Proteins are molecules, consisting of a large number of amino acids. The order in which the amino acids occur is called "sequence". Analysis of protein sequence is an integral part of basic health and life sciences. Availability of this information is a first step towards understanding protein function at the molecular level; it also serves as a protein fingerprint, allowing identification. An important role of the protein chemist has been to provide partial amino acid sequences to facilitate cloning of the corresponding geles. However, the focus of protein sequence analysis may shift to more function oriented questions, such as formation of complexes in the cell during signalling, cell cycle and differentiation. As many of those proteins are only available in minute quantities, it is imperative that analytical studies be carried out at the highest attainable levels of sensitivity. Traditionally, sequence analysis is done by stepwise removal of amino acids from the N-teminal end of the polypeptide, one residue at a time. The leaving residue is then converted to a more stable PTH-amino acid for identification by an analytical technique, usually HPLC. The entire process has been automated. Optimized commercial "sequencers" require 2-5 picomoles starting amount of peptide for analysis, almost one order of magnitude more than what can be isolated. Improvements will therefore be required. As existing sequencers have already been optimized to the limit, it is difficult to picture how this could be done without extensive modifications, i.e. miniaturization. Commercial instruments are not easily modified as they are 'boxed up' and inflexible, both in terms of hardware and software. A novel instrument must therefore be built. Our short-term objective is the development of an automated peptide sequencer for the "mid femtomole" range. We propose to do this by miniaturization of reaction vessels and analytical techniques, all in a very proximate arrangement. We foresee that the construction of a flexible, modularity built "micro-microsequencer" will also serve as a platform for long-term improvements / modifications to hardware and chemistry, leading to another 5-fold increase in sensitivity (20-100 femtomoles). One (two) order(s) of magnitude gained in polypeptide sequencing sensitivity will allow identification of proteins hitherto known only as 'activities' or 'spots on a 2DE-gel', and move the health and life sciences into new areas of inquiry. Specifically, we propose to construct an automated chemical sequencer, containing a miniaturized reaction cartridge, conversion flask and reagent/solvent flow path. Cyclically removed amino acids will be identified, initially, by microbore RP-HPLC and, later, by capillary LC. We propose to develop and/or optimize PTHamino acid separations on micro/capillary columns and couple them to the sequencer in close proximity and automated fashion. UV-detection will be used first and serve, in the medium-turn, as a bridge to even better sensitivities by utilizing amino acid derivatives of higher detectability. We propose therefore, to implement fluorescent-amino acid generating chemistries and accomplish micro/capillary-column chromatographic separation. The proposed instrument will also provide a unique tool for micro-delivery of reaction products and degradation products, from either cartridge or flask to non-chromatographic analytical instruments with sample volume restrictions (e.g. mass spectrometers, CZE,...). In turn, analytical versatility will create opportunities for further sequencing improvements, both in terms of sensitivity and speed. The significance of achieving the objectives will be our ability to analyze low abundant proteins, isolated with the most powerful fractionation techniques. Applications are ongoing in the areas of i) vesicle targetting and membrane fusion, and understanding the controlling machinery of synaptic transmission, and ii) interactions of negative cell cycle regulator s and their role as antiproliferative devices that, when disrupted, contribute to cancer. Technology transfer will be effected through publications, postdocs, graduate students, visitor trainees, and through the use in sequencing core facilities.
