18O Labeling:: a tool for proteomics

An evaluation of the proteolytic labeling and quantification of proteins for diagnostic purposes using trypsin and O-18-enriched H2O is presented. We demonstrate that comparative or relative quantitation can be performed effectively with this approach. We have developed a protocol that allows the conservation of the labeled peptides in natural abundance water without fear of back-exchange providing that pH is sufficiently low to quench the catalytic activity of trypsin, but not so low as to promote chemical back-exchange. Because the labeling efficiency depends on the nature of the peptide, a simple linear relationship between the relative O-16/O-18 digest buffer mixture content (x) and labeling efficiency (y) does not exist; rather it follows a probability based y = x(2) relationship. As such, the extent of peptide labeling using O-16/O-18 digest buffer mixture ratios may deviate significantly from that expected based on a linear relationship. The evaluation of the relative Ziptip efficiency indicated a loss in sample recovery as the peptide concentration was reduced using normal conditions, suggesting that there is a limit below which there are diminishing returns. In addition, the adsorptive losses due to Speedvac dry down and recovery indicated modest (20%) losses that may vary widely (0-50%) from peptide to peptide. The in-solution digestion efficiency of standard protein mixtures as a function of concentration revealed a linear decrease with decreasing concentration. This is consistent with enzyme kinetic effects and emphasizes a potential quantitation error that could arise when evaluating differential expression based on peptide detection. The results from our studies demonstrate the power of O-18 labeling as an optimization tool for proteomics process development. Copyright (C) 2001 John Wiley & Sons, Ltd.