Sequential Ion/Ion Reactions for Large Peptide and Whole Protein Characterization

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DESCRIPTION (provided by applicant): The ability to sequence and identify proteins, map their sites of post-translational modification (PTM), and assess their abundances is central to modern biology. Mass spectrometry (MS) is the gold standard technology by which this information is obtained. Serving as the centerpiece, tandem MS (MS/MS) is a principal component. Electron transfer dissociation (ETD), a relatively new MS/MS dissociation method, has generated significant excitement for its compatibility with previously intractable peptide/protein classes. Five years ago m/z range, mass accuracy, and mass resolution considerably restricted the application of ETD. Our initial RO1 proposal successfully eliminated this limitation by coupling ETD to the orbitrap mass analyzer. The resulting system routinely analyzes peptides and proteins, with and without labile PTMs, with a high-fidelity readout (orbitrap). As a result, it realized many of our anticipated outcomes and created numerous unforeseen opportunities. Just in the PI's laboratory, the latter set includes data-dependent selection of dissociation method (i.e., Decision Tree), discovery of the unique chemical compositions of z-type ions, internal spectral calibration using ETD reagents, activated-ion ETD, and several biological applications. By 2008, the commercial implementation of our technology began to reach researchers across the globe-nearly 300 to date-enabling access to numerous previously intractable problems such as mapping Arg methylation sites, increasing coverage of low molecular weight proteins, providing unambiguous PTM site assignment, and screening glycopeptide libraries, among many others. We detail two new aims that build upon the high impact results of our initial funding period. Aim 1, how do we broaden the utility of ETD for biomedical research? Aim 2, what is the role of gas- phase purification in quantitative proteomics? We continue with a balance of instrumentation, method, informatic, and applied projects constructed upon the widely used ETD-orbitrap platform we described 3.5 years ago.