De novo sequencing and disulfide mapping of a bromotryptophan-containing conotoxin by fourier transform ion cyclotron resonance mass spectrometry

Sudarslal Sadasivan Nair, Carol L. Nilsson, Mark Emmett, Tanner M. Schaub, Konkallu Hanumae Gowd, Suman S. Thakur, K. S. Krishnan, Padmanabhan Balaram, Alan G. Marshall

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

T-1-family conotoxins belong to the T-superfamily and are composed of 10-17 amino acids. They share a common cysteine framework and disulfide connectivity and exhibit unusual posttranslational modifications, such as tryptophan bromination, glutamic acid carboxylation, and threonine glycosylation. We have isolated and characterized a novel peptide, Mo1274, containing 11 amino acids, that shows the same cysteine pattern, -CC-CC, and disulfide linkage as those of the T-1-family members. The complete sequence, GNWCCSARVCC, in which W denotes bromotryptophan, was derived from MS-based de novo sequencing. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), infrared multiphoton dissociation, and collision-induced dissociation served to detect and localize the tryptophan bromination. The bromine contributes a distinctive isotopic distribution in all fragments that contain bromotryptophan. ECD fragmentation results in the loss of bromine and return to the normal isotopic distribution. Disulfide connectivity of Mo1274, between cysteine pairs 1-3 and 2-4, was determined by mass spectrometry in combination with chemical derivatization employing tris(2-carboxyethyl)-phosphine, followed by differential alkylation with N-ethylmaleimide and iodoacetamide. The ECD spectra of the native and partially modified peptide reveal a loss of bromine in a process that requires the presence of a disulfide bond.

Original languageEnglish (US)
Pages (from-to)8082-8088
Number of pages7
JournalAnalytical Chemistry
Volume78
Issue number23
DOIs
StatePublished - Dec 1 2006
Externally publishedYes

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Conotoxins
Cyclotron resonance
Disulfides
Mass spectrometry
Fourier transforms
Bromine
Ions
Cysteine
Tryptophan
Electrons
Carboxylation
Glycosylation
Iodoacetamide
Amino Acids
Peptides
Ethylmaleimide
Alkylation
Normal distribution
Threonine
Glutamic Acid

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

De novo sequencing and disulfide mapping of a bromotryptophan-containing conotoxin by fourier transform ion cyclotron resonance mass spectrometry. / Nair, Sudarslal Sadasivan; Nilsson, Carol L.; Emmett, Mark; Schaub, Tanner M.; Gowd, Konkallu Hanumae; Thakur, Suman S.; Krishnan, K. S.; Balaram, Padmanabhan; Marshall, Alan G.

In: Analytical Chemistry, Vol. 78, No. 23, 01.12.2006, p. 8082-8088.

Research output: Contribution to journalArticle

Nair, SS, Nilsson, CL, Emmett, M, Schaub, TM, Gowd, KH, Thakur, SS, Krishnan, KS, Balaram, P & Marshall, AG 2006, 'De novo sequencing and disulfide mapping of a bromotryptophan-containing conotoxin by fourier transform ion cyclotron resonance mass spectrometry', Analytical Chemistry, vol. 78, no. 23, pp. 8082-8088. https://doi.org/10.1021/ac0607764
Nair, Sudarslal Sadasivan ; Nilsson, Carol L. ; Emmett, Mark ; Schaub, Tanner M. ; Gowd, Konkallu Hanumae ; Thakur, Suman S. ; Krishnan, K. S. ; Balaram, Padmanabhan ; Marshall, Alan G. / De novo sequencing and disulfide mapping of a bromotryptophan-containing conotoxin by fourier transform ion cyclotron resonance mass spectrometry. In: Analytical Chemistry. 2006 ; Vol. 78, No. 23. pp. 8082-8088.
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