Application of Covalently Modified Glass Fibre Supports to Microsequence Analysis

Abstract

Polybrene, is generally accepted as a protein carrier in gas-liquid phase sequencing [1]. The modifier covers the negatively charged glass surface with a polymeric matrix of quaternary ammonium ions bridged by hydrophobic hexamethylene and trimethylene groups. This may give the support the potential of binding basic proteins as well as acidic and SDS-treated proteins by enabling multiple ionic and hydrophobic interactions. Although the problem of protein immobilisation seemed to be solved with the introduction of polybrene there are a number of serious drawbacks. The polybrene coated glass fibre filter has to be purified before sample application by performing 3 to 5 degradation cycles. This precycling procedure normally reduces the major background peaks (DPTU, DMPTU) to a practicable level but the remaining UV-absorbing contaminants often interfere when detecting PTH-amino acids at high sensitivity. This is illustrated in Fig. 1A, where the UV-traces of the third precycling step of a polybrene coated glass fiber filter with no added polypeptide are shown. Both DPTU and DMPTU are still giving signals equivalent to about 20–100 picomoles of PTH-amino acid. Another drawback is the unpredictable and relatively low initial yield which is reported for sperm whale myoglobin between 25% [2] and 78% [3].