Cervicovaginal lavage (CVL)supernatants were heat inactivated for 30 minutes at 56°C before further processing. Total protein concentrations were determined using the Pierce Coomassie Plus (Bradford) Protein Assay (Thermo Scientific, Rockford, IL, USA). Sample protein content and volume were normalised with 25mM ammonium bicarbonate (ABC). Soluble proteins were precipitated using an equal volume of ice cold 30% (w/v) TCA in acetone and incubated at -20⁰C for 2 hours. Samples were centrifuged at 12,000g for 10 minutes (4⁰C) to pellet proteins. Pellets were washed three times with ice cold acetone and allowed to air dry. Further sample processing was performed as previously described (Armstrong et al, 2014) with minor modifications. Briefly, protein pellets were resuspended in 25 mM ABC, 0.05%(w/v) rapigest (Waters), reduced and alkylated. Digestion was performed with proteomic-grade trypsin (Sigma-Aldrich, St. Louis, MO, USA) at a protein:trypsin ratio of 50:1. Rapigest was precipitated by addition of trifluoroacetic acid to a final concentration of 0.5% (v/v). Peptide mixtures were analyzed by on-line nanoflow liquid chromatography using the nanoACQUITY-nLC system (Waters MS technologies) coupled to an LTQ-Orbitrap Velos (ThermoFisher Scientific, Bremen, Germany) mass spectrometer equipped with the manufacturer’s nanospray ion source. The gradient of the analytic column (nanoACQUITY UPLCTM BEH130 C18 15cm x 75µm, 1.7µm capillary column) consisted of 3-40% acetonitrile in 0.1% formic acid for 90 min then a ramp of 40-85% acetonitrile in 0.1% formic acid for 5min.