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Knowledge regarding compositions of proteomes at the proteoform level enhances insights into cellular phenotypes. A strategy is described herein for discovery of proteoform-specific information about cellular proteomes. This strategy involved analysis of data obtained by bottom-up mass spectrometry of multiple protein OGE separations on a fraction by fraction basis. The strategy was exemplified using five matched sets of lysates of uninfected and human respiratory syncytial virus-infected A549 cells. Template matching demonstrated that 67.3% of 10475 protein profiles identified focused to narrow pI windows indicative of efficacious focusing. Furthermore, correlation between experimental and theoretical pI gradients indicated reproducible focusing. Based on these observations a proteoform profiling strategy was developed to identify proteoforms, detect proteoform diversity and discover potential proteoform regulation. One component of this strategy involved examination of the focusing profiles for protein groups. A novel concordance analysis facilitated differentiation between proteoforms, including proteoforms generated by alternate splicing and proteolysis. Evaluation of focusing profiles and concordance analysis were applicable to cells from a single and/or multiple biological states. Statistical analyses identified proteoform variation between biological states. Regulation relevant to cellular responses to human respiratory syncytial virus was revealed. Western blotting and Protomap analyses validated the proteoform regulation. Discovery of STAT1, WARS, MX1, and HSPB1 proteoform regulation by human respiratory syncytial virus highlighted the impact of the profiling strategy. Novel truncated proteoforms of MX1 were identified in infected cells and phosphorylation driven regulation of HSPB1 proteoforms was correlated with infection. The proteoform profiling strategy is generally applicable to investigating interactions between viruses and host cells and the analysi

The receptor tyrosine kinase EphA2 is frequently upregulated in cancer, where it plays an intriguing dichotomous role that may lend itself to therapeutic intervention. However, much remains to be understood about how ephrin-mediated activation of EphA2 signaling can reverse its tumour-promoting ligand-independent effects and instead inhibit migration and invasion. We used quantitative phosphoproteomics to analyze EphA2 signaling in PC-3 prostate cancer cells with the aim to further elucidate its downstream signaling and pinpoint phosphorylation-dependent cellular processes that may explain its dichotomous role. Stable isotope labeling by amino acids in cell culture (SILAC) combined with liquid chromatography-coupled mass spectrometry (LC-MS/MS) was used to quantify changes in relative phosphosite abundance in response to EphA2 activation. We identified 113 significantly regulated phosphosites comprising 73 phosphoproteins, including many novel and several previously described downstream effector proteins of EphA2 signaling. More than two thirds of the regulated phosphosites showed reduced phosphorylation in response to EphA2 activation. This is consistent with EphA2 activation inhibiting oncogenic signaling pathways, including Akt and ERK-MAPK signaling. Protein association analysis using the STRING database showed that a large proportion of these differentially regulated proteins are downstream targets of Akt and ERK-MAPK, respectively. Gene ontology enrichment analysis found our dataset to be strongly enriched in proteins associated with the cytoskeleton and its regulation. Overall, our data suggest that diminished phosphorylation of cytoskeletal regulators downstream of Akt and ERK-MAPK signaling may be a major mechanism by which stimulation of EphA2 can alter cell morphology and inhibit cell motility.