Improved automated solid-phase microsequencing of peptides using DABITC

✍ Scribed by Johann Salnikow; Arnold Lehmann; Brigitte Wittmann-Liebold

• Publisher: Elsevier Science
• Year: 1981
• Tongue: English
• Weight: 747 KB
• Volume: 117
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(81)90803-4

✦ Synopsis

Attachment studies with EDC (1 -ethyl-3-dimethylaminopropyl carbodiimide) have revealed that peptides, ranging from 4 to 31 residues, can be linked to aminopropyl glass with satisfactory yield, if optimal conditions are obeyed. The attachment yields are, however, significantly lower with peptides possessing carboxyl terminal lysine. High-sensitivity solid-phase sequencing of immobilized peptides can be performed if DABITC (4-N,N-dimethylaminoazobenxene 4'-isothiocyanate), an Edman reagent with a covalently linked chromophore, is used in conjunction with phenyl isothiocyanate. Technical improvements have been introduced by utilization of intermittent nitrogen flushes, pumping of the DABITC reagent in a stepwise manner, and incorporation of an automated conversion device based on instant aequous dilution of the trifluoroacetic acid efIluent. Solid-phase microsequencing constitutes a method of general applicability for peptides in the 1-to IO-nmol range, with up to 30-35 identifiable degradation steps.