Our results demonstrated that high-pressure assisted in-gel tryptic digestion compares with the standard in-gel tryptic digestion with respect to number of peaks detected, number of peptides identified, and sequence coverage. In all three parameters, PCT digestion performed at least as well, and sometimes better, than the standard digestion approach. For example, PCT provided sequence coverage ranging from 49percent to 66percent as compared to 38percent to 59percent obtained with standard digestion. This approach also reduced the extended time (about 16 hrs) required by the standard in-gel digestion to 45 minutes. The quantitation aspect of our study revealed good spot-to-spot and lane-to-lane reproducibility, with relative errors less than +-10 and 15percent, respectively. We have shown that this high pressure assisted, label-free approach can be used for the rapid, relative quantitation of proteins after 1DE separation. We have also demonstrated that this method can be used to determine the relative quantities of co-migrating proteins using influenza virus preparations. We are currently evaluating the applicability of this approach for absolute quantitation of proteins after 1DE separation.
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