Femtomole peptide mapping by derivatization, high-performance liquid chromatography, and fluorescence detection

A highly sensitive peptide mapping method using derivatization and fluoresc ence detection is described. Bovine cytochrome c was digested using a buffe r compatible with the derivatization that followed. The derivatization was performed with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The peptide mapping of the tagged digest was conducted with both HPLC and capillary LC (CLC) systems. A capillary LC-electrospray ionization mass spectrometer (M S) was set up for measuring the molecular weights of the tagged peptides. O ptimization was made of the conditions used for digestion, derivatization, and mapping. MS measurements of the tagged peptides suggested that there wa s only one derivatization product produced from all peptides (except one) a nd that all the identified peptides were fully tagged. Peptide mapping of t he tagged digest reviews a larger number of peptides, covering almost the e ntire sequence, Peptide mapping of a 20 fmol amount of tagged digest was re adily performed with the CLC system. By using derivatization and fluorescen ce detection, the sensitivity of peptide mapping could be improved 2000 tim es compared to that observed with uv detection of untagged peptides. (C) 20 01 Academic Press.