A simplified method for peptide de novo sequencing using (18)O labeling.

Incorporation of an O-18 atom into a peptide C-terminus by proteolytic cleavage in the presence of (H2O)-O-18 is one of the most effective ways of enhancing tandem mass spectrometry (MS/MS)-based de novo sequencing. Incorporation is usually accomplished by procedures including vacuum-assisted drying of tryptic peptides extracted from gels, their subsequent reconstitution in a (H2O)-O-16/(H2O)-O-18 mixture and re-treatment with trypsin. In the present work, we propose a simplified procedure for O-18 incorporation into tryptic peptides by adding (H2O)-O-18 and trypsin to the original digest solution. In comparison to published methods, the proposed protocol for peptide de novo sequencing brings significant advantages in analysis and workflow with no deterioration in method performance. We show that labeling by this simplified method leads to a highlighting of the y-ion fragment series in the peptide matrix-assisted laser desorption/ionization (MALDI)-MS/MS data, which facilitates MS/MS data interpretation. We also prove that eliminating acid extraction of peptides from gels does not result in a decrease in sequence coverage or a qualitative loss of particular peptides detectable by MALDI-MS. The method was examined by MALDI-MS/MS on bovine serum albumin and recombinant histidine kinase CKI1 from Arabidopsis thaliana, and was verified by de novo sequencing of tryptic peptides originating from Apodemus sylvaticus salivary proteins.