Initial characterization of the human seminal plasma proteome in a fertile normogonadic man by top-down strategy
Domenico Milardi1, Federica Vincenzoni3, Giuseppe Grande2, Giampietro Antonella1, Alfredo Pontecorvi2, Massimo Castagnola3, Laura De Marinis2 & Riccardo Marana1
Human seminal plasma contains a large array of proteins required for the normal physiology of spermatozoa and fertilization. To provide informations about the physiological mechanisms of male fertility we performed proteomic studies on human seminal plasma by a top-down approach. A semen sample was collected in a fertile normospermic man (sperm concentration 60×106 per ml; progressive motility 58%; normal morphology 45%). Hormonal blood assay was performed: testosterone 4.5 ng/ml (n.r. 3.58.0), estradiol 25 pg/ml (n.r. 2040), LH 4.1 UI/l (n.r. 2.510.0), FSH 3.2 UI/l (n.r. 2.58). An aliquot of seminal plasma was mixed (1:40) with aqueous trifluoroacetic acid (TFA/H2O 0.2% v/v), and centrifuged. The upper acidic supernatant was analyzed by an Ultimate 3000 Nano/Micro-HPLC apparatus (Dionex, Sunnyvale, CA, USA) equipped with an FLM-3000-Flow manager module coupled to an LTQ Orbitrap XL apparatus (Thermo Fisher). The column was a Dionex C18 with 3 μm particle diameter. The chromatography eluents were A TFA/H2O 0.056% (v/v) and B CH3CN+0.050% TFA. The applied gradient was linear from 0 to 50% of solvent B in 60 min, at flow rate of 4.5 μl/min. The LTQ-Orbitrap mass spectrometer was operated in data dependent mode in which each full MS scan (60 000 resolving power) was followed by three MS/MS scans. The most abundant molecular ions were dynamically selected and fragmented by collision-induced dissociation (CID) using a normalized collision energy of 35%. Tandem mass spectra were searched against the Swiss-Human.fasta database using SEQUEST (Proteome Discoverer software, ThermoFisher). The results were filtered using the following criteria: XCorr versus charge 1.8, 2.5, for 2+, 3+ ions; mass accuracy 3 ppm; high value peptide confidence. A total of 63 proteins were identified in seminal plasma. Based on the molecular function and biological process of the proteins, 18 were classified as sperm structural, 16 as secreted (14 of vescicular-prostatic and 2 of epidydimal origin), 15 as testicular or sperm regulatory, 5 as sperm movement proteins. Five proteins didnt present known molecular function. This report is the first identification, based on new approach and stringent criteria, of seminal plasma proteome in a man. Proteomics may give new insight about the molecular mechanisms of reproduction and may permit the identification of molecular markers in infertile patients.