Generic approach for absolute quantitation of large undigested peptides in plasma using a particular 2D-LC-MS/MS set-up

摘要

A generic approach for the absolute quantification of large undigested peptides in plasma at picomolar levels is described. Five peptides were studied: BNP (brain natriuretic peptide), Substance P, PTH (parathyroid hormone 1-34), C-peptide and alpha-MSH (melatonin stimulating hormone). Plasma samples were extracted by a 2 step procedure: protein precipitation using one volume of acetonitrile followed by ultra-filtration of supernatants on a MW cut-off of 30 KDa membrane. Large volumes (e.g. 1mL) of plasma extracts were injected on a particular LC set-up. The peptides were trapped on a 1X10 mm C8 column using a 10x on-line dilution. Then, the peptides were back-flushed and a second on-line dilution (2x) was applied during the transfer step. The refocalized peptides were resolved on a 0.3 mm i.d. C18 analytical column. This comprehensive protocol could be applied for sensitive quantification of most endogeneous or therapeutic peptides.