N-terminal and internal sequence determination of microgram amounts of proteins separated by isoelectric focusing in immobilized pH gradients

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Abstract
Isolation of microgram amounts of proteins and submicrogram quantities of peptides in a form suitable for sequence analysis is a key step in high sensitivity protein sequencing technology. Recently, methods have been developed which allow the direct, high yield, recovery of microgram amounts of sequenceable proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis. In the present publication, we describe an extension of these methods to obtain N-terminal or internal sequence information from proteins separated by isoelectric focusing in polyacrylamide gels containing immobilized pH gradients. For N-terminal sequence analysis, separated proteins were electrophoretically transferred (electroblotted) onto chemically-modified glass fiber sheets, a support compatible with Edman degradation chemistry. Transferred protein bands were detected on the support, cut out and directly inserted into the cartridge of a gas-phase protein sequenator. For internal sequence analysis, separated proteins were electrophoretically transferred onto nitrocellulose. Protein bands were detected by staining, cut out and the proteins subjected to enzymatic digestion directly on the support. The resulting cleavage fragments (peptides) were released into the supernatant where they were recovered and separated by narrow-bore reversed-phase high performance liquid chromatography for sequence analysis. The potential of this methodology is illustrated by the comparative peptide mapping of isoforms of bovine carbonic anhydrase.

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