On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins

We have used on-line sample clean-up, concentration, and chromatography wit h electrospray ionization mass spectrometry (ESI-MS), to characterize and d etermine the presence of disulfide bonds in recombinant full-length rat bra in calbindin D-28K and two deletion mutants of the protein, one lacking EF- hand 2 (calbindin Delta 2) and the other lacking EF-hands 2 and 6 (calbindi n Delta 2,6). The molecular weights of the expressed proteins dissolved in biological buffers were determined with high accuracy using a low-flow, pre ssurized chamber infusion system, that allows on-line protein clean-up by r emoving buffers/salts incompatible with ESI-MS. The molecular weight determ inations showed that the amino-terminal methionine residues had been cleave d during the expression and isolation of the recombinant proteins. Approxim ately 85-90% of the protein sequences were confirmed by on-line HPLC-ESI-MS analysis of peptides generated by a lysyl endoproteinase C digestion. Comp arisons of ESI-MS spectra of native and reduced calbindin D-28K and Delta 2 show that the full length- and Delta 2 mutant-protein contain one disulfid e bond. Molecular mass determinations of calbindin Delta 2,6 showed that th is protein contains a highly active cysteine residue that covalently binds a mercaptoethanol group, or forms a homodimer via a disulfide bond. The res ults show surprising differences amongst the deletion mutants of calbindin D28K With respect to the formation of disulfide bonds. These differences ar e not readily detected by other techniques and show that ESI-MS is a powerf ul, rapid method by which to detect disulfide linkages for intact proteins. Copyright (C) 1999 John Wiley & Sons, Ltd.