AUTOMATED ANALYTICAL SYSTEM FOR THE EXAMINATION OF PROTEIN PRIMARY STRUCTURE

This paper describes an automated analytical system for the examinatio n of protein primary structure in which (i) the target protein is firs t purified by immunoaffinity chromatography, (ii) subsequent chromatog raphic and chemical reaction steps in the sequencing process are direc tly coupled, (iii) buffer exchange between these unit operations is ac hieved while the protein is absorbed on a mixed bed of strong ion exch ange sorbents, (iv) proteolysis occurs in an immobilized trypsin colum n having a 10-1000 fold-excess of enzyme, (v) the tryptic digest is di rectly transferred to a perfusion dilute capture column where it is co ncentrated and rapidly desalted, and (vi) peptides eluted from the dil ute capture column and analytical microbore and capillary perfusion re versed-phase chromatography columns are analyzed by either single-stag e mass spectrometry (MS) or tandem MS/MS, Protein structure variants w ere easily recognized, and in the case of hemoglobin (Hb) S, the site of variation from Hb A(o) was verified.