Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS
Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. With the skyrocketing rate of AAV research and the prevalence of many new engineered capsids being investigated in preclinical and clinical trials, capsid characterization plays a vital role in serotype confirmation and...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-12-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050122001334 |
| _version_ | 1811241501269688320 |
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| author | Anh K. Lam Junping Zhang Dylan Frabutt Patrick L. Mulcrone Lei Li Lifan Zeng Roland W. Herzog Weidong Xiao |
| author_facet | Anh K. Lam Junping Zhang Dylan Frabutt Patrick L. Mulcrone Lei Li Lifan Zeng Roland W. Herzog Weidong Xiao |
| author_sort | Anh K. Lam |
| collection | DOAJ |
| description | Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. With the skyrocketing rate of AAV research and the prevalence of many new engineered capsids being investigated in preclinical and clinical trials, capsid characterization plays a vital role in serotype confirmation and quality control. Further, peptide mapping the capsid proteins might inevitably be a future requirement by regulatory agencies since it is a critical step in good manufacturing practice (GMP) for biotherapeutic characterization. To overcome many challenges that traditional methods like SDS-PAGE and western blots carry, liquid chromatography and mass spectrometry (LC-MS) allows high resolution and sensitivity with great accuracy in characterizing the AAV capsid proteins. Our optimized LC-MS method provides quick sample preparation, a fast and high-throughput 4-min run, and high sensitivity, which allows for very efficient characterization of wild-type and engineered capsids. This study also reports the usage of LC-MS/MS peptide mapping of AAV capsid proteins to determine the most accessible lysine residues targeted by chemical modifications. Our detailed protocols are anticipated to promote the development and discovery of AAV variants with high accuracy and efficiency. |
| first_indexed | 2024-04-12T13:37:07Z |
| format | Article |
| id | doaj.art-19ab892021054159a919044b17f3e279 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-04-12T13:37:07Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-19ab892021054159a919044b17f3e2792022-12-22T03:30:57ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012022-12-0127185194Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MSAnh K. Lam0Junping Zhang1Dylan Frabutt2Patrick L. Mulcrone3Lei Li4Lifan Zeng5Roland W. Herzog6Weidong Xiao7Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USADepartment of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USADepartment of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USADepartment of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USADepartment of Chemistry, Georgia State University, Atlanta, GA 30302, USADepartment of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USADepartment of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USADepartment of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Corresponding author. Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA.Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. With the skyrocketing rate of AAV research and the prevalence of many new engineered capsids being investigated in preclinical and clinical trials, capsid characterization plays a vital role in serotype confirmation and quality control. Further, peptide mapping the capsid proteins might inevitably be a future requirement by regulatory agencies since it is a critical step in good manufacturing practice (GMP) for biotherapeutic characterization. To overcome many challenges that traditional methods like SDS-PAGE and western blots carry, liquid chromatography and mass spectrometry (LC-MS) allows high resolution and sensitivity with great accuracy in characterizing the AAV capsid proteins. Our optimized LC-MS method provides quick sample preparation, a fast and high-throughput 4-min run, and high sensitivity, which allows for very efficient characterization of wild-type and engineered capsids. This study also reports the usage of LC-MS/MS peptide mapping of AAV capsid proteins to determine the most accessible lysine residues targeted by chemical modifications. Our detailed protocols are anticipated to promote the development and discovery of AAV variants with high accuracy and efficiency.http://www.sciencedirect.com/science/article/pii/S2329050122001334AAVcapsid characterizationLC-MSpeptide mappingengineered capsidsgene therapy |
| spellingShingle | Anh K. Lam Junping Zhang Dylan Frabutt Patrick L. Mulcrone Lei Li Lifan Zeng Roland W. Herzog Weidong Xiao Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS Molecular Therapy: Methods & Clinical Development AAV capsid characterization LC-MS peptide mapping engineered capsids gene therapy |
| title | Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS |
| title_full | Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS |
| title_fullStr | Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS |
| title_full_unstemmed | Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS |
| title_short | Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS |
| title_sort | fast and high throughput lc ms characterization and peptide mapping of engineered aav capsids using lc ms ms |
| topic | AAV capsid characterization LC-MS peptide mapping engineered capsids gene therapy |
| url | http://www.sciencedirect.com/science/article/pii/S2329050122001334 |
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