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 µl of plasma down to 10.0 ng/ml (~0.3 nM). An inhouse prepared (18)O-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(®) without the use of antibodies. When appropriate precautions are taken, the (18)O-labeled peptide is a practical and economical alternative to custom synthesis.
Keywords: LC–MS/MS; Nanobody; antibody-free; biopharmaceutical; enzymatic digestion; high sensitivity; multidimensional SPE; oxygen exchange internal standard.