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 characteriz ation of proteins separated by two-dimensional polyacrylamide gel electroph oresis (2D-PAGE). Proteins were digested in a gel in a buffer solution cont aining 50% O-18-labeled water, and mixtures of O-18/O-16-labeled peptides w ere analyzed by nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). This method was evaluated using horse skelet al muscle myoglobin as the model protein in SDS gel. The high resolution of FT-ICR MS minimized the overlapping of peptide peaks and facilitated ident ification of the C-terminal peptide, which was done by observing the undisr upted isotope peak pattern. As well, with its low ppm-level high mass accur acy, it can rapidly and reliably identify the in-gel-separated protein and determine its C-terminal by peptide mass fingerprinting alone. Therefore, t his method should be applicable to routine and high-throughput proteome stu dies. Here, the method was applied to the analysis of rat liver proteins se parated by ED-PAGE. The C-termini of eight proteins were successfully ident ified out of 10 randomly picked Coomassie brilliant blue-stained spots. The feasibility and limitations of this approach are reported in this paper.