Cryopreserved Seminal Plasma Proteomic Analysis

For proteomic analysis, seminal plasma was separated after cryopreservation. To prepare the samples for proteomic analysis, samples were thawed, and seminal plasma was separated from the sperm pellet by centrifugation at 3,000 g for 30 minutes to ensure complete removal of the cellular components. Seminal plasma samples were pooled and dissolved in 98% acetonitrile containing 0.1% trifluoroacetic acid followed by lyophilization at -80°C under vacuum for 2 days. The lyophilized sample was used to estimate the protein content. The samples were first precipitated in cold acetone, solubilized in 6 M urea, reduced with dithiothreitol and alkylated with iodoacetamide. The samples were subsequently diluted to give a urea concentration <2 M and then digested with trypsin. The tryptic digested products were subjected to a C18 clean up and then brought up in 50 μL of 1% acetic acid.