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Increasing Sequence Coverage in Top-Down Proteomics

 

 

For comprehensive disulfide bond assessment in top down proteomics, the use of electrochemically based reduction of the S-S bonds*) results in significant faster and easier  workflows. The reduction is based on a purely instrumental approach by placing an electrochemical cell in-line with the LC/MS system. The resulting EC-CID or EC-ETD spectra contain much more information about the different ions thereby increasing the sequence coverage up to a 3 fold.

*) US Patent application 13/608,907

 

 

 

Schematics Instrument set-up:
EC/MS system consisting of infusion pump, electrochemical cell and MS (A), and LC/EC/MS system consisting of (U)HPLC, electrochemical cell and MS (B). In both set-ups the µ-PrepCell (red circle) is used for electrochemical reduction of the bonds, controlled by ROXY.

 

 

 

 

Location / Assessment of Disulfide Bonds
Partial reduction enable the location of disulfide bonds within proteins and peptides as well as studies on their accessibility. 

 

CID Spectra of Somatostatin:
3 µmol/L somatostatin in 1% formic acid and 10% ACN, infusion EC/MS. CID of native (A) and reduced (B) somatostatin. In the native spectra (A), only one abundant fragment ion is detected, meanwhile in the reduced spectra (B), almost full series of y- and b-type ions were detected enabling unequivocal peptide identification.
Ref: A. Kraj et al., Analytical and Bioanalytical Chemistry, Sept. 2013, for PDF,

 

 

 

Increasing Sequence Coverage
Complete reduction is beneficial to increase the sequence coverage in CID and ETD. The resulting EC-CID or EC-ETD spectra contain many additional fragment ions thereby increasing the sequence coverage up to a 3 fold.

 

ETD Spectra of Oxytocin
Doubly protonated native (A) and on-line reduced (B) oxytocin. The on-line EC reduction of the disulfide bridge results in a more comprehensive characterization of the tocin-ring. The number of sequence informative fragment ions doubled from 8 to 16 and the total number of (assigned) fragment ions was significant higher for the reduced form.
Ref: S. Nicolardi et al., JASMS, Sept. 2013, for PDF

 

 

 

 

 

 

 

 

 

 



Application Notes

  • 210_006_02 - Reduction of Disulfide Bonds in Proteins
  • 210_010_01 - Monitoring the Electrochemical Reduction of Disulphide Bonds
  • 210_009_01 - Electrochemical Reduction of Biopharmaceuticals for HDXMS
  • 210_002_02 - Automated Screening on REDOX Reactions

Application Notebook


24077854 - A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection.
Kraj A, Brouwer HJ, Reinhoud N, Chervet JP.; Anal Bioanal Chem. 2013 Sep 29. [Epub ahead of print]

24018861 - On-Line Electrochemical Reduction of Disulfide Bonds: Improved FTICR-CID and -ETD Coverage of Oxytocin and Hepcidin.
Nicolardi S, Giera M, Kooijman P, Kraj A, Chervet JP, Deelder AM, van der Burgt YE.; J Am Soc Mass Spectrom. 2013 Sep 10. [Epub ahead of print]

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