ABSTRACT
This chapter describes a combination of automated gas phase Edman chemistry and on-line phenythiohydantoin (PTH) analysis that is suitable for sequencing of both proteins and peptides down to the low picomole level. It describes the instrumentation used for automated Edman degradation in the studies, in the Applied Biosystems Model 470A Gas Phase Protein/Peptide Sequencer. The use of gas phase coupling and cleavage reagents in Protein/Peptide Sequencer provides retention of the sample on a solid support without necessity of covalent chemical attachment to the support. The standard gas phase sequencer programs are designed to trap and stabilize the dehydro product with dithiothreitol (DTT) delivered to the conversion flask just before transfer of the anilinothiazolinone (ATZ) from the cartridge. ATZ-Gly converts to PTH-Gly somewhat slowly, the reaction being only 80 to 85% complete during the standard conversion conditions in the gas phase sequencer. The weakest component may be sample preparation, the Edman chemistry itself, or PTH analysis.
