Protein Cleavage, Disulfide Bond Reduction,

DNA Adduct Formation Using Electrochemistry/MS

BSPR/EBI Conference 2011

12th – 14th July

Agnieszka Kraj Antec, The Netherlands

Outline

• Applications overview • Principle of Electrochemistry • Reactions • Instrumentation • Electrochemistry in Proteomics • Conclusions

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

Reduction Oxidation

Principle of Electrochemistry (EC) upfront MS

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

Electrochemistry (EC) upfront MS Instrumental set-up

Electrochemistry in Proteomics

• peptide bond cleavage • disulfide bond reduction • surface oxidation • desalting

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

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.

Electrochemical Disulfide Bond Reduction

Electrochemical disulfide bond reduction

Insulin

Non reduced Cell OFF

Reduced Cell ON

Chain B

Chain B

Chain A

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).

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+] combination correspond to background formylation of the protein

Poster 42, Online Electrochemical Desalting of Proteins Mohamed Benama

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

DNA, nucleosides, etc.

Electrochemistry in Genomics

Electrochemistry in Genomics

Figure

8

0

1.0

0

inte

ns

ity

3000 E [mV]

0 500 1000

0.8

0.6

0.4

0.2

1500 2000 2500

1.0

inte

ns

ity /

ma

xiu

mu

m i

nte

ns

ity

3000 E [mV]

0 500 1000

0.8

0.6

0.4

0.2

1500 2000 2500

... 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

Figure

8

0

1.0

0

inte

ns

ity

3000 E [mV]

0 500 1000

0.8

0.6

0.4

0.2

1500 2000 2500

1.0

inte

ns

ity /

ma

xiu

mu

m i

nte

ns

ity

3000 E [mV]

0 500 1000

0.8

0.6

0.4

0.2

1500 2000 2500

... 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

Electrochemistry in Genomics

22

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.

Acknowledgements:

Mohamed Benama University of Bristol

Simon Lambert