Interfacing reversed-phase nanoHPLC with ICP-MS and on-line isotope dilution analysis for the accurate quantification of selenium-containing peptides in protein tryptic digests

摘要

An interface between nanoHPLC and ICP-MS was developed. It allowed the stable introduction into an ICP of mobile phases containing up to 90% of acetonitrile at flow rates of less than 500 nL min(-1). The on-line post-column addition of an isotopically enriched spike at flow rates of less than 4 mL min(-1) enabled isotope dilution quanti. cation of heteroatom containing analytes while the consumption of the labelled isotope was low. The coupled system was applied to the accurate, sensitive and specific determination of selenopeptides in nanolitre volumes ( 11 nL) of a tryptic digest of selenomethionyl calmodulin ( 17 002 Da). The peptides were separated by reversed phase nanoLC (340 nL min(-1) flow rate) whereas ICP collision cell MS was used for the simultaneous detection of 80 Se (analyte) and Se-76 (spike). The absolute detection limit was 40 fg ( 80 Se), a factor of 2 less than ever reported for a capillary HPLC-ICP-MS coupling. The sensitivity was constant during the chromatogram, regardless of the percentage of acetonitrile in the mobile phase. The selenium recovery was 103 +/- 4%. For selenopeptide analysis the sum of Se determined in each of the peaks equalled the total Se injected on the column. Since the tryptic peptides, miscleaved and/or oxidized peptides, incompletely digested protein and undigested protein could be determined in one run, the method allowed the precise evaluation of the efficiency and quality of tryptic digestion using several nanolitres of sample only.