ABSTRACT
The use of isotopically labeled endonuclease digestion products allows for the relative quantification of ribonucleic acids (RNAs). This approach utilizes ribonucleases such as RNase T1 to mediate the incorporation of 18O onto the 3'-terminus of the endonuclease digestion product from a solution containing heavy water (H2 18O). The accuracy and precision of relative quantification are dependent on the efficiency of isotope incorporation and minimizing any possible 18O to 16O back-exchange before or during mass spectral analysis. Here, we have investigated the stability of 18O-labeled endonuclease digestion products to back-exchange. In particular, the effects of pH, temperature and presence of RNase on the back-exchange process were examined using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We have found that back-exchange depends on the presence of the RNase--back-exchange was not observed once the enzyme was removed from the sample. With RNase present, at all pH values examined (from acidic to basic pH), back-exchange was detected at incubation above room temperature. The rates and extent of back-exchange were similar at all pH values. In contrast, back-exchange in the presence of RNase was found to be especially sensitive to incubation temperature--at temperatures below room temperature, minimal back-exchange was detected. However, back-exchange increased as the incubation temperature increased. Based on these findings, appropriate sample-handling and sample storage conditions for isotopically labeled endonuclease digestion products have been identified, and these conditions should improve the accuracy and precision of results from the relative quantification of RNAs obtained by this approach.
