Identification and C-Terminal Characterization of Proteins from Two-Dimensional Polyacrylamide Gels by a Combination of Isotopic Labeling and Nanoelectrospray Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

We propose a novel method for the identification and C-terminal characterization of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins were digested in a gel in a buffer solution containing 50% 18O-labeled water, and mixtures of 18O/16O-labeled peptides were analyzed by nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). This method was evaluated using horse skeletal muscle myoglobin as the model protein in SDS gel. The high resolution of FT-ICR MS minimized the overlapping of peptide peaks and facilitated identification of the C-terminal peptide, which was done by observing the undisrupted isotope peak pattern. As well, with its low ppm-level high mass accuracy, it can rapidly and reliably identify the in-gel-separated protein and determine its C-terminal by peptide mass fingerprinting alone. Therefore, this method should be applicable to routine and high-throughput proteome studies. Here, the method was applied to the analysis of rat liver proteins separated by 2D-PAGE. The C-termini of eight proteins were successfully identified out of 10 randomly picked Coomassie brilliant blue-stained spots. The feasibility and limitations of this approach are reported in this paper.