Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 109

Mass spectrometry (MS) is a technique that can identify the chemical constituents of complex and simple mixtures. It has emerged as a powerful tool for detecting and quantifying thousands of proteins in biological samples, including viruses and virus-infected cells.

A mass spectrometer ionizes the chemical constituents of a mixture and then sorts the ions based on their mass-to-charge ratio. Identification of the components is done by comparison with the patterns generated by known materials.

The total protein content of a cell or a virus particle is called the proteome. Human cells have been estimated to contain from 500,000 to 3,000,000 proteins per cubic micrometer, encoded by ∼20,000 open reading frames, and their products are further diversified by transcriptional, posttranscriptional, translational, and posttranslational regulation. The cell proteome may be further altered during virus infection. The proteome of virus particles is far less complex, but the very largest viruses can still contain hundreds of proteins. Mass spectrometry can be used to identify proteins and their concentrations in cells and in virus particles and also to reveal protein localization, protein-protein interactions, and posttranslational modifications in infected and uninfected cells.

Mass spectrometry may be combined with biochemical and genomic techniques to provide global views of viral reproduction cycles. For example, changes in proteins secreted by host cells upon virus infection can be readily characterized by performing mass spectrometry on supernatants from infected cells. Another application is to identify protein-protein interactions in virus-infected cells: a promiscuous biotinylating enzyme can be directed to a subcellular compartment, where it biotinylates adjacent molecules. These can be purified by attachment to streptavidin-containing beads and identified by mass spectrometry. Integration of mass spectrometry with some of the methods described above for genome analysis can be used to identify proteins that participate in the regulation of gene expression.

At one time the mass spectrometer was a very expensive instrument restricted to chemistry laboratories. Recent advances in the instrumentation, including cost reduction, as well as sample preparation and computational biology have propelled this technology into the virology research laboratory.