Objective: To estimate technical variations due to protein extraction, digestion, and liquid chromatography (LC) with tandem mass spectrometry (MS) analysis and to validate this technique for proteomic analysis of head and neck squamous cell carcinoma (HNSCC).
Design: Proteins from 2 HNSCC cell lines, FADU and SCC-25, were isolated, cleaved with cyanobromide followed by enzymatic digestion with endoproteinase lysine and trypsin. Peptide mixtures were separated by ion exchange chromatography and analyzed using LC-MS. Extraction and digestion of proteins from each cell line was repeated 3 times, and each extract was analyzed 3 times, producing 9 data sets for each cell line. Data analysis was performed using in-house software to build arrays with mass, retention time, and intensities. Statistical analysis was done to determine the technical and biological variations.