Characterization of Protein Impurities and Site-Specific Modifications Using Peptide Mapping with Liquid Chromatography and Data Independent Acquisition Mass Spectrometry

2009 ◽  
Vol 81 (14) ◽  
pp. 5699-5708 ◽  
Author(s):  
Hongwei Xie ◽  
Martin Gilar ◽  
John C. Gebler
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Peptide Mapping ◽  
Site Specific ◽  
Data Independent Acquisition

scholarly journals Fast and efficient digestion of adeno associated virus (AAV) capsid proteins for liquid chromatography mass spectrometry (LC-MS) based peptide mapping and post translational modification analysis (PTMs)

2021 ◽  
Author(s):  
Felipe Guapo ◽  
Lisa Strasser ◽  
Silvia Millan-Martin ◽  
Ian Anderson ◽  
Jonathan Bones
Keyword(s):  
Mass Spectrometry ◽  
Gene Therapy ◽  
Liquid Chromatography ◽  
High Resolution Mass Spectrometry ◽  
Peptide Mapping ◽  
Amino Acid Sequences ◽  
Capsid Proteins ◽  
Sequence Coverage ◽  
Adeno Associated Virus

Adeno-associated virus (AAV) represent a widely used delivery mechanism for gene therapy treatments currently being developed. The size and complexity of these molecules requires the development of sensitive analytical methods for detailed product characterization. Among the quality attributes that need to be monitored, characterization of the AAV capsid protein amino acid sequences and any associated post translational modifications (PTM) present should be performed. As commonly used for recombinant protein analysis, LC-MS based peptide mapping can provide sequence coverage and PTM information to improve product understanding and the development and deployment of the associated manufacturing processes. In the current study, we report a fast and efficient method to digest AAV5 capsid proteins in only 30 minutes prior to peptide mapping analysis. The performance of different proteases in digesting AAV5 was compared and the benefits of using nanoflow liquid chromatography for separation prior to high resolution mass spectrometry to obtain 100% sequence coverage are highlighted. Characterization and quantitation of PTMs on AAV5 capsid proteins when using pepsin as a single protease is reported, thereby demonstrating the potential of this method to aid with complete characterization of AAV serotypes in gene therapy development laboratories.

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