High-sensitivity quantitation of a Nanobody (R) in plasma by single-cartridge multidimensional SPE and ultra-performance LC-MS/MS

作者：Bronsema Kees J^*; Bischoff Rainer; Bouche Marie Paule; Mortier Kjell; van de Merbel Nico C

来源：Bioanalysis, 2015, 7(1): 53-64.

DOI：10.4155/bio.14.234

• antibody-free
• biopharmaceutical
• enzymatic digestion
• high sensitivity
• LC-MS
• MS
• multidimensional SPE
• Nanobody
• oxygen exchange internal standard

Background: A major challenge in protein quantitation based on enzymatic digestion of complex biological samples and subsequent LC-MS/MS analysis of a signature peptide is dealing with the high complexity of the matrix after digestion, which can reduce sensitivity considerably. Results: Using single cartridge multidimensional SPE, sufficient selectivity was introduced to allow quantitation in 50 mu l of plasma down to 10.0 ng/ml (0.3 nM). An inhouse prepared O-18-labeled signature peptide was used as the internal standard. The procedure was validated for human and rabbit plasma. Conclusion: The developed SPE procedure allowed the sensitive and selective LC-MS/MS quantitation of the Nanobody((R)) without the use of antibodies. When appropriate precautions are taken, the O-18-labeled peptide is a practical and economical alternative to custom synthesis.

• 出版日期2015