We developed a cytonuclear fractionated TPCA workflow, enabling the probing of global protein-protein interaction (PPI) networks with spatial resolution. We show that fractionation improves downstream PPI prediction, as well as increases the number of PPIs that can be detected. We apply this new workflow to study the spatiotemporal dynamics of host and viral proteins during infection with a mutant strain of HSV-1, HSV-1 RF.