High Resolution Separations for Intact Proteins (pp. 419-480)

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The routine analysis of intact proteins in basic research and clinical settings requires new separations platforms to reduce both the complexity of mixtures as well as the sample amount required. To satisfy these requirements, individual separations strategies must be optimized to reduce sample loss and increase resolution. In TR&D 1, we combine chromatographic and gel-based separation approaches to radically improve the capability of top-down proteomics. We place the new capabilities described in this TR&D at the epicenter of many of the driving biomedical projects within the National Resource for Translational and Developmental Proteomics. In two Specific Aims, we present new chromatographic methods for intact protein separations and a novel online gel-based separation for top-down proteomics called “GelSpray”. Specific Aim 1a is purposefully constructed to be available within the first year of the resource, and therefore represents an incremental advance lacking the level of innovation offered in Specific Aims 1b and 2. The development of new separations platforms that are intelligently interfaced with new and improved mass spectrometric instrumentation is required by diverse DBPs to increase the depth of characterization of which topdown proteomics is capable. By more accurately separating complex mixtures prior to mass analysis, new and more lucid insights into basic biology and translational research will be returned to collaborators. Further, improvements in throughput and reductions in sample loss will make possible the scale of study required to extract meaningful results in clinical applications where sample size is small, higher numbers of technical replicates increase depth and confidence of quantitative results (c.f. TR&D 3), and sample numbers are large. In sum, these efforts are required by all of the proposed DBPs that demand high fidelity readouts of the protein content of a sample as well as quantitative information regarding protein abundance.