Purification and Preparation of Chemicals for Microsequencing With the Edman Degradation

Abstract

When the first automated protein sequencer based on the Edman degradation was described in 1967 (1), relatively large amounts (>100 nmol) of proteins were required for sequence analysis. Recent improvements in sequencer design and the introduction of high-performance liquid chromatography (HPLC) analysis of phenylthiohydantoin (PTH) amino acids allow the Edman chemistry to be performed with as little as a few picomoles of proteins or peptides. However, successful microsequencing is possible only if the chemicals used in the instruments are free of contaminants that interfere with the Edman chemistry or with the subsequent PTH analysis. With a few exceptions, the following methods for obtaining chemicals of the required degree of purification for microsequencing using the spinning-cup (2,3) or gas-phase (4,5) sequencers, as appropriate, were employed successfully at the California Institute of Technology for several years. In the case of those chemicals used in the gas-phase sequencer, chemicals prepared by these or even more stringent methods are available commercially from Applied Biosystems.