Abstract
This chapter presents the sequence determinations of proteins and peptides at the nanomole and subnanomole level with a modified spinning cup sequenator. The chapter describes the improvements and gives practical criteria that critically assess the instrument performance. The modifications described is performed on a Beckman 890C sequenator but in principle can be applied to other instruments by allowing for changes from one model to another. The components that should be removed from the sequenator are (a) entire low-vacuum system including all lines, manifolds, and connections, (b) high-vacuum line and valve, (c) reagent, solvent, and waste diaphragm valves , (d) three-way valves and manifold that operates diaphragm valves, and (e) reagents and solvents and their connections except for vent and pressurized lines. The replacement components that follow are described by systems and require machine and glass shop fabrication work. The chemistry and operational description of an automated sequencer are discussed. One of the applications of the instrument described is the sequence determination of human leukocyte interferon, which is currently available in the nanomole range. Representative results from these studies are discussed. An important aspect of the sequencing strategy in the low nanomole to subnanomole range is the analysis of the PTH amino acids by reverse phase HPLC. These chromatograms illustrate the low backgrounds and the resulting high sensitivity, which can be reproducibly obtained with the modified sequenator.
Published Version
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