Genotypic differences greatly influence susceptibility and resistance to disease. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network properties, rather than simply as the result of individual genomic variations1. Genome sequencing efforts have identified numerous germline mutations associated with cancer predisposition and large numbers of somatic genomic alterations2. However, it remains challenging to distinguish between background, or “passenger” and causal, or “driver” cancer mutations in these datasets. Human viruses intrinsically depend on their host cell during the course of infection and can elicit pathological phenotypes similar to those arising from mutations3. To test the hypothesis that genomic variations and tumour viruses may cause cancer via related mechanisms, we systematically examined host interactome and transcriptome network perturbations caused by DNA tumour virus proteins. The resulting integrated viral perturbation data reflects rewiring of the host cell networks, and highlights pathways that go awry in cancer, such as Notch signalling and apoptosis. We show that systematic analyses of host targets of viral proteins can identify cancer genes with a success rate on par with their identification through functional genomics and large-scale cataloguing of tumour mutations. Together, these complementary approaches result in increased specificity for cancer gene identification. Combining systems-level studies of pathogen-encoded gene products with genomic approaches will facilitate prioritization of cancer-causing driver genes so as to advance understanding of the genetic basis of human cancer.

Despite intense, continued interest in global analyses of signaling cascades through mass spectrometry-based studies, the large-scale, systematic production of phosphoproteomics data has been hampered in-part by inefficient fractionation strategies subsequent to phosphopeptide enrichment. Here we explore two novel multidimensional fractionation strategies for analysis of phosphopeptides. In the first technique we utilize aliphatic ion pairing agents to improve retention of phosphopeptides at high pH in the first dimension of a twodimensional RP-RP. The second approach is based on the addition of strong anion exchange as the second dimension in a three-dimensional reversed phase ( Reversible phosphorylation plays a central role in the regulation of normal cell physiology. The strong links between aberrant signaling and human disease, along with the potential for specific inhibition of disrupted kinase activity, continue to drive efforts aimed at systematic and largescale analysis of phosphorylation in cells and tissues. Shortly after introduction of immobilized metal affinity chromatography (IMAC) 1 as an enrichment tool prior to mass spectrometry (MS) analysis (1-3), several laboratories demonstrated the feasibility of phosphopeptide identification and quantitation en masse (4 -7). In the ensuing years, despite widespread proliferation of improved and innovative (8 -14) phosphoproteomics methods, the field struggled with low specificity and poor reproducibility within and across protocols and laboratories. These limitations effectively made dynamic range a secondary issue for the majority of studies. Over the past ca. 5 years, the performance of phosphopeptide enrichment protocols and related methods has stabilized; in fact several groups (15-23) have successfully coupled phosphopeptide enrichment with online or offline fractionation schemes to achieve, in some cases, over 10,000 phosphopeptide identifications. Although these strategies provide for larger phosphosite catalogs, closer inspection reveals that the analytical efficiency, as measured by the number of phosphopeptide identifications per microgram of biological lysate consumed, has remained surprisingly consistent at Ϸ1-10 phosphopeptides/g across a wide range of sample types (Table I). One explanation is that the physicochemical properties of phosphopeptides render them less amenable to fractionation by commonly used techniques. For example, although the combination of strong cation exchange (SCX) with reversed phase (RP) has been tremendously successful for 1 The abbreviations used are: IMAC, immobilized metal affinity chromatography; AML, Acute Myeloid Leukemia; CAD, collisionally activated dissociation; ESI, electrospray ionization; FDR, False Discovery Rate; FL, FLT3 ligand; FLT3, FMS-like tyrosine kinase 3; LC/MS, liquid chromatography/mass spectrometry; Lm-OVA, Recombinant Listeria monocytogenes expressing chicken ovalbumin; ITD, internal tandem duplication; MS/MS, mass spectrometry/mass spectrometry or tandem mass spectrometry; NTA, nitrilotetra...

We tested whether surface a7 nicotinic acetylcholine receptor expression is dependent on an endogenous chaperone named Resistance to Inhibitors of Cholinesterase 3 (RIC3) by comparing RIC3 protein in rat GH4C1 and human SH-EP1 cells, which express strikingly different surface receptor levels following a7 transfection. Cloned rat RIC3 exists in at least two isoforms because of an ambiguous splice site between exons 4 and 5. Both rat isoforms permit surface a7 expression in SH-EP1 and human embryonic kidney (HEK) cells measured by a-bungarotoxin binding. Contrary to expectations, endogenous RIC3 protein expression determined by immunoblots did not differ between untransfected GH4C1 or SH-EP1 cells. siRNA against rat RIC3 exon 4 and shRNA against exons 2, 5 and 6 knocked down transfected rat RIC3 expression in SH-EP1 cells and simultaneously blocked toxin binding. However, no RNAi construct blocked binding when co-transfected with a7 into GH4C1 cells. shRNA against rat exons 2 and 5 knocked down rat RIC3 protein transfected into GH4C1 cells with a time course suggesting a protein half-life of a few days. These results suggest GH4C1 cells may possess unknown chaperone(s) allowing high surface a7 expression in the absence of known RIC3 splice variants.