomics
intensity / maxiumum intensity<br />
intensity<br />
/ maxiumum intensity<br />
intensity / maxiumum intensity<br />
intensity<br />
/ maxiu<br />
0.2<br />
Electrochemistry in Genomics<br />
0<br />
0 500 1000<br />
0.4<br />
... acetaminophen<br />
... guanosine<br />
1500 2000 2500<br />
E [mV]<br />
3000<br />
1.0<br />
0.8<br />
B<br />
1.0<br />
0.8<br />
... acetaminophen dimer<br />
... guanosine dimer<br />
... adduct<br />
0.6<br />
0.4<br />
0.2<br />
... acetaminophen<br />
... guanosine<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
0 500 1000<br />
1500 2000 2500<br />
E [mV]<br />
3000<br />
... acetaminophen dimer<br />
Guanosine C<br />
... guanosine dimer + APAP<br />
E < 1200mV<br />
... adduct<br />
Guanosine + APAP<br />
0 500 1000<br />
1500 2000 2500<br />
E [mV]<br />
3000<br />
0<br />
1200mV < E < 1800mV<br />
APAP<br />
Guanosine + APAP 1800mV < E<br />
0 500 1000<br />
E < 1200 mV<br />
(1) guanosine + APAP<br />
no product detected<br />
(2) guanosine + APAP<br />
1500 2000 2500<br />
E [mV]<br />
1200 mV < E < 1600 mV<br />
— APAP<br />
1600 mV < E<br />
(3) guanosine + APAP – APAP<br />
+ Guanosine – Guanosine<br />
+ APAP – Guanosine<br />
3000<br />
no product detected<br />
APAP-APAP<br />
APAP-APAP<br />
+ guanosine-guanosine<br />
+ APAP – guanosine
Electrochemistry in Genomics
Summary<br />
• EC/MS shows great potential in proteomics:<br />
• <strong>disulfide</strong> <strong>bond</strong> <strong>reduction</strong><br />
• protein (), peptide <strong>bond</strong> cleavage<br />
• surface oxidation<br />
• desalting<br />
• drug – protein binding<br />
• EC/MS is used successfully in mimicking of DNA damage and covalent<br />
adduct formation<br />
EC/MS represents a powerful technique for fast<br />
study of natures REDOX reactions.<br />
22
Acknowledgements:<br />
Mohamed Benama<br />
University of Bristol<br />
Simon Lambert
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