Accelerated 18O-labeling in urinary proteomics

Proteolytic 18O-labeling of peptides has been studied and optimized in order to improve the labeling efficiency and to accelerate the process without increasing the degree of incomplete labeling. Using peptides generated from tryptic digested bovine serum albumin (BSA) and cytochrome c as model proteins, it was shown that complete labeling was achieved after 2 h at pH 6. To increase the sample throughput in a bottom-up proteomic setup, tryptic digestion of proteins in-solution was replaced with tryptic digestion using immobilized trypsin. As a result, an integrated approach was made possible, where both digestion (pH 8) and 18O/16O-labeling of the resulting peptides (pH 6) were done using immobilized trypsin beads. This simplified the sample handling and reduced the overall reaction time significantly: the setup enabled tryptic digestion and 18O/16O-labeling without sample transfer steps within 3.5 h with average 18O/16O-ratios of 0.96 ± 0.13 in aqueous buffer. The initial results were confirmed with a more complex matrix, by spiking urine with the model proteins, yielding results comparable with the ratios obtained in buffer. Satisfying ratios were also achieved regarding urinary proteins identified in a full scale bottom-up experiment. Average 18O/16O-peptide ratios of 0.83 ± 0.13 and 0.91 ± 0.27 indicated good performance in a highly relevant matrix for biomarker discovery.