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LC/MS/MS Analysis of N-Terminal Protein Adducts with Improved Sensitivity: A Comparison of Selected Edman Isothiocyanate Reagents

Authors
Publisher
International Journal of Analytical Chemistry
Publication Date
Disciplines
  • Biology

Abstract

This study provides a basis for a new and straightforward method for LC/MS/MS-based screening of N-terminal protein adducts. This procedure is denoted the “FI 𝑅 E procedure” as fluorescein 𝑖 sothiocyanate (FITC) gave superior sensitivity by LC/MS/MS when measuring adducts ( 𝑅 ) of electrophilic compounds with a modified Edman procedure. The principles of the FI 𝑅 E- p r o c e d u r e are that adducts to N-terminal amino acids selectively are detached and measured from of proteins after derivatisation by isothiocyanate Edman reagents. In this study, FITC, 4-N,N-dimethylaminoazobenzene 4  -isothiocyanate (DABITC) and 4-dimethylamino-1-naphthyl isothiocyanate (DNITC) were used to synthesize thiohydantoin analytes from valine and N-methylvaline. The sensitivity by LC/MS/MS was enhanced by up to three orders of magnitude as compared to phenyl isothiocyanate and higher as compared to pentafluorophenyl isothiocyanate. The FITC reagent will enable measurements of low background adduct levels. Synthesized analytes were characterised with, for example, 1 H NMR, 1 3 C NMR, LC/MS/MS, and UV.

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