On-line procedures for alkylation of cysteine residues with 3-bromopropylamine prior to protein sequence analysis

Abstract

We have previously shown that 3-bromopropylamine offers several advantages over other alkylating reagents in the modification and subsequent identification of cysteine residues by protein sequencing. We describe here simple on-sequencer procedures for alkylating cysteines in proteins which employ the reduction of cystines in proteins with tri-n-butylphosphine and concomitant alkylation of the resulting cysteines with 3-bromopropylamine. Addition of an aqueous acetone wash to a modified reaction cycle on the Applied Biosystems 477A sequencer removes excess 3-bromopropylamine. As a result, very little background in the first step of the sequence analysis is seen. Under these conditions, cysteines are readily modified and identified during sequencing. Moreover, very little preview of the next amino acid is observed, which indicates that the N-terminal amino acid is not appreciably alkylated by 3-bromopropylamine. On-sequencer methods have been developed for proteins spotted onto glass fiber filters and proteins electroblotted onto polyvinylidene difluoride membranes.