Precolumn Isotope Dilution Analysis in nanoHPLC-ICPMS for Absolute Quantification of Sulfur-Containing Peptides

摘要

A novel generic approach based on precolumn isotopedilution nanoHPLC-ICPMS analysis was developed for the accurate absolute quantification of sulfur-containing peptides. A ~(34)S-labeled, species-unspecific sulfur spike (sulfate), noninteracting with analyte peptides under the optimized HPLC condition, was added directly to the chromatographic eluents. Thus a generic sulfur standard permanently present during analysis was used for peptide quantification. Interference-free detection of the ~(32)S and ~(34)S isotopes in ICPMS was achieved by eliminating O_(2~(+)) ions in a collision cell using Xe gas at 130 (mu)L min~(-1). The detection limit for sulfur was 45 (mu)g L~(-1) which corresponded to 1-2 pmol of individual peptides. The method was validated by the analysis of a standard peptide solution showing high accuracy (recovery 103percent) and good precision (RSD 2.1percent). The combination of nanoHPLC-ICP IDMS with nanoHPLC-ESI MS/MS allowed the precise quantification and identification of sulfur-containing peptides in tryptic digests of human serum albumin and salt-induced yeast protein (SIP18) at the picomole level.