Michelle Kennedy   Michelle Kennedy Ph.D., is a graduate student in Ileana Cristea’s lab at Princeton University. Her research focuses on utilizing mass spectrometry to understand how viruses exploit viral and host proteins to manipulate the subcellular environment for productive replication. In particular, she enjoys the challenge of extracting biological meaning from large datasets, and she leverages programming (specifically in Python) to not only analyze her own data, but also to develop tools that allow others to visualize and analyze their own results.

Leveraging Skyline to Develop and Analyze Data from a Targeted Mass Spectrometry Assay for Pan-herpesvirus Protein Detection

The presence and abundance of viral proteins within host cells are part of the essential signatures of the cellular stages of viral infections. Viral proteins are either brought into host cells by infectious particles or expressed at specific steps of the replication cycle. However, methods that can comprehensively detect and quantify these proteins are still limited, particularly for viruses with large protein coding capacity. By taking advantage of the targeted mass spectrometry tools available in the Skyline interface, we have designed and experimentally validated a targeted mass spectrometry assay for monitoring human viruses representing the three Herpesviridae subfamilies—herpes simplex virus type 1 (HSV-1), human cytomegalovirus (HCMV), and Kaposi’s sarcoma-associated herpesvirus (KSHV).Our assays target hundreds of peptides covering 50-80% of the predicted proteomes for each of these viruses and span proteins that are representative of different temporal protein expression classes as well as virion components (e.g. capsid, envelope, tegument, non-structural). Furthermore, we have demonstrated the applicability of these assays for 1) capturing the temporal cascades of viral replication, 2) detecting proteins throughout a range of virus concentrations, 3) assessing the effects of clinical therapeutic agents, 4) characterizing the impact of sirtuin-modulating compounds, and 5) studies using different laboratory and clinical viral strains. Altogether, our study provides a reproducible framework for monitoring the progression of herpesvirus infections under different conditions and it benefitted heavily from the accessible environment afforded by Skyline for data analysis and integration.