An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry
Protein deamidation can severely alter the physicochemical characteristics and biological functions of protein therapeutics. Cobratide is a non-addictive analgesic with wide clinical acceptance. However, the Asn residue at position 48 from the N-terminus of the cobratide amino acid sequence (N48) te...
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MDPI AG
2022-09-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/27/19/6154 |
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| author | Bo Liu Lu Huang Rongrong Xu Huihong Fan Yue Wang |
| author_facet | Bo Liu Lu Huang Rongrong Xu Huihong Fan Yue Wang |
| author_sort | Bo Liu |
| collection | DOAJ |
| description | Protein deamidation can severely alter the physicochemical characteristics and biological functions of protein therapeutics. Cobratide is a non-addictive analgesic with wide clinical acceptance. However, the Asn residue at position 48 from the N-terminus of the cobratide amino acid sequence (N48) tends to degrade during purification, storage, and transport. This characteristic could severely affect the drug safety and clinical efficacy of cobratide. Traditional methods for quantitating deamidation reported in previous research are characterised by low efficiency and accuracy; the quality control of cobratide via this method is limited. Herein, we developed an improved <sup>18</sup>O-labelling method based on the detection of a unique peptide (i.e., the protein fragment of cobratide containing the N48 deamidation hotspot after enzymolysis) using an Orbitrap high-resolution mass spectrometer to quantify deamidated cobratide. The limits of detection and quantification of this method reached 0.02 and 0.025 μM, respectively, and inter- and intra-day precision values of the method were <3%. The accuracy of the <sup>18</sup>O-labelling strategy was validated by using samples containing synthesised peptides with a known ratio of deamidation impurities and also by comparing the final total deamidation results with our previously developed capillary electrophoresis method. The recoveries for deamidation (Asp), deamidation isomerisation (iso-Asp), and total deamidation were 101.52 ± 1.17, 102.42 ± 1.82, and 103.55 ± 1.07, respectively. The robustness of the method was confirmed by verifying the chromatographic parameters. Our results demonstrate the applicability of the <sup>18</sup>O-labelling strategy for detecting protein deamidation and lay a robust foundation for protein therapeutics studies and drug quality consistency evaluations. |
| first_indexed | 2024-03-09T21:26:40Z |
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| last_indexed | 2024-03-09T21:26:40Z |
| publishDate | 2022-09-01 |
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| series | Molecules |
| spelling | doaj.art-856c15ab0f5848aaa9db39de9d7680932023-11-23T21:06:55ZengMDPI AGMolecules1420-30492022-09-012719615410.3390/molecules27196154An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap Mass SpectrometryBo Liu0Lu Huang1Rongrong Xu2Huihong Fan3Yue Wang4National Institutes for Food and Drug Control, 31st Huatuo Rd., Daxing Dist., Beijing 102629, ChinaNational Institutes for Food and Drug Control, 31st Huatuo Rd., Daxing Dist., Beijing 102629, ChinaNational Institutes for Food and Drug Control, 31st Huatuo Rd., Daxing Dist., Beijing 102629, ChinaNational Institutes for Food and Drug Control, 31st Huatuo Rd., Daxing Dist., Beijing 102629, ChinaNational Institutes for Food and Drug Control, 31st Huatuo Rd., Daxing Dist., Beijing 102629, ChinaProtein deamidation can severely alter the physicochemical characteristics and biological functions of protein therapeutics. Cobratide is a non-addictive analgesic with wide clinical acceptance. However, the Asn residue at position 48 from the N-terminus of the cobratide amino acid sequence (N48) tends to degrade during purification, storage, and transport. This characteristic could severely affect the drug safety and clinical efficacy of cobratide. Traditional methods for quantitating deamidation reported in previous research are characterised by low efficiency and accuracy; the quality control of cobratide via this method is limited. Herein, we developed an improved <sup>18</sup>O-labelling method based on the detection of a unique peptide (i.e., the protein fragment of cobratide containing the N48 deamidation hotspot after enzymolysis) using an Orbitrap high-resolution mass spectrometer to quantify deamidated cobratide. The limits of detection and quantification of this method reached 0.02 and 0.025 μM, respectively, and inter- and intra-day precision values of the method were <3%. The accuracy of the <sup>18</sup>O-labelling strategy was validated by using samples containing synthesised peptides with a known ratio of deamidation impurities and also by comparing the final total deamidation results with our previously developed capillary electrophoresis method. The recoveries for deamidation (Asp), deamidation isomerisation (iso-Asp), and total deamidation were 101.52 ± 1.17, 102.42 ± 1.82, and 103.55 ± 1.07, respectively. The robustness of the method was confirmed by verifying the chromatographic parameters. Our results demonstrate the applicability of the <sup>18</sup>O-labelling strategy for detecting protein deamidation and lay a robust foundation for protein therapeutics studies and drug quality consistency evaluations.https://www.mdpi.com/1420-3049/27/19/6154cobratidedeamidation impurityhigh-resolution mass spectrometry<sup>18</sup>O-labelling |
| spellingShingle | Bo Liu Lu Huang Rongrong Xu Huihong Fan Yue Wang An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry Molecules cobratide deamidation impurity high-resolution mass spectrometry <sup>18</sup>O-labelling |
| title | An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry |
| title_full | An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry |
| title_fullStr | An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry |
| title_full_unstemmed | An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry |
| title_short | An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry |
| title_sort | improved isotope labelling method for quantifying deamidated cobratide using high resolution quadrupole orbitrap mass spectrometry |
| topic | cobratide deamidation impurity high-resolution mass spectrometry <sup>18</sup>O-labelling |
| url | https://www.mdpi.com/1420-3049/27/19/6154 |
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