Identifying the hereterodimer partner of KRT9 protein for epidermolytic palmoplantar keratoderma using co-immunoprecipitation coupled with mass spectrometer

Human sole skin sample was collected from resection margins of skin primary malignant melanoma. Most of the sample was stored in liquid nitrogen before being processed for immunoprecipitation. Only epidermis part was used for this study. Skin epidermis was minced and lysed in ice-cold solubilization buffer (P0013, Beyotime). After debris was removed, part of cleared supernatant was transfered to immunoprecipitation using prepared beads bound with mouse monoclonal anti-keratin 9 antibody (ab19124, Abcam) or normal mouse IgG (074-1807, KPL). Proteins that co-precipitated with antibody-bead complex were washed with Tris-buffered saline. Then the beads were resuspended in sample buffer and heated for 5 min at 105℃ to elute proteins. The protein samples were centrifuged and collected for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Protein bands of interest were excised, reduced with dithiothreitol, alkylated with iodoacetamide, and destained. The gel pieces were digested, and the supernatant was withdrawed for further liquid chromatography-tandem mass spectrometry analysis. Peptide sequences and intensity were analyzed using Thermo Scientific™ Q Exactive mass spectrometer. Protein identification was performed using the Mascot search engine (http://www.matrixscience.com/) by searching LC-MS-MS fragmentation patterns from each peptide against the Swiss-Prot database in Uniprot (https://www.uniprot.org/). Qualified protein sequences that meet the peptide sequencing criterion were retained for afterwards analysis.