SLeX-BSA and LeX-BSA IP sperm protein-summary (MALDI-TOF-MS/MS)

Identification of Sialyl-Lewis(x) (SLeX)/ Lewis(x) (LeX)-binding proteins from human sperm membrane protein extracts using SLeX/LeX-BSA affinity chromatography followed by MALDI-TOF-MS/MS. SLeX-binding proteins on the plasma membrane of capacitated spermatozoa were identified by our established chromatographic method using SLeX-BSA neoglycoprotein affinity column followed by mass spectrometric analysis as described. In brief, capacitated spermatozoa (100×106) were washed thrice in EBSS. Non-integral, peripheral membrane-associated proteins on spermatozoa were removed by incubation of the washed spermatozoa in 1 M NaCl in PBS with gentle stirring for 10 minutes at 25ºC. The spermatozoa were then collected by centrifugation at 600× g for 10 minutes before extraction of the sperm plasma membrane proteins by the ProteoExtract Native Membrane Protein Extraction Kit (Merck, Kenilworth, NJ) according to the manufacturer’s instructions. The insoluble fraction was discarded after centrifugation at 15,000× g for 40 minutes. The supernatant was diluted in a solution of MOPS-NaOH buffer (pH 7.3) containing 0.2% Triton X-100 and 6 mM MnCl2. SLeX-BSA (Dextra, Reading, UK) conjugated sepharose beads (GE Healthcare) were used to precipitate the SLeX-binding protein from the extracted membrane protein fractions. LeX-BSA, which did not bind to human spermatozoa was used as a control.