Princeton University Cristea Lab - HSV-1, HCMV, KSHV herpesvirus PRM assay

A scalable, targeted mass spectrometry-based assay for monitoring the progression of herpesvirus infections
Data License: CC BY 4.0 | ProteomeXchange: PXD025879 | doi:
  • Organism: Human betaherpesvirus 5, Herpes simplex virus 1, Kaposi's sarcoma-associated herpesvirus, Homo sapiens
  • Instrument: Q Exactive HF
  • SpikeIn: No
  • Keywords: PRM, herpesvirus, HSV-1, HCMV, KSHV
  • Lab head: Ileana Cristea Submitter: Michelle Kennedy
Herpesviruses are large, double-stranded DNA viruses that infect up to 100% of the population and represent a significant public health burden. Throughout their finely controlled lifecycles, herpesviruses express between 70 and 250 viral proteins with expression patterns that are characterized by a temporal cascade whereby subsequent expression of viral genes depends on the prior expression of other viral genes for successful infection. Considering their substantial protein coding capacity, the development of a method to systematically monitor viral protein levels throughout infection is not readily attainable using antibody or RNA-based approaches. Here, we present targeted mass spectrometry-based assays that comprehensively quantify viral protein levels for viruses that span the alpha-, beta-, and gamma-herpesvirus subfamilies: herpes simplex virus type I (HSV-1), human cytomegalovirus (HCMV), and Kaposi sarcoma-associated herpesvirus (KSHV), respectively. Our parallel reaction monitoring (PRM)-based assays measure 59 (HSV-1), 90 (HCMV), and 62 (KSHV) viral proteins that represent all temporal classes of gene expression as well as different components of the virion. We then applied our assays to assess the effects of clinically relevant as well as other potential antiviral agents on viral protein levels. We also showed that these assays are applicable to a wide variety of viral strains and infection models.
Experiment Description
Signature peptides unique to HSV-1, HCMV, and KSHV proteins were manually curated for each virus by performing an iterative process of exploratory, data-dependent MS analyses of infected samples and experimental validation of peptide detection and reliability by scheduled PRM analysis on a Thermo Q Exactive HF. Peptide spectral libraries were built with Proteome Discoverer 2.4, and PRM data was analyzed in Skyline.
Sample Description
Human fibroblast cells (MRC5) were infected HSV-1 or HCMV at an MOI of 3. For KSHV, iSLK.219 cells harboring latent KSHV were reactivated with sodium butyrate and doxycycline. Whole-cell lysates were collected at time points corresponding to stages of virus gene expression, genome replication, and virion assembly/egress. HCMV and KSHV samples (from cell culture): Frozen cell pellets were resuspended in lysis buffer and lysed by repeated steps of incubation at 95°C for 3 min. followed by sonication in a cup-horn sonicator for 20 pulses. Proteins were then reduced and alkylated and extracted by methanol-chloroform precipitation, and digested with trypsin. The resulting peptides were desalted using the StageTip method and resuspended at 0.75μg/μl for peptide LC-MS/MS analysis. HSV-1 samples (from cell culture): Due to a smaller amount of available starting sample and to demonstrate assay applicability to other peptide preparation methods, HSV-1 samples were prepared using S-Trap following the manufacturers protocol. Pooled elutions from the S-Trap column were resuspended at 0.75μg/μl for peptide LC-MS/MS analysis. HSV-1 samples (frontal lobe tissues): Frontal lobe brain tissues from HSV-1 (strain 17)-infected mice were collected at 8 days post-infection. Pooled tissues were homogenized using a Dounce homogenizer and lysed by repeated steps of incubation at 95°C for 3 min. followed by sonication in a cup-horn sonicator for 20 pulses. Lysates were then further homogenized using a Polytron and centrifuged to remove any remaining insoluble particles. Next, samples were reduced and alkylated and proteins were extracted by methanol-chloroform precipitation. Peptides were generated by trypsin digest on an S-trap column and pooled elutions from the column were resuspended at 0.75μg/μl for peptide LC-MS/MS analysis.
Created on 2/3/22, 6:06 PM
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