The combination of large-scale protein separation techniques, sophisticated mass spectrometry, and systems bioinformatics has led to the establishment of proteomics as a distinct discipline within the wider field of protein biochemistry. Both discovery proteomics and targeted proteomics are widely used in biological and biomedical research, whereby the analytical approaches can be broadly divided into proteoform-centric top-down proteomics versus peptide-centric bottom-up proteomics. This chapter outlines the scientific value of top-down proteomics and describes how fluorescence two-dimensional difference gel electrophoresis can be combined with the systematic analysis of crucial post-translational modifications. The concept of on-membrane digestion following the electrophoretic transfer of proteins and the usefulness of comparative two-dimensional immunoblotting are discussed.
Keywords: Comparative proteomics; DIGE; Difference gel electrophoresis; Mass spectrometry; Top-down proteomics; Two-dimensional gel electrophoresis.
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