An On-Line Isocratic HPLC System for the Analysis of PTH-Amino Acids on A Gas-Phase Sequencer

Abstract

Since the description of a gas phase sequencer in 1981 by Hewick et al.1, the protein sequencing field has increasingly used this methodology for routine sequence analysis of peptides and proteins in the low nanomole to low picomole range. The commercially available instrument from Applied Biosystems has greatly simplified automated Edman chemistry, including the conversion of anilinothiazolinone (ATZ) to phenythiohydantoin (PTH) derivatives of amino acids. The analysis of the PTH derivatives is routinely performed by high performance liquid chromatography (HPLC) using various reverse phase columns. Investigators have utilized either gradient2-4 or isocratic5-6 modes for the separation of the 20 common PTH derivatives. Routinely, the sample collected from the sequencer is dried in a Speed-Vac, reconstituted in a small volume (10–50 uL of acetonitrile containing an internal standard), and injected onto an HPLC. Investigators may inject 10–100? of the sample, depending on the required sensitivity of the analysis. If, as is sometimes the case, the sample must be reanalyzed, it is recommended that no more than 40–60% of the sample be analyzed in a single HPLC run. Most laboratories allow a sequencer run to proceed overnight, workup the 10–15 cycles accumulated at one time, and load these samples onto a HPLC equipped with an autosampler. Since it is often impossible to determine if a sequencer run is successful by examining a single or even two cycles, it may be late morning before it is realized that the evening’s run was successful or not. If successful, it may require many more hours of sample workup and HPLC time before it can be determined if the run is “over.” Thus we ask, have we reached the end of a peptide, or can we interpet any more cycles on a protein? Because of this delay between sample production by the sequencer and sample analysis by the HPLC, the efficiency of sequence analysis is reduced by at least a factor of two. It is clear that an on-line HPLC which analyzes the cycles as fast as they are produced would greatly increase the efficiency of microsequence analysis. Although such a system is now available from Applied Biosystems, when this work was started in late 1984 no commercial system was available. This report may be of interest for those who wish to consider the parameters involved in the development of an on-line system, or who wish to construct their own.