A novel protocol for in-depth analysis of recombinant adeno-associated virus capsid proteins using UHPLC-MS/MS.
Rapid Commun Mass Spectrom
; 36(6): e9247, 2022 Mar 30.
Article
en En
| MEDLINE
| ID: mdl-34951071
ABSTRACT
RATIONALE In-depth characterization of the three capsid viral proteins (VPs 1, 2, and 3) of adeno-associated viruses (AAVs) is immediately needed to ensure the consistency in gene therapy products and processes. These proteins are typically present at very low concentrations in matrices containing high concentrations of excipients and salts. Thus, there is a need for convenient methods for sample preparation before proteomic analysis. The aim of this study was to meet this need by developing a fast, reliable approach for isolating VPs in a manner enabling their efficient digestion and in-depth characterization using liquid chromatography-mass spectrometry (LC-MS). METHODS:
VPs from Anc80 were precipitated with different organic solvents, and the resulting precipitates were dissolved in either sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) or guanidine hydrochloride (Gu-HCl). The peptides obtained by the following enzymatic digestion by either trypsin or Asp-N were analyzed using LC-MS/MS.RESULTS:
We found that precipitation with chloroform/methanol/water results in fast, efficient preparation of VP samples, allowing 100% and 99.2% amino acid sequence coverage of VP1 for trypsin and Asp-N digestion, respectively. This also allowed complete sequence confirmation of VP1, VP2, and VP3 of Anc80, as well as characterization of the amino acid sequences of the N- and C-terminal regions of each VP, together with their post-translational modifications (PTMs).CONCLUSIONS:
The presented method enables fast, reliable, and relatively cheap sample preparation for identifying AAV serotypes and characterizing the heterogeneity of capsid viral proteins, including their PTMs.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Cromatografía Líquida de Alta Presión
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Dependovirus
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Proteínas de la Cápside
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Espectrometría de Masas en Tándem
Tipo de estudio:
Evaluation_studies
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Risk_factors_studies
Idioma:
En
Revista:
Rapid Commun Mass Spectrom
Año:
2022
Tipo del documento:
Article
País de afiliación:
Suiza