A Targeted Mass Spectrometry Assay for Detection of HIV Gag Protein Following Induction of Latent Viral Reservoirs

During early infection, HIV-1 establishes a reservoir of latently infected cells that persist during antiretroviral therapy. These reservoirs are considered the primary obstacle to eradicating HIV-1 from patients, and multiple strategies are being investigated to eliminate latently infected cells. Measuring the reservoir size using an affordable and scalable assay is critical as these approaches move into clinical trials: the current “gold-standard” viral outgrowth assay is costly, labor-intensive, and requires large numbers of cells. Here, we assessed whether selective reaction monitoring-mass spectrometry (SRM-MS) is sufficiently sensitive to detect latent HIV reservoirs following reactivation of virus. The Gag structural proteins were the most abundant viral proteins in purified virus and infected cells, and tractable peptides for monitoring Gag levels were identified. We then optimized a Gag immunoprecipitation procedure that permitted sampling of more than 107 CD4+ T cells, a requirement for detectin...