Project Summary Proposed here is research to develop a collection of technologies that will make it possible to read the complete amino acid sequence of proteins (forwards and backwards) on a chromatographic time scale at the femtomole level and to also characterize multiple posttranslational modifications that exist on the same protein molecule and together regulate its biological activity. This research is driven by four major innovations from my lab. These include development of (a) electron transfer dissociation (ETD) for fragmentation of peptides and intact proteins, (b) combined ETD and IIPT (ion-ion proton transfer) chemistry to obtain n- and c-terminal sequence information that identifies proteins, (c) front end ETD (FETD) that facilitates a 10-50 fold increase in sensitivity for intact proteins, and (d) a micro column enzyme reactor that is fully active in 8 M urea and can be employed to generate 3-10 KDa protein fragments from large proteins. Going forward, we will implement and optimize ETD and IIPT in combination with parallel ion parking (PIP). This will include the introduction of new ETD reagents and new wave forms to enhance the efficiency of parallel ion parking and new IIPT reagents to extend the usable mass range to m/z 4,000. Also proposed is research to incorporate the powerful micro-column enzyme reactor into a rotation-driven microfluidic disc system where the protease bed length, solvent flow in hundreds of nL/min, and digestion times on the order of hundreds of milliseconds can all be controlled and measured with high accuracy. By using the rotation-driven microfluidic disc system, we expect to increase sample throughput, lower sample quantities, and increase reproducibility of individual sample analyses. All of this will be applied to fully characterize (a) bispecific antibodies, (b) proteins in outer membrane vesicles secreted by the antibiotic resistant, gram negative bacteria, Neisseria gonorrhoeae, (c) plant proteins that are involved in growth or growth inhibition and are targets for O-GlcNAcylation and O-fucosylation, respectively, (d) posttranslational modifications that exist on histone chaperones during the cell cycle, and (e) posttranslational modifications that regulate methylation of Arg residues on histones H3 and H4.