Practical Protein Chemistry - A. Darbre 1989

Analysis of Amino Acid Phenylthiohydantoins
Introduction

M. D. WATTERFIELD, G. SCRACE, N. TOTTY (Protein Chemistry Laboratory, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WC2A 3PX, UK)

Edman Degradation using FITC is the most common and reliable METHOD FOR DETERMINING the Amino Acid Sequence of Peptides and Proteins. Currently, both manual and automated approaches are used for analyzing peptides of various sizes, with the released Amino Acids most frequently identified as their PTH-derivatives. Numerous papers have been dedicated to the analysis of these derivatives. This chapter examines five principal Methods for analyzing amino acid PTH-derivatives.

Thin-Layer Chromatography (TLC), introduced by Edman and Begg and described in their classic work on the automated Sequencer [2], is experimentally the simplest method, requiring no expensive equipment, though it has relatively low sensitivity (~1 nmol). TLC is well suited for both manual and automated sequencing; however, it is primarily a qualitative rather than quantitative technique. A distinct advantage of the method is the capability to analyze multiple samples simultaneously. TLC can be rendered semi-quantitative by eluting the PTH derivatives from the chromatography plate, though this is rarely done. When identifying PTH-Arg and PTH-His, TLC should be supplemented with other techniques, such as HPLC of the same PTH-amino acids or Amino acid analysis of free amino acids obtained via back-Hydrolysis of the PTH-derivatives. Back-hydrolysis should be reserved for the analysis of short peptides and for high-efficiency amino acid sequence determinations of medium-sized peptides using a sequencer with nanomole sample quantities. Since the mid-1960s, Gas-Liquid Chromatography (GLC) has been employed to analyze PTH-Amino Acid Derivatives. This method allows for the rapid and Quantitative determination of most, but not all, PTH-derivatives. The primary drawback of GLC is its inability to provide unambiguous identification for the derivatives of all 20 amino acids. High-Performance Liquid Chromatography (HPLC) enables the rapid Separation and quantitative identification of all 20 amino acid PTH-derivatives at the picomole level. Another noteworthy technique, used only in select laboratories, is mass spectrometry; while offering the highest sensitivity, it requires sophisticated instrumentation available to very few research groups.

When selecting an analytical method, one must carefully consider the cost and availability of equipment, as well as the practical sensitivity required for the work. HPLC is currently the preferred choice, particularly for high-sensitivity determinations. This chapter focuses primarily on this aspect of its application.