Massive parallel analysis of protein expression is a vibrant area of modern biomedical research. Comparisons between different proteomic systems can provide helpful information on prospective drug targets and on individual disease variations. This is not an easy task, however. Subtle differences in expression levels between identical species have to be probed. A sensitive detection method is necessary, along with a means of differentiating between counterpart proteins from distinct sources.

Researchers from the University of Maryland, led by Catherine Fenselau, have introduced an approach involving high-resolution mass spectrometry (MS) and selective tagging of tryptic protein fragments with ¹⁸O, for quantitative comparative proteomics. They demonstrated the approach by using a model system of the virion proteins from two adenoviral strains with closely related serotypes (Anal. Chem. 2001, 73, 2836– 2842)

Stable isotopic labeling was achieved during the proteolytic cleavage of one pool of proteins dissolved in “heavy” water by enzymatic incorporation of two ¹⁸O atoms into the carboxyl termini of the resulting peptides. At the same time, proteins from the second pool were cleaved analogously in normal water.

After proteolysis, the peptide mixtures were analyzed by MS, and the relative signal intensities of each corresponding peptide pair (differing by 4 Da) were determined. Accurate mass measurements allowed the peptides to be related to the precursor proteins from which they were derived, while relative MS signal intensities of paired peptides quantified the expression levels from each pool. The MS-determined ratios were in good agreement with the UV-measured concentrations of the tryptic peptides. This result suggests that MS characterization of ¹⁸O-labeled peptide fragments is a useful and reliable tool for quantitative proteomic studies. Several unpaired sets of isotopic peaks were observed in the mass spectra. These could be interpreted as differential posttranslational modification or mutation in proteins from counterpart proteome pools. Such cases are particularly important for the identification of new drug targets.

The Maryland scientists think that proteolytic ¹⁸O labeling will be especially helpful for studying proteins that are often problematic for traditional approaches, such as very small or very large proteins, or hydrophobic or basic ones. Only further experiments will tell if these hopes can be realized.