Aim: Immobilized metal ion affinity chromatography is widely employed for purifying polyhistidine-tagged recombinant proteins from cell lysates. The technique can be applied for quantification of therapeutic proteins in biological matrices by LC–MS/MS. Results: A protein reagent-free workflow was developed for quantifying polyhistidine-tagged proteins by LC–MS/MS. The workflow includes target protein enrichment by immobilized metal ion affinity chromatography, on-bead trypsin digestion and quantification of signature peptides by LC–MS/MS. It was applied to quantify a 6×His-tagged protein in a mouse pharmacokinetic study with assay sensitivity of 10.0 ng/ml and linearity up to 10,000 ng/ml. Conclusion: The protein reagent-free workflow developed herein can overcome reagent limitation and serve as a viable approach for quantifying polyhistidine-tagged therapeutic proteins to support discovery pharmacokinetic and pharmacodynamic studies.
Keywords:
- immobilized metal ion affinity chromatography
- LC–MS/MS
- polyhistidine-tagged proteins
- reagent-free workflow
- signature peptides
- stable isotope-labeled internal standard