Top-Down Proteomics with Intact Proteins
Characterization of Intact Proteins
Bottom-up proteomics (analyzing peptides that result from protein digestion) has demonstrated the capability for broad proteome coverage and good throughput, but is not ideally suited to the discovery and identification of modified proteins. Top-down proteomics (including subjecting intact protein ions to gas-phase dissociation) allows the study of modified proteins, but coverage, sensitivity, and throughput are still problematic.
Our approach is based on the combination of bottom-up with intact protein analyses to characterize modified proteins. Fractionation at the intact protein level is typically employed to reduce complexity and increase measurement dynamic range. Bottom-up measurements are used to identify the subset of proteins present in each fraction. These identifications are then used in combination with high-accuracy intact protein mass measurements (employing FTICR) to achieve protein and modified-protein identifications. Unidentified species are characterized using a conventional top-down analysis tool, electron capture dissociation (ECD). However, our approach dramatically reduced the number of candidates for ECD, and thus enables more efficient use of this technique, which has been proven not to be ideally suited to on-line combination with separations.
This capability can easily be combined with relative (e.g., 14N/15N stable isotope labeling) and absolute abundance measurements to discover differences in protein abundances between different cell states. Furthermore, species of interest can be characterized using FTICR-targeted MS/MS capability developed recently at PNNL.