Electrochemistry in Proteomics
Transcript of Electrochemistry in Proteomics
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Protein Cleavage, Disulfide Bond Reduction,
DNA Adduct Formation Using Electrochemistry/MS
BSPR/EBI Conference 2011
12th – 14th July
Agnieszka Kraj Antec, The Netherlands
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Outline
• Applications overview • Principle of Electrochemistry • Reactions • Instrumentation • Electrochemistry in Proteomics • Conclusions
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Electrochemistry upfront MS
Disulfide bond reduction
Peptide bond cleavage
Desalting
Oxidative damage of
DNA Signal
enhancement in MS
Metabolite synthesis
Drug metabolism
Drug ̶ protein binding
Oxidative tagging of proteins
Application Areas Electrochemistry/MS
Disulfide bond reduction
Peptide bond cleavage
Desalting
Drug ̶ protein binding
Oxidative tagging of proteins
Proteomics
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Reduction Oxidation
Principle of Electrochemistry (EC) upfront MS
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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 Da
Electrochemically Oxidizable Amino Acid
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Electrochemistry (EC) upfront MS Instrumental set-up
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Electrochemistry in Proteomics
• peptide bond cleavage • disulfide bond reduction • surface oxidation • desalting
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Tyrosine containing peptides: 1000mV
Mechanism of cleavage after Y and W
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
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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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Electrochemical disulfide bond reduction
Insulin
Non reduced Cell OFF
Reduced Cell ON
Chain B
Chain B
Chain A
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Electrochemical Reduction of Lactalbumin
Electrochemical reduction of the protein results in shift of charge state distribution suggesting conformational change of protein (S-S bridges reduction).
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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
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Electrochemical Desalting of Proteins
0 V 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
Poster 42, Online Electrochemical Desalting of Proteins Mohamed Benama
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Lysozyme NMR structure (1E8L, model 6) showing surface with underlying secondary structure, disulfides, and substrate binding site
Figure 3. Lysozyme FT-MS spectra showing slight over-oxidation at +2.1V. Satellite peaks present in spectra may be due to sulfate adducts.
Rel
ativ
e A
bu
nd
ance
(%
)
Mass/Charge
Figure 3. Lysozyme FT-MS spectra showing slight over-oxidation at +2.1V. Satellite peaks present in spectra may be due to sulfate adducts.
Rel
ativ
e A
bu
nd
ance
(%
)
Mass/Charge
Lysozyme FT-MS spectra showing slight over-oxidation at +2.1V. Satellite peaks present in spectra may be due to sulfate adducts.
Electrochemical Oxidation as a Surface Mapping Probe of Higher Order Protein Structure
McClintock et al., Anal. Chem. 2008, 80, 3304
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DNA, nucleosides, etc.
Electrochemistry in Genomics
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Electrochemistry in Genomics
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Figure
8
0
1.0
0
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ns
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3000 E [mV]
0 500 1000
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1500 2000 2500
1.0
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ma
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mu
m i
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3000 E [mV]
0 500 1000
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... adduct
... acetaminophen dimer ... guanosine dimer
... acetaminophen ... guanosine
C
A
B
(1) guanosine + APAP E < 1200 mV
no product detected
(2) guanosine + APAP 1200 mV < E < 1600 mV
APAP-APAP
(3) guanosine + APAP 1600 mV < E APAP-APAP
+ guanosine-guanosine
+ APAP – guanosine
/ m
axiu
mu
m i
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Figure
8
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... adduct
... acetaminophen dimer ... guanosine dimer
... acetaminophen ... guanosine
C
A
B
(1) guanosine + APAP E < 1200 mV
no product detected
(2) guanosine + APAP 1200 mV < E < 1600 mV
APAP-APAP
(3) guanosine + APAP 1600 mV < E APAP-APAP
+ guanosine-guanosine
+ APAP – guanosine
/ m
axiu
mu
m i
nte
ns
ity
Guanosine + APAP E < 1200mV no product detected
Guanosine + APAP 1200mV < E < 1800mV APAP — APAP
Guanosine + APAP 1800mV < E APAP – APAP
+ Guanosine – Guanosine
+ APAP – Guanosine
Electrochemistry in Genomics
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Electrochemistry in Genomics
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Summary
EC/MS shows great potential in proteomics: disulfide bond reduction protein (?), peptide bond cleavage surface oxidation desalting drug – protein binding
EC/MS is used successfully in mimicking of DNA damage and covalent
adduct formation
EC/MS represents a powerful technique for fast study of natures REDOX reactions.
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Acknowledgements:
Mohamed Benama University of Bristol
Simon Lambert
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