Book excerpt: Defining signaling and transcriptional regulators in a disease context is integral to the basic understanding of pathological functions of molecular pathways. Transforming growth factor-beta (TGF-ß) is a critical cytokine in wound healing and inflammatory responses, but over-activation contributes to pro-fibrotic disease processes. The goal of this work is to begin to define specific molecular facets of TGF-ß signaling in diabetic kidney fibrosis. State of the art mass spectrometric approaches were employed towards a label-free quantitative spectral counting method. These methods were applied in two contexts of TGF-ß signaling: 1) a murine model of diabetic kidney disease; and 2) a characterization of Notch4 intracellular domain-(ICD) protein complexes that we and others have shown to regulate TGF-ß signaling. In approach 1) kidney tubules were isolated from wild type and diabetic mice, proteins extracted were applied to 2-dimensional liquid chromatography fractionation followed by MS/MS (2D-LC-MS/MS) analysis and spectral counting. 476 significantly differentially abundant proteins were quantified. Of these, the SH2-SH3 adaptor GRAP (Grb2-related adaptor protein 2) was significantly elevated in diabetic tubule tissue. Findings were validated by immunohistochemistry and immunoblot. Upon further examination it was found that GRAP was elevated by TGF-ß stimulation and capable of enhancing a TGF-ß-based SMAD transcriptional reporter and enhanced secretion of pro-fibrotic matrix fibronectin in renal tubule cell cultures. These data indicate a novel role for GRAP in kidney tubule fibrosis via exacerbation of the TGF-ß pathway. Approach 2) employed affinity isolation of Notch4ICD followed by 2D-LC-MS/MS to define novel molecular partners imparting functional effects on Notch4 in proximal tubules. This approach identified the Elongin C (ElgC) ubiquitin ligase complex as a novel regulator of Notch4ICD in human proximal tubule cells. Further, ElgC regulates Notch4ICD transcriptional activity in a proteasome-dependent manner. Additionally, ElgC inhibits Notch4ICD dependent potentiation of SMAD-directed TGF-ß activity and fibronectin secretion. Co-expression of Notch4ICD and ElgC leads to the proteasome dependent degradation of a substantial portion of ectopic Notch4ICD. These findings indicate that ElgC mediates TGF-ß activity through regulating the expression levels and subsequent activity of Notch4ICD. In summary, proteomics approaches were utilized in an unbiased and targeted fashion to define novel mediators of TGF-ß signaling in kidney tubule cell biology and diabetic kidney disease.