This project is focused on developing improved instrumentation and protocols for identification and structural elucidation of large peptides and intact proteins. The proposed instrument is optimized for application to doubly charged positive ions and provides unique capabilities for MS-MS of intact proteins ionized by MALDI. Fragmentation mechanisms available include unimolecular fragmentation following excitation in the MALDI ion source (PSD), and electron transfer dissociation (ETD) from reactions of small negative ions with doubly charged intact proteins. The approach is based on recent advances in MALDI-TOF and TOF-TOF technology but requires addition of a second pulsed ion source and a new TOF analyzer for fragment ions that features up to 45 kV acceleration to achieve high performance on high mass, high energy ions. The ion optics are designed so that beams from the two sources can be merged within the collision region with the pulses accurately synchronized in time, and with the relative velocity of the beams accurately determined. For collisions between positive and negative ions the relative velocity can be set very close to zero, providing sufficient time for efficient ion chemistry to occur. A novel element in this instrument is an additional pulsed accelerator in the field-free space adjacent to the ion source that effectively reduces the velocity spread of selected ions and allows high resolution precursor selection simultaneously with high resolution measurements of fragment spectra. The first product resulting from this work is a new TOF-TOF that produces high-quality MS-MS spectra on intact proteins. This instrument generates very high quality MS-MS spectra at unprecedented speed and with very high sensitivity. In combination with a new LC interface with MALDI being developed in a separate project, an integrated LC-MS-MS system incorporating the new TOF-TOF will provide throughput more than an order of magnitude higher than any existing LC-MS-MS system, whether electrospray or MALDI, and will confidently identify many more proteins at low concentrations in complex biological samples. Because this instrument is designed for analysis of intact proteins, it will be particularly valuable for monitoring post-translational modifications that are frequently missed using bottom-up approaches for protein analysis.
Functional proteomics requires characterization of proteins at the phenotypic level rather than merely gene level identifications. It is necessary to characterize the entire peptide sequence of a protein and post-translational modifications must be fully detected. The MALDI-TOF-TOF with ETD developed in this project provides accurate molecular weight and complete, unambiguous sequence, including unusual amino acids and post-translational modifications, on proteins and large peptides present at trace levels in complex mixtures. Speed, sensitivity, and dynamic range are substantially superior to the performance presently possible with other technologies.