Strategy for the Quantitation of a Protein Conjugate via Hybrid Immunocapture-Liquid Chromatography with Sequential HRMS and SRM-Based LC-MS/MS Analyses
| Autor: | Yue Zhao, Guowen Liu, Jinping Gan, Xiling Yuan, Jon E. Peterson, Jim X Shen |
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| Rok vydání: | 2017 |
| Předmět: |
Male
Analyte Bioanalysis Fluorine Radioisotopes Tandem mass spectrometry Mass spectrometry 030226 pharmacology & pharmacy 01 natural sciences Analytical Chemistry Workflow 03 medical and health sciences 0302 clinical medicine In vivo Tandem Mass Spectrometry Animals Chromatography Chemistry 010401 analytical chemistry Selected reaction monitoring Peptide Fragments 0104 chemical sciences Fibronectins Macaca fascicularis Radiopharmaceuticals Ex vivo Conjugate Chromatography Liquid |
| Zdroj: | Analytical chemistry. 89(9) |
| ISSN: | 1520-6882 |
| Popis: | With the development of modern instrumentation and technologies, mass spectrometry based assays have played an important role in protein bioanalysis. We have developed a novel strategy by combining the "bottom-up" and "top-down" approaches using both high-resolution (HRMS) and selected reaction monitoring (SRM) based mass spectrometric detection to quantify a positron emission tomography (PET) detection tracer for an oncology marker. Monkey plasma samples were processed by immunocapture purification, followed by liquid chromatography (LC) with HRMS full scan analysis. Summed multiple charge states and multiple isotopes per charge state of the analyte were used during quantitation for optimized sensitivity. After the HRMS analysis, the remaining samples were digested by trypsin, followed by SRM detection. The HRMS approach provided the solution to a unique problem related to stability of the protein conjugate by quantifying the intact protein. The SRM method only measured a signature peptide generated from enzymatic digestion, but had a lower quantitation limit to meet the sensitivity requirement to assess the pharmacokinetics in a toxicology study. Both methods demonstrated good sensitivity, accuracy, precision and robustness, and the results revealed that there was no significant difference between the data sets obtained from both methods, indicating no in vivo or ex vivo degradation occurred in the incurred samples after dosing. This workflow not only provided the quantitative results for pharmacokinetic evaluation, but also revealed valuable in vivo stability information on the intact protein level. |
| Databáze: | OpenAIRE |
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