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

[摘要] 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 “FIRE procedure” as fluoresceinisothiocyanate (FITC) gave superior sensitivity by LC/MS/MS when measuring adducts (R) of electrophiliccompounds with a modifiedEdman procedure. The principles of the FIRE-procedureare 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-dimethylaminoazobenzene4′-isothiocyanate(DABITC) and 4-dimethylamino-1-naphthyl isothiocyanate (DNITC) were used to synthesizethiohydantoin analytes from valine and N-methylvaline. The sensitivity by LC/MS/MS was enhancedby up to three orders of magnitude as compared to phenyl isothiocyanate and higher as compared topentafluorophenyl isothiocyanate. The FITC reagent will enable measurements of low backgroundadduct levels. Synthesized analytes were characterised with, for example,H1NMR,C13NMR, LC/MS/MS, and UV.

[学科分类] 分析化学