Viral proteomics: The emerging cutting-edge of virus research

Zhou, ShengTao; Liu, Rui; Zhao, Xia; Huang, CanHua; Wei, YuQuan

The SILAC approach relies on pre-experimental labeling to analyze the expression level of proteins, and is limited to in vitro investigation. Conversely, chemical isotope labeling targets the proteins or peptides after lysis (post experimental labeling). Theoretically, this approach is applicable to both in vitro and in vivo samples. The amine -NH 2 , thiol-SH or -COOH reactive groups of proteins or peptides are isotopically labeled and the samples are mixed equally and analyzed. Widely used methods in this category are isotopecoded affinity tag (ICAT), which targets thiol groups, isobaric tags for relative and absolute quantification (iTRAQ), which targets all the amine groups in the peptides, and 18 O-labeling, which targets the carboxyl group of peptides during enzymatic hydrolysis [51] . For the ICAT approach, although affinity purification of cysteine-containing peptides significantly reduces the sample complexity, it also prevents the identification and quantitation of peptides containing cysteines and a trypsin-cleavable site. ICAT, 2DE, and multidimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC MS/MS) were used to identify the differential expression profile of proteins upon infection of Atlantic salmon with infectious hematopoietic necrosis virus. The majority of changes in expression levels of proteins, such as natural killer cell enhancement factor (NKEF), procathepsin L, superoxide-producing NADPH oxidase, and interferon-induced viral resistance protein Mx (IFI-Mx) appeared to be associated with the infectious process [54] . In 2006, Go et al. [55] analyzed the proteome of Drosophila cells infected by flock house virus using the stable isotope labeling method ICAT. This study characterized 1500 cellular proteins from the host, including 150 upregulated and 66 downregulated proteins. Functional classification revealed that these differentially expressed proteins belonged to cellular replication, apoptotic, and metabolic pathways. A more recent study surveyed the global differential protein expression in Vero E6 cells upon SARS-CoV infection. Different proteomic strategies, comprising 2D-GE-ESI-MS/MS using Cy3-Cy5 labeling for detection of bands and ICAT-labeling coupled with 2D-LC-MS/MS, defined the alterations in viral protein expression and host cell protein expression levels. The 2DE method identified 63 proteins belonging to 48 unique gene products. By contrast, the ICAT approach identified 322 proteins, with 119 upregulated proteins and 48 downregulated proteins, implying a higher sensitivity of ICAT in detecting differentially expressed gene products compared with the traditional 2DE method [56] .