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.
Kosaka
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Biomedical Research Laboratories, Sankyo Company, Ltd., Tokyo, Japan. kosaka@shina.sankyo.co.jp
Takazawa
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Nakamura
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D000595:Amino Acid Sequence; D000818:Animals; D017357:Cyclotrons; D015180:Electrophoresis, Gel, Two-Dimensional; D005583:Fourier Analysis; D006736:Horses; D007554:Isotopes; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry*; D051381:Rats
D007554:Isotopes; D011506:Proteins