Hormonal Study

A blood sample was collected at 8 am to determine levels of T, E2, dihydrotestosterone (DHT), sex hormone-binding globulin (SHBG), LH, FSH, free T3, free T4, TSH, PRL, and insulin-like growth factor 1 (IGF-1).

Semen Analysis and In-solution Digestion

In all patients, a standard semen analysis was performed, assessing the ejaculate volume and pH, sperm count, percentage of sperm motility and morphology, according to WHO (1999) classification.

Liquified semen samples were then centrifuged at 9,200 × g for 20 minutes to obtain the seminal plasma. After the centrifugation, an aliquot was checked under microscope to confirm that no spermatozoa were present. Seminal plasma was divided in 0.5-mL aliquots, and immediately frozen at −80°C until analysis. An aliquot of each seminal sample was subjected to in solution digestion protocol, as reported in Supplemental Data.

Proteomic Analysis

The samples obtained from the digestion procedure were resuspended in aqueous trifluoroacetic acid and analyzed with an Ultimate 3000 Nano/Micro-HPLC apparatus equipped with an FLM-3000-Flow manager module, coupled with an LTQ-Orbitrap XL hybrid mass spectrometer. High-resolution MS spectra were collected in data-dependent acquisition mode. The three most intense multiple-charged ions were selected and fragmented by using collision-induced dissociation and spectra were recorded in the Orbitrap.

Data Analysis

Tandem mass spectra were analyzed by the Thermo Proteome Discoverer 1.2 program, using SEQUEST cluster (University of Washington, Seattle, WA, licensed to Thermo Electron Corp.) as a search engine against uniprot-taxonomy-9606 human protein database. Protein information obtained by SEQUEST was then analyzed using the software tool for researching annotations of proteins (STRAP), a user-friendly, open-source C# application, to annotate each protein according to the gene ontology (GO) system. STRAP automatically obtains GO terms associated with proteins in a proteomics results identification list using the freely accessible UniProtKB and EBI GOA databases. Summarized in an easy-to navigate tabular format, STRAP includes meta-information on the protein, in addition to complimentary GO terminology (27). When an individual protein was known to be assigned to more than one GO annotation, all of the annotations were counted nonexclusively (Domenico et al., 2012).

I suggest you to go for 2D PAGE for semen protein analysis

You are suggested to use two-dimensional electrophoresis (2-DE) coupled with mass spectroscopy to analyze semen proteins.

Western Blotting by SDS-PAGE gel electrophoresis using specific 1st and 2nd antibody might work