Analysis of ISG15-Modified Proteins from A549 Cells in Response to Influenza Virus Infection by Liquid Chromatography-Tandem Mass Spectrometry

Interferon stimulated gene 15 kDa protein (ISG15) is the first ubiquitin-like protein identified, which plays vital roles in a variety of fields including viral infection and immunological regulation. In this study, liquid chromatography-tandem mass spectrometry was used to analyze ISG15-modified proteins in A549 cells in response to infection by influenza virus, which were enriched by immunoprecipitation. A total of 22 cellular host proteins were identified in A549 cells infected by influenza virus, including ubiquitin-like ISG15 protein, cyclin-T1, heat shock protein 71 kDa, caldesmon, eukaryotic translation initiation factor, and so on. Besides, non-structural protein (NS1) from influenza virus was also identified. Among the 22 host proteins identified, 6 were also identified in the control non-infected A549 cells, including annexin A1, fructose-bisphosphate aldolase A, ATP synthase subunit g, enolase, actin, and tubulin. Bioinformatics analysis revealed that the identified ISG15-modified host proteins induced by influenza virus infection could be classified into 9 protein classes: chaperone, oxidoreductase, enzyme modulator, transferase, nucleic acid binding, transcription factor, kinase, cytoskeletal protein, and structural protein. This study provides a specific and effective tool for analyzing ISG15-modified proteins at proteome level.

Graphical abstractCellular proteins modified by ISG15 are enriched by immunoprecipitation, and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Figure optionsDownload full-size imageDownload as PowerPoint slide