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Novel techniques for identification and characterization of proteins loaded on gels in femtomole amounts
K Gevaert1, H De Mol, J L Verschelde
1Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Belgium. Krgev@gengenp.rug.ac.be
Abstract:
A combination of techniques is presented allowing gel-purified protein identification in the femtomole range using matrix-assisted-laser-desorption-ionization mass spectrometry. The proteins are detected in the primary gel by a sensitive negative staining procedure, transferred, and concentrated in a secondary gel matrix. There, they are digested in the presence of H218O and their sequences are predicted (1) by peptide mass fingerprinting, (2) by comparing the post-source-decay (PSD) spectra with theoretical spectra of candidate isobaric peptides using a computer algorithm called MassFrag, and (3) by a manual readout of the 18O/16O-labeled fragmentation ions in the PSD spectra.