Comparison of the Spinning Cup and Gas-Phase Instruments
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The first automated Edman sequencer was described in 1967 (1). When the Beckman instrument became commercially available in 1969, the automated sequencer became the workhorse of protein chemists. The nominal operating range of the Beckman instrument was 100–500 nmol of protein. With the exception of a few minor changes, the Beckman instrument has remained largely unchanged over a period of 15 yr. Major changes in the reagent/solvent delivery system, design of the spinning cup housing and vacuum system, together with automatic conversion of 3-phenyl-2-thiohydantain (PTH) to 5-anilinothiazolinone (ATZ) derivatives were reported by Wittmann-Liebold (2,3). Hunkapiller and Hood (4) demonstrated that these changes, together with the use of polybrene to immobilize small amounts of protein or peptide, and identification of PTH-derivatives by reverse-phase HPLC (RP-HPLC) resulted in the ability to sequence low nanomolar to subnanomolar amounts of sample. The use of polybrene film to immobilize the sample was critical to the methodology, and was first introduced by Tarr (5). As demonstrated by Wittmann-Liebold (2), rigorous purification of reagents and solvents is also important for improved Edman chemistry (4,6).
KeywordsTryptic Peptide Initial Yield Double Sequence Beckman Instrument Background Peak