Exploring the processing of baculovirus P10 protein using next-generation protein sequencing
We are pleased to share a new case study produced in collaboration with Quantum-Si, now published on the OET and Quantum-Si website. The study describes how we used single-molecule protein sequencing to investigate the processing of P10, a highly abundant baculovirus protein that has been studied for more than 40 years.
Identifying a potential truncated form of P10
During infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV), we observed that the expected 10 kDa P10 band on SDS-PAGE gels was replaced by a band of approximately 8 kDa during the later stages of infection. Previous work suggested that P10 may be cleaved by viral cathepsin at amino acid 59, making proteolytic processing a possible explanation for the smaller band. However, subsequent experimental work did not confirm cleavage at this position.
Sequencing revealed differences in the C-terminal region of P10
To confirm the identity of the smaller band and investigate potential cleavage sites, we used Quantum-Si single-molecule protein sequencing. The 10 kDa band from day 4 post-infection and the 8 kDa bands from days 6 and 7 were excised from SDS-PAGE gels. The proteins were then digested with Lys-C, and the resulting peptides were analysed using the Quantum-Si Platinum® Pro instrument.
The peptide reads confirmed that both bands contained P10. However, the C-terminal peptide detected in the 10 kDa band was not detected in the 8 kDa band, indicating a difference in the C-terminal region of the smaller form.
Importantly, the peptide spanning the predicted cleavage site at amino acid 59 was still detected in the 8 kDa band, suggesting that cleavage does not occur at this position.
Together, these findings point towards a potential cleavage region downstream of amino acid 72. Bioinformatic analysis identified several putative cathepsin cleavage sites in this region, although further experimental validation is required to establish the precise processing site.
Understanding P10 processing informs the development of baculovirus expression systems
P10 is expressed during the late stages of baculovirus infection, but its precise molecular function and role during infection is still being discovered. Investigating how P10 is processed can help expand our understanding of baculovirus biology and virus–host interactions, while informing the continued development of OET's baculovirus expression systems.
This study also illustrates how single-molecule protein sequencing can help identify proteins within unexpected gel bands and investigate differences in protein sequence or processing. OET offers protein sequencing services using the Quantum-Si Platinum platform, alongside our protein expression, production and purification services.
The case study authors are Jaspreet S. Grewal, Leo P. Graves, Adam Chambers, Linda A. King and Robert D. Possee. We would like to thank Quantum-Si, Paru Oatey and Richard Broadhead for their collaboration.
If you would like to discuss how protein sequencing could support your research, please contact the OET team.
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