Quantification of protein posttranslational modifications using stable isotope and mass spectrometry I: principles and applications |
| |
Authors: | Jiang Xinzhao Grace Apostol Izydor Luo Quanzhou Lewis Jeffrey Keener Ronald Luo Shun Jerums Matthew Zhang Xin Wypych Jette Huang Gang |
| |
Institution: | Department of Analytical and Formulation Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA. |
| |
Abstract: | With the increased attention to quality by design (QbD) for biopharmaceutical products, there is a demand for accurate and precise quantification methods to monitor critical quality attributes (CQAs). To address this need we have developed a mass spectrometry (MS) based method to quantify a wide range of posttranslational modifications (PTMs) in recombinant proteins using stable isotope-labeled internal standard (SILIS). The SILIS was produced through metabolic labeling where 15N was uniformly introduced at every nitrogen atom in the studied proteins. To enhance the accuracy of the method, the levels of PTMs in SILIS were quantified using orthogonal analytical techniques. Digestion of an unknown sample mixed with SILIS generates a labeled and a nonlabeled version of each peptide. The nonlabeled and labeled counterparts coelute during RP-HPLC separation but exhibit a sufficient mass difference to be distinguished by MS detection. With the application of SILIS, numerous PTMs can be quantified in a single analysis based on the measured MS signal ratios of 15N-labeled versus the nonlabeled pairs. Several examples using microbial and mammalian-expressed recombinant proteins demonstrated the principle and utility of this method. The results indicate that SILIS is a valuable methodology in addressing CQAs for the QbD paradigm. |
| |
Keywords: | |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|