Practical Protein Chemistry - A. Darbre 1989

Amino acid sequence analysis at the micro level using a gas-phase peptide-protein sequencer
Introduction

R. M. HEWICK (Genetics Institute, 225 Longwood Avenue, Boston, Massachusetts 02115, U.S.A.), M. W. HUNKAPILLER (Applied Biosystems Inc., 850 Lincoln Center Drive, Foster City, California 94404, U.S.A.)

Automated Edman Degradation for determining the Amino acid sequences of Peptides and Proteins is most commonly performed using a liquid-phase (LP) Sequencer with a rotating cup Reactor [2]. In recent years, both the LP sequencer and its operating Procedures have been significantly improved [6, 9, 14–16]. By paying special attention to the meticulous purification of Reagents and Solvents—used both in the sequencer and in the High-Performance Liquid Chromatography (HPLC) system for analyzing cleaved Amino Acid Derivatives—it is now possible to determine extended amino acid sequences (>30 residues) starting with less than 1 nmol of peptide or protein quantities (<1 nmol) [7]. The main drawback of LP analysis is sample washout, particularly of hydrophobic peptides. The addition of Polybrene* during sequence analysis on the LP sequencer has helped overcome this issue [6, 14].

To eliminate sample loss during washing steps, a solid-phase (SP) analytical method was proposed [11]. Because the polypeptide under study is covalently attached to a chemically modified support matrix (based on Glass or polystyrene), sample loss during washing with organic solvents during amino acid Cleavage is prevented. The simple design of the SP sequencer's reactor Column facilitates miniaturization, thereby increasing sequencing sensitivity. At the same time, covalently attaching peptides and proteins to the support in a heterogeneous system is not straightforward, and yields at this stage are only 20–50%. The coupling reactions between the protein (or peptide) and the support matrix are carried out outside the sequencer; they are quite labor-intensive and time-consuming. Notably, planning the covalent attachment experiment depends to some extent on prior knowledge of the polypeptide's Amino Acid Composition. A significant disadvantage of the SP method is that gaps in sequence data occur at the points where the polypeptide binds to the matrix, resulting in incomplete sequence information; furthermore, the sample is entirely lost (washed out of the column) if the next amino acid to be cleaved happens to be the final attachment point between the peptide and the support.

* Polybrene: 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide. — Transl. note.

Recently, a new type of miniaturized sequencer—the gas-phase sequencer—was developed, which utilizes gaseous reagents to carry out the Edman reaction [4, 8]. The only liquids that come into contact with the protein (or peptide) film are phenyl isothiocyanate and organic solvents in which the protein is sparingly soluble. The washout of hydrophobic peptides and proteins associated with sodium dodecyl sulfate (SDS) is prevented by the addition of Polybrene.

The peptide of interest is applied to a porous filter and then dried, yielding a thin sample film within the reaction chamber of the gas-phase (GP) sequencer. The peptide remains tightly bound to the filter even without covalent attachment. Because the sample maintains the same physical state throughout the entire Edman degradation process, a miniaturized flow-through reaction vessel is used instead of the cumbersome and complex rotating cup system. The reduced size of the reactor leads to further reductions in sample consumption compared to the LP sequencer [7]. Operating a GP sequencer requires significantly fewer reagents, the Edman cleavage proceeds faster, and the instrument's design is simpler than that of LP devices.

This chapter discusses the most important Design Features of the GP sequencer, as well as methodological approaches successfully applied to microscale structural analysis (<100 pmol) of peptides eluted from SDS-polyacrylamide gels (SDS-PAGE).