An automated C-terminal sequencing method for analysis of proteins us ing an ABI 473A sequencer

Abstract

We developed a novel chemistry for C-terminal sequencing of proteins based on derivatization with acetylisothiocyanate to yield amino acid thiohydantoins (TH-AAs), and it was used for manual sequencing of biopharmaceutical products on a routine basis.This simple chemistry was automated using a ABI 473A N-terminal sequencer. All reagents (R1, trimethylsilylisothiocyanate; R3, alkaline thiocyanate for cleavage) and solvents required for sequencing were accommodated on the sequencer, which was modified to deliver liquid R2 (acetyl chloride) to the reaction vessel.The conversion flask was used for preparing the TH-AAs for analysis by online HPLC using a graphitized carbon (Hypercarb) column. Results obtained with model proteins and recombinant protein drugs suggest that at least three residues from the C terminus can be easily determined.The C-terminal heterogeneity in more than five types of recombinant immunoglobulin G was determined, and the differences in Gly-Lys ratios were consistent with changes observed in the isoelectric focusing profile of these antibodies. Because the chemistry uses only four reagents delivered to the reaction vessel and three to the conversion flask,we believe that the automated protocol can be easily adapted to any existing N-terminal sequencer.