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
✍ Scribed by Kenneth L. Johnson; Timothy D. Veenstra; James M. Londowski; Andy J. Tomlinson; Rajiv Kumar; Stephen Naylor
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 116 KB
- Volume
- 13
- Category
- Article
- ISSN
- 0269-3879
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✦ Synopsis
We have used on-line sample clean-up, concentration, and chromatography with electrospray ionization mass spectrometry (ESI-MS), to characterize and determine the presence of disulfide bonds in recombinant full-length rat brain calbindin D 28K and two deletion mutants of the protein, one lacking EF-hand 2 (calbindin D2) and the other lacking EF-hands 2 and 6 (calbindin D2,6). The molecular weights of the expressed proteins dissolved in biological buffers were determined with high accuracy using a low-flow, pressurized chamber infusion system, that allows on-line protein clean-up by removing buffers/salts incompatible with ESI-MS. The molecular weight determinations showed that the amino-terminal methionine residues had been cleaved during the expression and isolation of the recombinant proteins. Approximately 85-90% of the protein sequences were confirmed by on-line HPLC-ESI-MS analysis of peptides generated by a lysyl endoproteinase C digestion. Comparisons of ESI-MS spectra of native and reduced calbindin D 28K and D2 show that the full length-and D2 mutant-protein contain one disulfide bond. Molecular mass determinations of calbindin D2,6 showed that this protein contains a highly active cysteine residue that covalently binds a mercaptoethanol group, or forms a homodimer via a disulfide bond. The results show surprising differences amongst the deletion mutants of calbindin D 28K with respect to the formation of disulfide bonds. These differences are not readily detected by other techniques and show that ESI-MS is a powerful, rapid method by which to detect disulfide linkages for intact proteins.