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. 2021 Nov;32(21-22):1403-1416.
doi: 10.1089/hum.2021.009. Epub 2021 Jul 16.

Characterization of Adeno-Associated Virus Capsid Proteins with Two Types of VP3-Related Components by Capillary Gel Electrophoresis and Mass Spectrometry

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Characterization of Adeno-Associated Virus Capsid Proteins with Two Types of VP3-Related Components by Capillary Gel Electrophoresis and Mass Spectrometry

Hiroaki Oyama et al. Hum Gene Ther. 2021 Nov.

Abstract

Recombinant adeno-associated virus is a leading platform in human gene therapy. The adeno-associated virus (AAV) capsid is composed of three viral proteins (VPs): VP1, VP2, and VP3. To ensure the safety of AAV-based gene therapy products, the stoichiometry of VPs of AAV vector should be carefully monitored. In this study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, capillary gel electrophoresis (CGE), and liquid chromatography-UV-mass spectrometry (LC-UV-MS) were performed to evaluate the VP components of AAV1, AAV2, and AAV6. Two types of VP3-related components, VP3 variant and VP3 fragment, were identified. The VP3 variant was the N-terminal shorter VP3, of which the translation started at M211, not at the conventional initiation codon, M203. The VP3 variant could be generated by leaky scanning of the first initiation codon of VP3. We also showed that the VP3 variant was identified in a minor peak before VP3 in CGE measurement. Meanwhile, the VP3 fragment was the C-terminal cleaved VP3, of which the sequence of VP3 ended at D590 or D626, indicating that cleavage occurred between D590 and P591, or D626 and G627. The cause of the cleavage of the DP or DG sequence was hydrolysis due to low pH of the mobile phase and high temperature of the column oven in the LC system, which was necessary to clearly separate the peak of VPs. VP3 fragments, detected only in LC-UV-MS in small amount account with less than 3% of total peak area, should be included in the quantification of VP3. Finally, the relationship of VP stoichiometry determined by the above three methods was discussed. From this study, we proposed that the VP components of AAV should be complementarily evaluated by CGE and LC-UV-MS.

Keywords: adeno-associated virus; capillary gel electrophoresis; mass spectrometry; viral protein.

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Conflict of interest statement

No competing financial interests exist.

Figures

Figure 1.
Figure 1.
Evaluation of VP components by electrophoresis. (a) SDS-PAGE result of AAV1, AAV2, and AAV6. (b) CGE electropherograms of AAV1, AAV2, and AAV6. CGE, capillary gel electrophoresis; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; VP, viral protein.
Figure 2.
Figure 2.
MS/MS spectra of N-terminal peptides detected by peptide mapping analysis. (a) VP3N-terminal peptide of which translation started at M203. (b) VP3 variant N-terminal peptide of which translation started at M211. MS, mass spectrometry.
Figure 3.
Figure 3.
Detection of VP1, VP2, VP3, and VP3 variant by LC-UV-MS measurement. (a) LC chromatograms of AAV1, AAV2, and AAV6. (b) Deconvoluted mass spectra of separated peaks (Peaks 1, 2, and 3 in AAV1 and AAV6, and peaks 1 and 2 in AAV2). The peak number shown at the upper right corresponds to the number depicted in Fig. 3a. The label “p” denotes the phosphorylation and “o” oxidation. LC-UV-MS, liquid chromatography–UV–mass spectrometry.
Figure 4.
Figure 4.
Detection of VP3 fragments and evaluation of the effect of column holding time on the generation of VP3 fragments. (a) Deconvoluted mass spectra of peak 4 in AAV1 and AAV6. The peak number shown at the upper right corresponds to the number depicted in Fig. 3a. (b) LC chromatogram of AAV1 samples treated with different column holding time. The black, blue, and red lines represent the column holding time of 0, 30, and 60 min, respectively. (c) Traces of the peak area of VP components and fragments. The peak area is calculated from (b). Closed circles, diamonds, and squares correspond to VP3, VP2, and VP1, whereas cross marks and open circles represent the N-terminal region of VP3 fragments and C-terminal region of VP3 fragments, respectively. (d) Deconvoluted mass min in spectra of the peak eluted at around 4 min in (b). Deconvolution analysis was performed with the data of AAV1 treated with the column holding time of 60 min.
Figure 5.
Figure 5.
Sequence alignment of VP3 and reaction scheme of DP and DG fragmentation. (a) Sequence alignment of VP3 of AAV1-12 and AAVrh10 focusing on the N-terminal regions and cleavage regions at the DP or DG sequence. Residue number was assigned based on the VP1 sequence of AAV1. The VP3 variant and VP3 fragment experimentally confirmed in this study and previous studies, are shown in gray. (b) Reaction schemes of isomerization of an aspartic acid residue and hydrolysis of DX sequence. (c) Isomerization reactions of DP and DG sequence.

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