Fibroblasts were lysed in 100 mM ammonium bicarbonate and 1 M urea with ultrasonication (Branson Sonifier 250, Branson Ultrasonics Corp) at output control 3 and 30 % duty cycle for three cycles of five pulses, with 1 min on ice between each cycle. Lysates were centrifuged at 13,000 g for 30 min at 4°C. Protein concentration of the soluble fraction was measured by Bradford Protein assay (Bio-Rad) and 30 μg were used for SRM analysis. Relative quantification of peptides was carried out using a modified version of the SRM assay described in Fernández-Guerra et al. (2014 (link)). In brief all targeted proteins were monitored by detection of 2–5 tryptic peptides. Defined amounts of heavy labeled synthetic peptide analogs were spiked into the samples and used for relative quantification. The summed fragment ion peak areas for each peptide were normalized to the signal responses from the corresponding spiked heavy-labeled peptide standards. The means of the ratios for each peptide measured in control fibroblasts were set to 100% and the means for the patient fibroblast samples were expressed as the percentage of these. Samples from 3 independently grown control fibroblasts and 3 parallel cultures of patient fibroblasts were analyzed.
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