Princeton University Library Catalog

Absolute Proteome Quantification of Extracellular HSV-1 Virions Using Protein Concatemers

Author/​Artist:
Desanti de Oliveira, Beatriz [Browse]
Format:
Senior thesis
Language:
English
Advisor(s):
Cristea, Ileana [Browse]
Department:
Princeton University. Department of Molecular Biology [Browse]
Class year:
2013
Description:
65 pages
Restrictions note:
Walk-in Access. This thesis can only be viewed on computer terminals at the Mudd Manuscript Library.
Summary note:
Infecting 40-80% of the world’s population, herpes simplex virus type 1 (HSV-1) is one of the most common human pathogens. HSV-1 is responsible for a number of diseases of minor, moderate, and severe pathology, including oral sores, virally induced blindness, viral encephalitis, and disseminated infection of neonates. After infection, the virus replicates in epithelial cells, enters sensory neurons, and is transported to neuronal cell bodies where it establishes lifelong latency. Periodically HSV-1 reactivates, causing recurring infections and facilitating its dissemination. Although great progress has been made in characterizing both the structure and composition of this virus, a comprehensive quantitative analysis of all proteins found in HSV-1 virions is still lacking. Here I develop an approach and report a proof-of-concept absolute quantification of major structural viral proteins within the HSV-1 virion using stable isotope dilution mass spectrometry paired with the QconCAT technology, which generates artificial protein concatemers of internal standard peptides. Using strict criteria for selection of proteotypic peptide, two QconCATs genes were designed containing 88 tryptic peptides covering 45 viral proteins, which represents 90% of viral proteins within the mature HSV-1 virion. [13C6]Arg/Lys-labeled QconCATs were expressed in and purified from Escherichia coli. Mass spectrometric quantification of gradient-purified extracellular virions spiked with heavy-labeled QconCAT achieved valid isotope ratios for 41 out of 45 viral proteins. Consistent with known capsid protein stoichiometry, a VP5:VP23:VP19C ratio of 3:2:1 was observed. This is the first time that such an approach is developed for quantifying viral proteins. The application of this quantification strategy is not limited to HSV-1 virions, and can be extended to HSV-1 protein levels during the course of infection.