The study was approved by the Ethics Committee of Freiburg University and conducted according to the Declaration of Helsinki. Cell culture and stable isotope labelling in cell culture Primary dermal fibroblasts were isolated from the skin of the four patients and, as controls, from the foreskin of circumcised 3-, 4- and Menaquinone-7 9-year-old healthy boys (Sprenger et al, 2013). mutations (Kern et al, 2009). Very little information exists on the consequences of loss of C7 at the cellular level and in relation to the cellular microenvironment. Loss of the structural function of C7 perturbs its interaction with laminin-332, which provides dermal-epidermal anchorage and is also required for keratinocyte survival (Waterman et al, 2007). C7 deficiency was associated with increased TGF-1 and accumulation of dermal ECM proteins in skin (Fritsch et al, 2008). In three-dimensional cultures analysis to carry a signal peptide (SignalP; www.cbs.dtu.dk/services/SignalP/) (Petersen et al, 2011) were counted as extracellular (Henningsen et al, 2010). These measures yielded 660 extracellular proteins in the ECM and 740 in the CM from the total list of identified proteins, with 60% being detected in both fractions (Figure 1C). The filtered proteins were Menaquinone-7 analysed based on their Swiss-Prot (SP) and Protein Information Resource (PIR) keywords (Figure 1D). While membrane proteins were enriched in the ECM fraction, proteins with enzymatic activities, such as proteases and hydrolases, were enriched in the CM, indicating the different nature of the two compartments. As expected, the terms secreted, ECM, and signal peptide, among others, were common to both groups. To assess potential differences in the abundance of extracellular proteins in a physiological setting, we SILAC Rcan1 labelled skin fibroblasts of three healthy donors (Sprenger et al, 2010). CM and ECM were purified and the data processed as outlined above. In two biological replicates of ECM and CM, respectively, we quantified 863 potential extracellular proteins, of which 40% were annotated as being extracellular based on GO terms. We observed only minor donor-specific differences in ECM and CM, indicating that three samples were sufficient to capture proteome alterations in the used experimental settings (Figure 1E; Supplementary Tables S1 and S2). In all, 95% of proteins were in the interval of 0.75 (log2 SILAC ratios), not showing altered abundance in the different samples, and biological replicates showed good reproducibility Menaquinone-7 (mutations leading to a premature stop codon (Supplementary Figure S4; Table I). The ECMs of the three controls were combined to generate a Super-SILAC mix, minimising the interindividual influences of the healthy donors (Geiger et al, 2010). This mix was then spiked in equal amounts into the ECMs purified from medium and heavy labelled RDEB cells (Figure 2A). Furthermore, Super-SILAC samples were also generated of ECMs labelled control cells using the same procedure as for the patients. Subsequently, samples and data were Menaquinone-7 processed as outlined (Figures 2A, B and ?and1C).1C). The same workflow was performed for the CM. Since the Super-SILAC mix was used as a common standard, it was possible to directly compare quantitative differences between the four RDEB and the three control samples. We quantified 587 potential extracellular proteins (45% carrying extracellular’ GO terms) in a total set of 190 LC-MS/MS analyses comprising at least two biological replicates for all conditions (Supplementary Tables S3 and S4; Supplementary Figures S5 and S6). Of these, 154 proteins were identified only in CM samples (Supplementary Table S4). On average, 45.7% of the ECM proteins and 31.7% of the CM proteins of RDEB fibroblasts showed abundance differences larger than 0.75 Menaquinone-7 (log2 SILAC ratio), much larger proportions than.