<b>Proteomic profiles of seminal plasma in individuals with Secondary Infertility: insights into the involvement of oxidative stress</b>

Pooled samples from controls (n=10), patients with secondary infertility (n=10), and fertile men with high oxidative stress (n=10) were used for proteomic analysis. Equal protein concentrations (15 mg/mL) from each sample were used to normalize the groups and account for biological variability.Proteomic profiling of seminal plasma was conducted using a Finnigan LTQ linear ion trap mass spectrometer (LC-MS/MS system). Peptides were fractionated by injecting 5 μL into a high-performance liquid chromatography (HPLC) column (Phenomenex Jupiter C18 reversed-phase capillary chromatography column). Fractions were eluted in acetonitrile/0.1% formic acid at a flow rate of 0.25 μL/min and introduced online to the mass spectrometer. The micro-electrospray ion source was operated at 2.5 kV, and a full spectral scan was performed using the data-dependent multitask capabilities of the instrument to determine peptide molecular weights and amino acid sequences.Proteins were identified using various databases, including Proteome Discoverer version 1.4.1.288, Mascot (version 2.3.02; Matrix Science, London, UK), Sequest (version 1.4.0.288; Thermo Fisher Scientific, San Jose, CA, USA), and X! Tandem (The GPM, thegpm.org; version CYCLONE [2010.12.01.1]). Scaffold (version 4.0.6.1; Proteome Software Inc., Portland, OR, USA) was used for validation. Peptide identification utilised a confidence threshold of 95.0% (Eng et al., 1994), and protein identification adhered to stringent criteria with a 99.0% probability threshold, ensuring a false detection rate (FDR) below 1.0% and requiring at least two identified peptides per protein. The Protein Prophet algorithm (Serang and Noble, 2012) was used to assess the protein probabilities. Gene ontology (GO) terms from the National Centre for Biotechnology Information (NCBI; retrieved on 21 October 2013) was used to augment protein identification.