Automated analytical system for the examination of protein primary structure

This paper describes an automated analytical system for the examination of protein primary structure in which (i) the target protein is first purified by immunoaffinity chromatography, (ii) subsequent chromatographic and chemical reaction steps in the sequencing process are directly coupled, (iii) buffer exchange between these unit operations is achieved while the protein is absorbed on a mixed bed of strong ion exchange sorbents, (iv) proteolysis occurs in an immobilized trypsin column having a 10-1000 fold-excess of enzyme, (v) the tryptic digest is directly transferred to a perfusion dilute capture column where it is concentrated and rapidly desalted, and (vi) peptides eluted from the dilute capture column and analytical microbore and capillary perfusion reversed-phase chromatography columns are analyzed by either single-stage mass spectrometry (MS) or tandem MS/MS. Protein structure variants were easily recognized, and in the case of hemoglobin (Hb) S, the site of variation from Hb A0 was verified.