Updated publication reference for PubMed record(s): 30541839. The 240-kb Salmonella phage SPN3US genome encodes 264 gene products, many of which are functionally uncharacterized. We have previously used mass spectrometry to define the proteomes of wild-type and mutant forms of the SPN3US virion. In this study we sought to determine if this technique was suitable for the characterization of the SPN3US proteome during liquid infection. Mass spectrometry of SPN3US-infected cells identified 232 SPN3US and 1994 Salmonella proteins. SPN3US proteins with related functions, such as proteins with roles in DNA replication, transcription and virion formation, were coordinately expressed in a temporal manner. Mass spectral counts showed the four most abundant SPN3US proteins to be the major capsid, two head ejection proteins, and the functionally unassigned protein, gp22. This high abundance of gp22 in infected bacteria contrasted with its absence from mature virions, suggesting it might be the scaffold protein, an essential head morphogenesis protein yet to be identified in giant phages. These studies demonstrate the power of mass spectral analyses for facilitating the acquisition of new knowledge into the molecular events of viral infection.